CN101203231A - Methods, compositions and articles of manufacture for enhancing the viability of cells, tissues, organs and organisms - Google Patents

Abstract

The present invention concerns the use of oxygen antagonists and other active compounds for inducing stasis or pre-stasis in cells, tissues, and/or organs in vivo or in an organism overall, in addition to enhancing their survivability. It includes compositions, methods, articles of manufacture and apparatuses for enhancing survivability and for achieving stasis or pre-stasis in any of these biological materials, so as to preserve and/or protect them. In specific embodiments, there are also therapeutic methods and apparatuses for organ transplantation, hyperthermia, wound healing, hemorrhagic shock, cardioplegia for bypass surgery, neurodegeneration, hypothermia, and cancer using the active compounds described.

Description

Methods, compositions and articles of manufacture for enhancing the viability of cells, tissues, organs and organisms

Background of the invention

This application is related to U.S. Provisional Patent Applications 60/673,037 and 60/673,295, both filed April 20, 2005, and U.S. Provisional Patent Applications 60/713,073, filed August 31, 2005, US Provisional Patent Application 60/731,549 and US Provisional Patent Application 60/762,462, filed January 26, 2006, are hereby incorporated by reference in their entirety.

The Government may have rights in this invention pursuant to Grant No. GM048435 from the National Institute of General Medical Sciences (NIGMS).

1. Field of invention

The present invention relates generally to the fields of cell biology and physiology. More particularly, the present invention relates to methods, compositions and apparatus for enhancing the survivability and/or reducing damage to cells, tissues, organs and organisms, especially under adverse conditions (including but not limited to hypoxic or anaerobic conditions), using one or more substances (including substances that compete with oxygen). In certain embodiments, the present invention involves exposure of a subject to oxygen antagonists, protective metabolites, or other chemical compounds discussed herein, or precursors thereof (collectively " active compounds"), methods, compositions and devices for the treatment, prevention and diagnosis of diseases and conditions.

2. Description of related technologies

Stasis is a Latin term that means "stagnation". In the context of stasis in living tissue, the most common form of stasis involves the preservation of tissue for transplantation or repositioning. Typically, such tissues are submerged in physiological fluids, such as saline, and placed in a cold environment to reduce biochemical processes that lead to cell damage. This stagnation is incomplete and cannot be relied upon for long periods of time. In fact, the success of organ transplants and limb replacements is inversely correlated with the time it takes for an organ or limb to detach from the intact organism.

A more extreme form of stasis involves placing the entire organism in a state colloquially known as "arrest." Although still largely considered within the realm of science fiction, it has gained a bad reputation when the wealthy seek to be frozen after death in the expectation that future medical breakthroughs will allow them to be resurrected and cured of their deadly diseases. It is said that more than a hundred people have been refrigerated since the first attempt in 1967, and that more than a thousand have made legal and financial arrangements for human cold storage with one of several organizations, such as the Alcor Life Extension Foundation. Such methods involve administration of anti-ischemic drugs, cryopreservation and perfusion of whole organisms with cryosuspension fluids. This form of biostasis has not been demonstrated to be reversible.

The effect of inducing stagnation in a biological substance as contemplated by the compositions, methods or articles of manufacture described herein is characterized by inducing or initiating stagnation, in whole or in part, followed by a period of stasis followed by a return to normal or near normal Physiological state, or a state that a person skilled in the art considers to be better than the state of a biological substance that has never undergone stagnation. Stasis can also be defined as what it is not . Biostasis is not any of the following states: sleep, coma, death, being anesthetized, or grand malseizure.

There are numerous reports of individuals surviving from marked pulse and respiratory arrest after exposure to hypothermic conditions, usually in cold water immersion. Although not fully understood by scientists, it is possible that the ability to survive this condition stems from what is known as the "mammalian diving reflex." This reflex is thought to stimulate the vagus nervous system, which controls the lungs, heart, larynx, and esophagus in order to protect vital organs. Presumably, the cold water stimulates nerve receptors on the skin causing blood to shunt to the brain and on to the heart, and away from the skin, gastrointestinal tract, and extremities. At the same time, protective reflex bradycardia, or slowing of the heartbeat, conserves the body's diminished supply of oxygen. Unfortunately, the manifestation of this reflex is not the same in all people and is believed to be a factor in only 10-20% of cold water immersion cases.

Compositions and methods that do not rely solely or at all on low temperature and/or oxygen may be applicable in the field of organ preservation, as well as tissue or cell preservation. Cells and tissues are currently preserved at cryogenic temperatures, usually at temperatures substantially below freezing, such as in liquid nitrogen. However, dependence on temperature can cause problems because the instruments and reagents used to generate such low temperatures may not be readily available when needed, or they may need to be replaced. For example, tissue culture cells are often stored for a period of time in tanks containing liquid nitrogen; however, these tanks often require periodic replacement of the liquid nitrogen in the unit, or the liquid nitrogen will be depleted and temperature will not be maintained. Also, due to the freeze/thaw process, damage to cells and tissues can occur. Accordingly, improved techniques are needed.

Furthermore, the lack of ability to control cellular and physiological metabolism in whole organisms subjected to trauma such as amputation and hypothermia is a key shortcoming in the medical field. On the other hand, the anecdotal evidence discussed above strongly suggests that, if properly understood and regulated, it is possible to induce arrest in cells, tissues, and whole organisms. Therefore, there is a great need for improved methods for controlling metabolic processes, especially under traumatic conditions.

Summary of the invention

Accordingly, the present invention provides methods, compositions, articles of manufacture and apparatus for inducing arrest in cells, tissues and organs located in or derived from an organism, as well as in the organism itself. These methods, compositions, articles of manufacture, and apparatus are useful for protecting biological material, and for preventing, treating, or diagnosing diseases and disorders in living things. Moreover, these methods may induce arrest directly by themselves, or they may induce arrest by themselves, but by enhancing the ability of biological substances to enter arrest in response to injury or disease conditions, for example by reducing the time or level of injury or disease required to obtain arrest. , to act indirectly. This condition may be referred to as pre-stasis. Details of this application, as well as other uses, are described below.

The present invention is based in part on studies using compounds which have been determined to have a protective function, and are thus useful as protective agents. Furthermore, the overall results of studies involving different compounds indicate that compounds with available electron-donor centers are particularly effective in inducing arrest or pre-arrest. Moreover, these compounds induce reversible stagnation, meaning they are not so toxic to a particular biological substance that it dies or decomposes. It is further contemplated that the present invention may be used to enhance the viability and/or prevent or reduce damage to biological material that may be subjected to or subjected to adverse conditions.

In particular embodiments, the methods of the invention are used to induce arrest or pre-arrest in biological matter, e.g. cells, tissues, organs and/or organisms, following injury (e.g. traumatic injury) or following onset or progression of a disease, In order to protect the biological material from harm associated with the injury or disease before, during or after treatment of the injury or disease. In additional embodiments, the methods of the present invention are used to induce or promote arrest or pre-arrest in biological matter prior to undergoing a damaging event (e.g., elective surgery) or prior to disease onset or progression, in order to protect said biological matter from Injury related to adverse conditions such as injury or disease. Such methods are generally referred to as "pretreatment" with the active compound. Pretreatment includes methods wherein the biological material is provided with an active compound before and during, and before, during and after the biological material is subjected to adverse conditions (e.g., onset or progression of injury or disease), and wherein the biological material is provided only after the biological material is subjected to adverse conditions Methods of providing active compounds to the biological material were previously provided.

According to various embodiments of the methods of the present invention, arrest may be induced by treating the biological substance with an active compound that itself directly induces arrest, or alternatively, by treating the biological substance with an active compound that does not itself induce arrest, but instead, promotes or enhances the response of the biological substance to another Stimuli, such as, but not limited to, injury, disease, hypoxia, excessive bleeding, or treatment of biological substances with additional active compounds to achieve stasis required capacity or reduce the time required to do so are induced by treatment of biological substances.

In a particular embodiment, the treatment with the active compound induces "pre-arrest", which refers to a state of hypometabolism in which biological substances must transition to arrest. Pre-arrest is characterized by a reduction in metabolism within the biomaterial that is less than that defined as arrest. In order to achieve stasis with active compounds, the biomass must necessarily pass through a stepwise hypometabolic state transition in which the oxygen consumption and CO ₂ production in the biomass are reduced by less than a factor of two. Such a continuous process, in which metabolism or cellular respiration is reduced to less than twofold by active compounds, can be described as a "pre-arrested" state.

When stagnation involves a two-fold reduction in _CO2 production or _O2 consumption (i.e., to 50% or less), these parameters are directly measured in the biomass by methods known to those skilled in the art (wherein a lower than 2-fold ) indicates pre-stagnation. Accordingly, certain measurements of carbon dioxide and oxygen levels in the blood, as well as other markers of metabolic rate familiar to those skilled in the art, including but not limited to blood _pO2 , _VO2 , _pCO2 , pH, and lactate levels, can be used in the present invention Monitor for onset or progression of pre-arrest. Pre _- arrest may be associated with at least 10%, 15%, 20% _, 25%, 30%, A 35%, 40%, 45% or 50% reduction in _CO2 emission, which refers to the amount of _CO2 released from the lungs, was associated _. Also, in various embodiments, pre-arrest is characterized by a reduction in one or more indicators of metabolic activity of less than or equal to 1%, 2%, 5%, 10% compared to normal physiological state , 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 49%. In additional embodiments, pre-arrest is characterized by its ability to enhance or facilitate entry into arrest in response to another stimulus (where the other stimulus may include prolonged treatment with the same active agent), or it enhances the viability of the biological substance Or the ability to protect a biological substance from injury, the onset or progression of disease, or hemorrhage, especially hemorrhage that can lead to irreversible tissue damage, hemorrhagic shock, or lethality.

While the methods of the present invention, which are explicitly exemplified herein by way of example, may involve the induction of "arrest", it should be understood that these methods can be readily adapted to induce "pre-arrest", and such methods of inducing pre-arrest are contemplated by the present invention. Moreover, the same active compounds used to induce arrest can also be used to induce pre-arrest by providing them to the biological substance at a lower dose than that used to induce arrest and/or for a shorter time than that used to induce arrest. This is achieved at the time of induced stagnation.

In certain embodiments, the present invention involves exposing a biological material to an amount of an agent so as to effect stagnation of the biological material. In some embodiments, the present invention relates to a method for inducing arrest in biological material in vivo, the method comprising: a) identifying an organism in which arrest is desired; and, b) exposing said organism to an effective amount of an oxygen-antagonizing Agents or other active compounds are used to induce stagnation in biological substances in vivo. Inducing "stagnation" in biological matter means that the matter is alive, but it has one or more of the following characteristics: the rate or amount of carbon dioxide produced by the biological matter is reduced by at least twofold; the rate or amount of oxygen consumed by the biological matter is reduced At least a two-fold reduction (i.e., 50%); and a reduction in movement or activity of at least 10% (applicable only to motile cells or tissues, such as sperm cells or the heart or extremities, or when arrest is induced in a whole organism) (collectively referred to as "Indicators of Cellular Respiration"). In certain embodiments of the invention, it is contemplated that the rate of oxygen consumption by the biomass is about, at least about, or at most about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10 -, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 100-, 150-, 200-, 250-, 300-, 350-, 400-, 450-, 500-, 600-, 700-, 800-, 900-, 1000-, 1100-, 1200-, 1300-, 1400-, 1500-, 1600-, 1700- , 1800-, 1900-, 2000-, 2100-, 2200-, 2300-, 2400-, 2500-, 2600-, 2700-, 2800-, 2900-, 3000-, 3100-, 3200-, 3300, 3400- , 3500-, 3600-, 3700-, 3800-, 3900-, 4000-, 4100-, 4200-, 4300-, 4400-, 4500-, 5000-, 6000-, 7000-, 8000-, 9000-, or 10000 -fold or more reduction, or may arise from any range therein. Alternatively, it is contemplated that embodiments of the present invention may use about, at least about, or at most about , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% or more reduction in biomass oxygen consumption rate is discussed. It is contemplated that any assay for measuring oxygen consumption may be employed, and a typical assay will involve utilizing a closed environment and measuring the difference between the oxygen put into the environment and the oxygen remaining in the environment after a period of time. It is further contemplated that carbon dioxide production can be measured to determine the amount of oxygen consumption by the biomass. Thus, there may be a reduction in carbon dioxide production, which corresponds to the reduction in oxygen consumption discussed above.

In the methods of the invention, stagnation or pre-stagnation is transient and/or reversible, meaning that the biomass no longer exhibits the characteristics of stagnation at some later point in time. In some embodiments of the invention, compounds unsuitable as oxygen antagonists are administered instead of oxygen antagonists. It is contemplated that the methods discussed with respect to oxygen antagonists can be applied to any compound that is an oxygen antagonist, a protective metabolizer, a compound having the structure of formula I, II, III or IV, any other compound discussed herein, or a salt or precursor thereof. body. Compounds that carry out any of the methods of the invention and qualify as oxygen antagonists, protective metabolizers, compounds having the structure of Formula I, II, III or IV, or salts or precursors thereof, will be considered "active compounds". In particular embodiments, induction of arrest is desired, in which case the compound may be referred to as an "activity arrest compound". It is contemplated that in some embodiments of the invention, the method is achieved by inducing arrest. For example, a method of treatment may involve the induction of arrest, in which case the active compound is an activity arrest compound. It is specifically contemplated that, in the embodiments in which active compounds are discussed, the invention includes, and may be limited to, oxygen antagonists.

In certain embodiments of the invention, biological material is treated with an active compound which does not induce stagnation itself (at least not at the level and/or time period provided), but rather induces the biological material to enter A pre-arrested state with therapeutic benefit and enhanced ability of biological substances to achieve arrest in response to other stimuli such as injury, disease state, or treatment with other active compounds or with the same active compound if longer or in greater doses.

The term "biological matter" refers to any living biological material (in preferred embodiments mammalian biological material), including cells, tissues, organs and/or organisms, and any combination thereof. It is contemplated that stasis can be induced in a part of an organism (eg, a cell, a tissue, and/or an organ or organs), whether that part remains within or removed from the organism, or leaves the whole organism in a state of arrest. Furthermore, both homogeneous and heterogeneous cell populations may be the subject of embodiments of the present invention, taking into account both cellular and tissue aspects. The term "in vivo biological material" refers to biological material within the body, ie still within or attached to an organism. Furthermore, the term "biological matter" is to be understood as a synonym for the term "biological material". In certain embodiments, it is contemplated that one or more cells, tissues or organs are isolated from the organism. The term "isolated" may be used to describe such biological material. Consider that stagnation can be induced in isolated biological material.

An organism or other biological substance in need of stasis is one in which the stasis of all or part of the organism can yield a direct or indirect physiological benefit. For example, a patient at risk for hemorrhagic shock could be deemed to require arrest, or a patient who would undergo coronary artery bypass surgery could benefit from protecting the heart from ischemia/reperfusion injury. Other applications are discussed throughout this application. In some cases, a biological or other biological substance is identified or determined to require stasis based on one or more tests, screenings, or assessments that indicate a condition or disease, or risk of a condition or disease, that may be prevented or treated by performing the stasis. Alternatively, consideration of the patient's medical history or family medical history (asking the patient) may yield information on the need for stasis of the organism or other biological substance. It will be apparent to those skilled in the art that one application of the invention would be to reduce the overall energy requirements of biological materials by inducing stasis.

Alternatively, organisms or other biological matter will require active compounds to enhance viability. For example, a patient may be in need of treatment for injury or disease or any other application discussed herein. Based on the methods discussed in the preceding paragraphs, for example by considering a patient's medical or family medical history, they may be determined to be in need of enhanced viability or treatment.

The term "oxygen antagonist" refers to a substance that competes with oxygen for its utilization by biological matter that requires oxygen to live ("oxygen-utilizing biological matter"). Oxygen is commonly used or required by a variety of cellular processes that produce a major source of energy readily available to biological matter. The oxygen antagonist effectively reduces or eliminates the amount of oxygen available to the oxygen-utilizing biological species, and/or the amount of oxygen available to the oxygen-utilizing biological species. In one embodiment, an oxygen antagonist can directly effect its oxygen antagonism. In another embodiment, the oxygen antagonist can achieve its oxygen antagonism effect indirectly.

Direct oxygen antagonists compete with molecular oxygen for binding to molecules (eg, proteins) that have an oxygen binding site or ability to bind oxygen. Antagonism can be competitive, noncompetitive or anticompetitive, as known in the art of pharmacology or biochemistry. Examples of direct oxygen antagonists include, but are not limited to, carbon monoxide (CO), which competes with oxygen for binding to hemoglobin and to cytochrome c oxidase.

In the absence of direct competition for oxygen binding to oxygen-binding molecules, indirect oxygen antagonists affect the availability or delivery of oxygen to cells that use oxygen to generate energy (eg, in cellular respiration). Examples of indirect oxygen antagonists include, but are not limited to, (i) carbon dioxide, which reduces hemoglobin (or other globulin, such as myoglobin) binding to oxygen-utilizing animals through a process known as the Bohr effect capacity of oxygen in the blood or hemolymph, thereby reducing the amount of oxygen delivered to the oxygen-utilizing cells, tissues, and organs of the organism, thereby reducing the availability of oxygen to oxygen-utilizing cells; (ii) carbonic acid Anhydrase inhibitors (Supuran et al., 2003, which is incorporated by reference in its entirety), which increase the concentration of carbon dioxide by inhibiting the hydration of carbon dioxide in the lungs or other respiratory organs, thereby reducing hemoglobin (or other globulin, such as myoglobin) bind to the oxygen in the blood or hemolymph of an oxygen-utilizing animal, thereby reducing the amount of oxygen delivered to the organism's oxygen-utilizing cells, tissues, and organs, thereby reducing the utilization of oxygen Cellular availability of oxygen; and (iii) molecules that bind oxygen and shield oxygen from binding to or make oxygen unavailable to bind to oxygen binding molecules, including but not limited to oxygen chelators, antibodies, and the like.

In some embodiments, the oxygen antagonist is both a direct oxygen antagonist and an indirect oxygen antagonist. Examples include, but are not limited to, compounds, drugs or agents that directly compete for oxygen binding to cytochrome C oxidase and are also capable of binding and inhibiting the enzymatic activity of carbonic anhydrase. Thus, in some embodiments, an oxygen antagonist inhibits or reduces the amount of cellular respiration occurring in a cell, eg, by binding to a site on cytochrome C oxidase that would otherwise bind oxygen. Cytochrome C oxidase specifically binds oxygen and then converts it to water. In some embodiments, such binding to cytochrome c oxidase is preferably releasable and reversible (e.g., has an in vitro dissociation constant _Kd of at least 10 ⁻² , 10 ⁻³ or 10 ⁻⁴ M, and has An in vitro dissociation constant K _d of not more than 10 ⁻⁶ , 10 ⁻⁷ , 10 ⁻⁸ , 10 ⁻⁹ , 10 ⁻¹⁰ or 10 ⁻¹¹ M). In some embodiments, oxygen antagonists are assessed by measuring ATP and/or carbon dioxide output.

The term "effective amount" means an amount that achieves the stated result. In certain methods of the invention, an "effective amount" is, for example, an amount that induces arrest in a biological material in need of arrest. In other methods, an "effective amount" is, for example, an amount that induces pre-arrest in a biological material in need of arrest or in need of enhanced viability. In additional embodiments, an "effective amount" may refer to an amount that increases the viability of an organism or other biological material. This can be determined (or assumed) based on a comparison or pre-comparison with untreated biological material or biological material treated with a different dose or regimen that does not result in viability.

It is understood that when arrest is induced in a tissue or organ, an effective amount is that amount that induces arrest in the tissue or organ as determined by the collective amount of cellular respiration of the tissue or organ. Thus, for example, if the level of cardiac oxygen consumption (a collection of cells of the heart) is reduced by at least about 2-fold (i.e., 50%) after exposure to a particular amount of a certain oxygen antagonist or other activity arresting compound, it should be understood that the particular The amount is an effective amount to induce arrest in the heart. Similarly, an effective amount of an agent that induces arrest in an organism is that amount that is assessed with respect to a common or aggregate level of a particular parameter of arrest. It should also be understood that when inducing arrest in an organism, an effective amount is one that generally induces arrest in the entire organism, unless only specific parts of the organism are targeted. It is also understood that an effective amount may be an amount sufficient to induce arrest by itself, or it may be an amount sufficient to induce arrest in combination with other agents or stimuli, such as another active compound, injury or disease state.

The concept of an effective amount of a particular compound, in some embodiments, relates to how much available oxygen can be utilized by biological matter. Typically, stagnation can be induced when about 100,000 ppm or less of oxygen is present in the absence of any oxygen antagonists (room air has about 210,000 ppm of oxygen). Oxygen antagonists are used to alter how much oxygen is available. At an oxygen concentration of 10 ppm, stagnation was induced. Thus, stagnation can be induced despite the fact that the actual oxygen concentration to which the biomass is exposed may be higher, or even substantially higher, than 10 ppm due to the competitive effect of oxygen antagonists with the oxygen-metabolizing proteins necessary for oxygen binding in the biomass. In other words, an effective amount of an oxygen antagonist reduces the available oxygen concentration to the point where the oxygen present is not available. This occurs when the amount of oxygen antagonist reduces the effective oxygen concentration below the _Km for oxygen binding to essential oxygen metabolizing proteins (ie equivalent to 10 ppm oxygen). Thus, in some embodiments, the oxygen antagonist reduces the effective concentration of oxygen by about or at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 60-, 70-, 80-, 90-, 100-, 150-, 200-, 250-, 300-, 350- , 400-, 450-, 500-, 600-, 700-, 800-, 900-, 1000-, 1100-, 1200-, 1300-, 1400-, 1500-, 1600-, 1700-, 1800-, 1900 -, 2000-, 2100-, 2200-, 2300-, 2400-, 2500-, 2600-, 2700-, 2800-, 2900-, 3000-, 3100-, 3200-, 3300, 3400-, 3500-, 3600 -, 3700-, 3800-, 3900-, 4000-, 4100-, 4200-, 4300-, 4400-, 4500-, 5000-, 6000-, 7000-, 8000-, 9000-, or 10000-fold or more , which can originate from any of these ranges. Alternatively, it is contemplated that embodiments of the present invention may be discussed in terms of a reduction in effective oxygen concentration of about, at least about, or at most about 50, 51, 52, 53, 54, 55, 56, 57 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, or any range derivable therein. It should be understood that this is another way of indicating decreased cellular respiration.

Also, in some embodiments, arrest can be measured indirectly by a decrease in the core body temperature of an organism. It is contemplated that a decrease in core body temperature of about, at least about, or at most about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 mm or more, or any range that may be derived therefrom. In some embodiments of the invention, hypothermia can be induced, such as moderate hypothermia (at least 10 mm hypothermia) or severe hypothermia (at least 20 mm hypothermia).

Furthermore, effective amounts can be expressed as concentrations with or without qualification for the length of exposure. In some embodiments, it is generally contemplated that in order to induce stasis or achieve other stated goals of the invention, the biological material is exposed to an oxygen antagonist or other reactive compound for about, at least about, or at most about 5, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55, 60 seconds, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days , 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, December, 1, 2, 3, 4, 5, 6, 7 , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years, and any combination or range derivable therein. It is further contemplated that the amount of time may be indeterminate depending on the reason or purpose of administering the oxygen antagonist or other active compound. Thereafter, the biological material can continue to be exposed to the oxygen antagonist or other active compound, or, in other embodiments of the invention, the biological material can no longer be exposed to the oxygen antagonist or other active compound. This latter step can be accomplished by removing or effectively removing the oxygen antagonist or other reactive compound from the presence of the biological material in which it is desired to stagnate, or the biological material can be removed from the environment containing the oxygen antagonist or other reactive compound. Finish. Furthermore, any active compound may be exposed or provided to the biological material continuously (exposed over an uninterrupted period of time), intermittently (exposed over multiple periods) or periodically (exposed over regular periods of time). Based on this difference, the dosages of the active compounds may be the same or they may vary. In some embodiments, by every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, December, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years, or any range derivable therein, provided to or exposed to biological material with active compound 1, 2, 3, 4 , 5, 6, 7, 8, 9 or 10 times to provide the active compound periodically.

Also, in some embodiments of the invention, the biological material is exposed to or provided with the active compound for a sustained period of time, wherein "for a period of time" means a period of at least about 2 hours. In other embodiments, the biological material may be exposed to or provided with the active compound in a sustained manner for more than one day. In this case, the active compound is provided to the biological material in a continuous continuous manner. In certain embodiments, the biological material is exposed to or provided with the active compound continuously, intermittently (exposure over multiple periods), or periodically (exposure in a repeated regular pattern). , 4, 5, 6, 7, 8, 9, 10, 11, 12 or more hours (or any range that may be derived therefrom), 2, 3, 4, 5, 6, 7 days and/or 1, 2, 3, 4, 5 weeks and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, December and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years (or any range that may derive therein).

In some embodiments, the organism can be treated before and during; before, during and after; during and after; A substance is exposed to or provides an active compound. Such exposure may or may not be continuous.

The doses of active compound administered in these different ways may be the same or they may vary.

Also, in certain embodiments, the active compound may be provided in a continuous, sustained manner at levels considered "low", meaning levels less than those that cause metabolic flexibility such as visible CBT, heart rate, or _CO2 or _O2 consumption or The level at which the amount of drop is generated.

In certain embodiments, in an unfavorable physiological branch such as apnea ("a period of time during which breathing is so markedly reduced that the subject takes 10% or fewer breaths"), lack of observable skeletal muscle Prior to locomotion, dystonia and/or hyperactivity, biological substances are exposed to or provided with active compounds, eg metabolites, in amounts exceeding what was previously understood as the maximum tolerated dose. Such amounts may be particularly relevant in increasing viability in some embodiments of the invention, eg, for increasing the chance of surviving adverse conditions such as those that would induce death from hemorrhagic shock.

The active compounds of the present invention induce a physiological state that enhances viability in organisms in need of enhanced viability and comprise a set of observable physiological changes in response to an effective dose of the active compound, which changes may include hyperpnea, One, more, or both of apnea and concomitant or subsequent loss of neuromuscular tone or voluntary control of movement and sustained heartbeat. A transient and measurable change in arterial blood color may also be observed. Hyperpnea refers to rapid shallow breathing. Apnea refers to the cessation or reduction of breathing as described above.

In certain embodiments, the subject becomes apneic, which is manifested by cessation of breathing followed by apneic breathing after a short time. In rats, this occurs after about 20 seconds. It is thus contemplated that a subject induced to apnea may exhibit 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% of the number of breaths following exposure to the active compound. Thereafter, the subject may have occasional breaths, which may be considered apneic breaths. In certain embodiments of the invention, the apnea lasts until the subject is no longer exposed to the active compound.

In some embodiments of the invention, an effective amount can be expressed as _LD50 , which refers to "median lethal dose" which means the administered dose that kills half of _the animal population (causes 50% mortality). Also, in other embodiments, the effective amount may be independent of the weight of the biomass ("weight-independent"). For example, in rodents and humans, the _LD50 of _H2S gas is about 700 ppm before adverse physiological effects appear. Also, in some embodiments of the invention, increased viability generally refers to longer survival, which is an embodiment of the invention.

The invention also relates to a method for inducing apnea in an organism, which method comprises administering a biologically effective amount of an active compound. In certain embodiments, the organism also does not exhibit any skeletal muscle movement induced by the active compound. It is specifically contemplated that the organism may be a mammal, including a human. In other embodiments, the effective amount exceeds an amount considered lethal. In further embodiments, the concentration may be lethal, yet the duration of exposure may be about, at least about, or at most about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 seconds, 1 , 2, 3, 4, 5 minutes, or longer (or any range that may be derived therefrom or other time-wise period specified in this disclosure). In particular embodiments, the mammal is exposed to at least about 600 ppm of reactive gaseous compounds, such as _H2S .

Also, in certain embodiments, there is a step of identifying animals in need of treatment. In further embodiments, there is the step of observing apnea in the organism. In still further embodiments, the methods involve obtaining a blood sample from an organism and/or assessing the color of the organism's blood. It has been observed that exposure to _H2S changes the color of blood from mammals; the color changes from bright red to a darker red wine color and then brick red. Assessing color can be accomplished visually without the need for an instrument or machine, while in other embodiments an instrument, such as a spectrophotometer, can be used. Also, blood samples can be obtained from organisms and other types of analysis can be performed on them. Alternatively, a blood sample may not be required, but blood may be evaluated without a sample. For example, a modified pulse oximeter that shines IR or visible light through a finger can be used to monitor blood color changes.

In certain embodiments, exposing a biological substance to an effective amount of an active compound that does not cause arrest or pre-arrest. In some embodiments, there may be no evidence of reduction in oxygen consumption or carbon dioxide production in the presence of the active compound.

In additional embodiments, the organism may be exposed to the active compound while sleeping. Also, as discussed above, the exposure may be regular, such as daily (meaning at least one exposure per day).

It is specifically contemplated that in some embodiments the active compound is provided to the subject via a nebulizer. This can be applied with any embodiment of the invention. In some cases, nebulizers are used to manage hemorrhagic shock. In a further embodiment, the active compound is provided to the subject as a single dose. In particular instances, the single dose or multiple doses are doses that will induce apnea in a subject. In some embodiments, the subject is administered at least about 1,000, 2,000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 11,000, 12,000 or more ppm of _H2S gas. The exposure time can be any time discussed herein, including about or about at most 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 minutes or less (or any time that can be derived therefrom any range).

In a further embodiment, the rate of metabolism of the biological substance may be altered following exposure to the active compound. In certain embodiments, the RQ ratio (CO ₂ production/O ₂ consumption) of the biomass is altered following exposure to the active compound. This can occur after initial or repeated exposure or after acute exposure. In some embodiments, the RQ ratio decreases after exposure. Said reduction may be about, at least about or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80% or more, or Any reduction in scope may result therefrom. The reduction may be the result of increased _O2 consumption, a decrease in _CO2 production relative to _O2 consumption.

In some embodiments, no physiological changes are observed in the biological material exposed to the active compound, except that its RQ ratio changes after exposure. Accordingly, in some embodiments of the invention, the methods involve measuring changes in the RQ ratio in a subject. This can occur before and/or after exposure to the active compound.

Thus, in some embodiments of the invention, stasis is induced, and a further step in the methods of the invention is to maintain the relevant biological material in stasis. This can be accomplished by continued exposure of the biological material to oxygen antagonists or other reactive compounds and/or exposure of the biological material to non-physiological temperatures or other oxygen antagonists or other reactive compounds. Alternatively, the biological material can be placed in a preservative or solution, or exposed to normoxic or hypoxic conditions. It is contemplated that the biomass can be held in a stagnant state for about, at least about, or at most about 30 seconds, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months , 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years and may originate therein any combination or range. It is also contemplated that other changes in the environment may be made in addition to or instead of changing the temperature, such as pressure changes or enabling cryoprotection or cryopreservation environments (eg, glycerol-containing environments).

It will be appreciated that different lengths of arresting time may be required for "arresting" with respect to whole animals versus "arresting" for cells or tissues. Thus, for a human subject, such as a subject undergoing surgical treatment, a subject being treated for malignant hyperthermia, or a trauma patient, a lag time of up to 12, 18 or 24 hours is generally contemplated. For non-human animal subjects, eg, non-human animals transported or held for commercial purposes, a period of 2 or 4 days, 2 or 4 weeks or longer of arrest is contemplated.

The term "exposing" is used in its ordinary sense to denote subjecting a biological substance to an oxygen antagonist or other reactive compound. In some embodiments, this can be accomplished by contacting the biological material with an oxygen antagonist or active compound. In other embodiments, this is accomplished by contacting the biological material with an active compound that may or may not be an oxygen antagonist. In the context of cells, tissues or organs in vivo, "exposing" can further mean "uncovering" these substances so that they can come into contact with oxygen antagonists or other active compounds. For example, this can be done surgically. Exposure of biological substances to oxygen antagonists or other reactive compounds can be accomplished by incubation in or with antagonists (including immersion), perfusion or infusion with antagonists, injection of biological substances with oxygen antagonists or other reactive compounds, or exposure to oxygen antagonists. Agents or other active compounds are applied to biological substances. Also, inhalation or ingestion of the oxygen antagonist or other active compound is contemplated, or any other route of drug administration to use the oxygen antagonist or other active compound, if arrest of the whole organism is desired. Also, the term "provide" means "supply" according to its usual and ordinary meaning. It is contemplated that the compound can be provided to the biological material in one form and can be converted by a chemical reaction to its form as the active compound. According to the present invention, the term "providing" includes the term "exposure" in the context of the term "effective amount".

In some embodiments, an effective amount is characterized as a sublethal dose of an oxygen antagonist or other active compound. In the context of inducing stasis of cells, tissues or organs (not whole organisms), "sublethal dose" means a single administration of less than half of the oxygen antagonism that would cause the death of cells in at least a majority of the biological matter within 24 hours of administration Oxygen antagonist or active compound in the amount of agent or active compound. If arrest of the whole organism is desired, "sublethal dose" means a single administration of an oxygen antagonist or active compound that is less than half the amount of oxygen antagonist that would cause death of the organism within 24 hours of administration. In some embodiments, an effective amount is characterized as a near-lethal dose of the oxygen antagonist or active compound. Similarly, in the context of inducing stasis of cells, tissues or organs (not whole organisms), "near-lethal dose" means that within 25% of a single administration of an inhibitor that will cause at least a majority of cells to die within 24 hours of administration. amount of oxygen antagonist or active compound. If stasis of the whole organism is desired, "near-lethal dose" means that a single administration of an oxygen antagonist or active compound is within 25% of the amount of inhibitor that would cause death of the organism within 24 hours of administration. In some embodiments, the sublethal dose is administered by administering a predetermined amount of the oxygen antagonist or active compound to the biological material. In particular, this can be achieved with any active compound.

It is also contemplated that, in some embodiments, an effective amount is characterized as a supralethal dose of an oxygen antagonist or other active compound. In the context of inducing stasis of cells, tissues or organs (not whole organisms), "supraletal dose" means a single administration of at least 1.5 times (1.5x) that will cause at least most of the cells in the biological The amount of active compound that dies. If stasis of the whole organism is desired, "supraletal dose" means a single administration of at least 1.5 times the amount of active compound that would cause death of the organism within 24 hours of administration. Specifically contemplated, supralethal doses may be about, at least about, or at most about 1.5x, 2x, 3x, 4x, 5x, 10x, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, 100x, 150x, 200x, 250x, 300x, 400x, 500x, 600x, 700x, 800x, 900x, 1000x, 1100x, 1200x, 1300x, 1400x, 1500x, 1600x, 1700x, 1800x, 1900x, 2000x, 3000x, 4000x, 5000x, 800x 9000x, 10,000x or more, or any range derivable therein, is the amount of active compound that will cause the death of at least a majority of the cells in the biological material (or the whole organism) within 24 hours of application.

The amount of active compound provided to the biological material can be about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260 ,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500 ,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750 , 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000mg , mg/kg or mg/m2 or any range derivable therein. Alternatively, said amounts may be expressed as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 , 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 ,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300 ,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,520,530,540 ,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790 , 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 mM or M or may be derived therefrom any range.

In some embodiments, an effective amount is administered by monitoring the amount of oxygen antagonist or other active compound administered alone or in combination with monitoring the duration of administration of the oxygen antagonist or other active compound , monitoring the physiological response of biological substances to the administration of oxygen antagonists or other active compounds (such as pulse, respiration, pain response, movement or activity, metabolic parameters such as cellular energy production or redox state, etc.), and reducing, interrupting or stopping the administration The compound, when the lower or upper limit of the change in the response is measured, and so on. Moreover, these steps may additionally be employed in any of the methods of the present invention.

Tissues that are in stagnation or have undergone stagnation can be used in many applications. For example, they may be used for infusion or transplantation (therapeutic applications, including organ transplantation); for research purposes; for screening assays to identify, characterize or produce other compounds that induce arrest; for testing samples from which tissues are obtained (diagnostic applications); for preserving or preventing damage to tissues in an organism to be returned to the tissue source (prophylactic applications); and for preserving or preventing damage to them during transport or storage. Details of such applications, as well as other uses, are described below. The term "isolated tissue" means tissue that is not located within an organism. In some embodiments, the tissue is all or part of an organ. The terms "tissue" and "organ" are used according to their usual and ordinary meanings. Although tissue is composed of cells, it is understood that the term "tissue" refers to a collection of similar cells that form a defined type of structural material. Also, organs are special types of tissues.

The present invention relates to a method for inducing arrest in isolated tissue comprising: a) identifying a tissue in which arrest is desired; and b) exposing the tissue to an effective amount of an oxygen antagonist to induce arrest.

The compositions, methods and articles of manufacture of the invention are useful on biological material that is to be transferred back to the biological material source (autologous) donor organism or to a different recipient (heterologous) subject. In some embodiments, biological material is obtained directly from a donor organism. In other embodiments, the biological material is placed in culture prior to exposure to the oxygen antagonist or other active compound. In some cases, biological material is obtained from donor tissue that has undergone extracorporeal membrane oxygenation, a technique performed to help preserve biological material, prior to removal of the biological material. Furthermore, the method comprises administering or transplanting a biological substance in which stasis is induced to a living recipient organism.

In some embodiments, the organ or tissue to be retrieved and then transplanted is exposed to an oxygen antagonist or other active compound while still in the donor subject. It is contemplated that in some cases the donor's vasculature is used to expose the organ or tissue to oxygen antagonists or other active compounds. This can be done if the heart is still beating or if a pump, catheter or syringe is available to administer the oxygen antagonist or other active compound into the vasculature for delivery to the organ or tissue.

The methods of the invention also involve inducing arrest in isolated tissue comprising incubating the tissue with an oxygen antagonist or active arresting compound that produces hypoxic conditions for an effective amount of time for the tissue to enter arrest.

Cells that are in arrest or that have undergone arrest can be used in many applications. For example, they can be used for infusion or transplantation (therapeutic applications); for research purposes; for screening assays to identify, characterize or produce other compounds that induce arrest; for testing samples from which cells are obtained (diagnostic applications) ; for preserving or preventing damage to cells in an organism to be returned to the cell source (prophylactic use); and for preserving or preventing damage to cells during transport or storage. Details of this application, as well as other uses, are described below.

The present invention relates to a method for inducing arrest in one or more cells separated from an organism, the method comprising: a) identifying cells in which arrest is desired; and b) exposing said cells to an effective amount of an oxygen antagonist or other activity arresting compounds to induce arrest.

Consider that a cell can be any oxygen-utilizing cell. Cells can be eukaryotic or prokaryotic. In certain embodiments, the cells are eukaryotic. More specifically, in some embodiments, the cells are mammalian cells. Mammalian cells contemplated for use in the present invention include, but are not limited to, those from humans, monkeys, mice, rats, rabbits, hamsters, goats, pigs, dogs, cats, ferrets, cows, sheep and horses.

Also, the cells of the invention may be diploid, but in some cases the cells are haploid (sex cells). Also, the minutiae can be polyploid, aneuploid or anucleated. Cells may be derived from specific tissues or organs such as: heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, artery, cornea, blood, small intestine, large intestine, brain, spinal cord, smooth muscle , skeletal muscle, ovaries, testes, uterus and umbilical cord. Furthermore, cells can also be characterized as one of the following cell types: platelets, myeloid cells, erythrocytes, lymphocytes, adipocytes, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, skeletal muscle cells, endocrine cells, glial cells , neurons, secretory cells, barrier function cells, contractile cells, absorptive cells, mucosal cells, limbal cells (from the cornea), stem cells (totipotent, pluripotent, or multipotent) , unfertilized or fertilized oocytes or sperm cells.

The present invention also provides methods, compositions for increasing the viability of and/or reducing damage to biological material under adverse conditions by reducing metabolic demand, oxygen demand, temperature, or any combination thereof in the biological material of interest and instruments. In some embodiments of the invention, the viability of a biological material is enhanced by providing it with an effective amount of a protective metabolizing agent. The agent enhances viability by preventing or reducing damage to the biological material, arresting death or senescence of all or a portion of the biological material, and/or prolonging the lifespan of all or a portion of the biological material, relative to biological material not exposed to the agent . Alternatively, in some embodiments, an agent prolongs the survival of a tissue and/or organism that would not otherwise survive without the agent.

A "protective metabolizer" is contemplated as a substance or compound capable of reversibly altering the metabolism or enhancing the viability of a biological material to which it is exposed or in contact.

In certain embodiments, the protective metabolizing agent induces arrest in the treated biological matter; while, in other embodiments, the protective metabolizing agent itself does not directly induce arrest in the treated biological matter. Protective metabolic agents, as well as other active compounds, can enhance the viability and/or reduce damage to biological material without inducing stagnation in said biological material itself, but by reducing cellular respiration and corresponding metabolic activity to less than about 50 % reduction in oxygen consumption or carbon dioxide production. Furthermore, such compounds may cause biological matter to enter or reach arrest more rapidly, easily or efficiently in response to an injury or disease state, for example by inducing the biological matter to enter a pre-arrested state.

Viability includes viability of the substance when it is subjected to adverse conditions - ie, under conditions where there may be adverse and irreversible damage or damage to the biological substance. Unfavorable conditions include, but are not limited to: when the oxygen concentration in the environment is reduced (hypoxic or anaerobic, such as at high altitudes or underwater); when biological matter cannot receive those oxygens (such as during ischemia), which can Caused by: i) reduced blood flow to organs (eg, heart, brain, and/or kidneys) due to vascular blockage (eg, myocardial infarction and/or stroke), ii) occurs during heart/lung bypass surgery extracorporeal blood shunting (for example, "pumphead syndrome" in which heart or brain tissue is damaged as a result of cardiopulmonary bypass surgery) or iii) blood loss due to trauma (for example, hemorrhagic shock or surgery); body temperature Too low, where the biological material is subjected to temperatures below physiological, either due to exposure to a cold environment or due to the cryogenic state of the biological material, such that it cannot sustain sufficient oxygenation of the biological material; high temperature, where the biological material is subjected to more than physiological Temperature, which is due to exposure to a thermal environment or hyperthermic state of biological material such as caused by malignant fever; pathology of excess heavy metals, such as iron disorders (genetic as well as environmental) such as hemochromatosis, acquired iron overload, Sickle cell anemia, juvenile hemochromatosis, dietary hemosiderosis, thalassemia, porphyria cutanea tarda, iron hypererythroblastic anemia, iron deficiency anemia, and anemia of chronic disease. It is contemplated that protective metabolic agents are oxygen antagonists in certain embodiments of the invention. It is also contemplated that in certain other embodiments, the oxygen antagonist is not a protective metabolic agent. In other embodiments of the invention, one or more compounds may be used to increase or enhance the viability of biological matter; reversibly inhibit the metabolism and/or activity of biological matter; reduce the oxygen demand of biological matter; Damage to biological matter under adverse conditions; preventing or reducing damage or injury to biological matter; preventing aging or senescence of biological matter, and providing therapeutic benefits as described throughout the application with respect to oxygen antagonists. It is contemplated that the embodiments involving the induction of arrest are also applicable to these other embodiments. Accordingly, any of the embodiments discussed with respect to stagnation may be implemented in terms of these other embodiments.

The active compounds used to induce stagnation, or any of these other embodiments, may cause or provide their desired effect, in some embodiments, only provide the desired effect while they are in the environment of the biological matter (i.e. no sustained effect) and/ Or they can provide these effects beyond 24 hours after the biological substance is no longer exposed to them. Furthermore, this may also be the case when combinations of active compounds are used.

In certain embodiments, the biological material is exposed to an amount of an oxygen antagonist or other active compound that reduces the rate or amount of carbon dioxide produced by the biological material by at least 2-fold, and by about, at least about, or at most about 3-, 4- -, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 100-, 200-, 300-, 400-, 500-fold or more, or any range derivable therein. Alternatively, it is contemplated that embodiments of the present invention may use about, at least about, or at most about , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, or any range derivable therein, reduction in the rate or amount of carbon dioxide produced by biomass is discussed. In still further embodiments, the biological material is exposed to an oxygen antagonist or other active compound in an amount that reduces the rate or amount of oxygen consumed by the biological material by at least 2-fold, and by about, at least about, or at most about 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, 50-, 100-, 200- , 300-, 400-, 500-fold or more, or any range that can be derived therefrom. Alternatively, it is contemplated that embodiments of the present invention may use about, at least about, or at most about , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, or any range that may result from a reduction in the rate or amount of oxygen consumed by biomass is discussed. In still further embodiments, the exposure of the biological material to is reduced by at least 10%, and by about, at least about, or at most about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99 or 100% or may be derived from any range of motion or activity therein amount of oxygen antagonist or other active compound. As with other embodiments, these characteristics and parameters are relevant to any biological substance induced into a stasis state. Therefore, if arrest is induced in the heart of an organism, these parameters will be assessed in terms of the heart and not the whole organism. With regard to organisms, a decrease in oxygen consumption on the order of about 8-fold is a stagnation known as "hibernation". Furthermore, it is understood in this application that a decrease in oxygen consumption on the order of about 1000-fold may be considered "stagnation". It should be understood that embodiments of the invention involving arrest can be accomplished at the hibernation or stasis level, as desired. It should be understood that "-fold reduction" is relative to the amount of reduction; for example, if a non-hibernating animal consumes 800 units of oxygen, the hibernating animal consumes 100 units of oxygen.

Also, in some embodiments of the invention, methods are provided for reducing cellular respiration, which may or may not be as high as needed to achieve stasis. In the method of the present invention about, at least about or at most about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 , 85, 90, 95 or 100% reduction in oxygen consumption. This can also be expressed and assessed in any cellular respiration index.

It is contemplated that the biological material may be exposed to one or more oxygen antagonists or other reactive compounds more than once. It is considered that the biological substance can be exposed to one or more active compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times, which means that when the biological substance is exposed to multiple , with intermittent periods (in terms of exposure to active compounds) in between.

It is also contemplated that the active compounds may be administered before, during, after, or any combination of these, the onset or progression of a deleterious lesion or disease state. In certain embodiments, pretreatment of biological material with an active compound is sufficient to enhance viability and/or reduce damage from deleterious damage or disease damage. Pretreatment is defined as exposing the biological material to an active compound prior to the onset or detection of harmful or disease damage. Following pretreatment, exposure can be terminated at or near the onset of injury, or continued after initiation of injury.

In certain embodiments, methods involving prior exposure to an active compound (i.e., pretreatment) are used to treat conditions in which harmful or diseased lesions are 1) predetermined or preselected, or 2) It is possible to predict in advance. Examples of qualifying condition 1 include, but are not limited to, major surgical procedures in which blood loss may occur spontaneously or as a result of the procedure, in which oxygenation of blood may be compromised, or in which vascular delivery of blood may be reduced (eg, after coronary bypass Cardiopulmonary bypass surgery in the setting of graft (CABG) surgery), or in the treatment of an organ donor prior to removal of the donor organ for transport and transplantation into a recipient in need of organ transplantation. Examples that qualify for condition 2 include, but are not limited to, medical conditions where the risk of injury or disease progression is inherent (e.g., in unstable angina, post angioplasty, hemorrhagic aneurysm, hemorrhagic stroke, following major trauma or blood loss), or a medical condition in which the risk can be diagnosed with a medical diagnostic test.

Exposure to an active compound may enhance viability or reduce injury when exposure to harmful lesions or disease lesions occurs after initiation or detection to obtain a therapeutic effect. Exposure to active compounds may be transient or chronic. Exposure duration may be only as long as required to achieve an indicator of arrest activity or pre-arrest (eg, blood _pCO2 , _pO2 , pH, lactate or sulphohemoglobin levels or body temperature), or it may be longer. In certain embodiments, the exposure occurs following a traumatic injury (including iatrogenic and/or non-iatrogenic injury) to an organism and is used to induce arrest or pre-arrest in the whole organism or tissue injured therein, In order to prevent or minimize injury, such as ischemia and reperfusion injury, before, during and/or after treatment.

In one embodiment, the invention includes a method of protecting a mammal from surgically induced cellular damage comprising providing the mammal with hydrogen sulfide or other reactive compound in an amount sufficient to induce the mammal into pre-arrest prior to surgery. Surgery may be elective, planned, or emergency surgery, such as cardiopulmonary surgery. Hydrogen sulfide can be administered by any means available in the art, including, for example, intravenously or by inhalation.

In another embodiment, the present invention includes a method of protecting a mammal from cellular damage caused by a disease or adverse medical condition comprising providing the mammal with sufficient to induce the mammal before the onset or progression of the disease or adverse medical condition Hydrogen sulfide or other reactive compounds in pre-stagnation or stagnation quantities. This embodiment can be used in a variety of different diseases and adverse medical conditions including, for example, unstable angina, post angioplasty, aneurysm, hemorrhagic stroke or shock, trauma or blood loss.

In a particular embodiment, the invention relates to the prevention of an organism, such as a mammal, from hemorrhagic death or from irreversible tissue damage caused by hemorrhage by providing the mammal with hydrogen sulfide or other reactive compound in an amount sufficient to prevent the hemorrhagic death of the animal. method. In certain other embodiments, the organism can go into hemorrhagic shock, but not die from excessive bleeding. The terms "bleeding" and "bleeding" are used interchangeably to refer to any loss of blood from a blood vessel. It includes, but is not limited to, internal and external bleeding, bleeding caused by injury (which may be from an internal source or from an external physical source, such as from a gunshot, stab wound, physical trauma, etc.).

Moreover, additional embodiments of the invention relate to preventing death or irreversible tissue damage caused by blood loss or other lack of oxygenation of cells or tissues, such as lack of adequate blood supply. This may be, for example, caused by actual loss of blood, or may be caused by preventing cells or tissue from being perfused (eg, reperfusion injury), causing blockage of blood to cells or tissue, locally or globally lowering blood pressure in an organism, reducing blood pressure in The amount of oxygen carried or the result of a condition or disease that reduces the number of oxygen-carrying cells in the blood. Conditions and diseases that may be involved include, but are not limited to, blood clots and embolisms, cysts, growths, tumors, anemia (including sickle cell anemia), hemophilia, other blood clotting disorders (e.g., von W. Leebrand disease, ITP) and atherosclerosis. These conditions and diseases also include those that, as a result of injury, disease or condition, produce a substantially hypoxic or anaerobic condition to cells or tissues.

In some cases, a total sublethal dose or a total non-lethal dose is administered to the biological substance. As discussed above, with respect to the induction of arrest in biological material other than the whole organism, "sublethal total dose" means the amount of active compound administered by multiple administrations, which is less than the total amount that will cause death of at least most of the cells within 24 hours of a single administration. half the amount of active compound. In other embodiments, an effective amount is characterized as a near-lethal dose of an oxygen antagonist or other active compound. Likewise, "near-lethal total dose" means the amount of multiple administrations of an oxygen antagonist or other active compound that is within 25% of the amount of active compound that would cause death of at least a majority of cells within 24 hours of a single administration. Furthermore, "total supraletal dose" refers to the amount of active compound for multiple administrations that is at least 1.5 times the amount of active compound that would cause death of at least a majority of cells (or the whole organism) within 24 hours of a single administration. It is contemplated that multiple doses may be administered in order to induce arrest throughout the organism. The definitions of "sublethal total dose", "near-lethal total dose" and "supraletal total dose" can be extrapolated based on the individual doses discussed earlier for stasis in the whole organism.

Biological material can be exposed to or contacted with more than one oxygen antagonist or other active compound. The biological material may be exposed to at least one active compound, including 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oxygen antagonists or other active compounds, or any range derivable therein. For various active compounds, the term "effective amount" refers to the total amount of active compounds. For example, biological material can be exposed to a first active compound and then exposed to a second active compound. Alternatively, biological material may be exposed to more than one active compound simultaneously or in an overlapping fashion. Furthermore, it is contemplated that more than one active compound may be included or admixed together, for example, in a single biological material exposed composition. It is thus contemplated that, in some embodiments, combinations of active compounds are employed in the compositions, methods and articles of manufacture of the invention.

Biological material may be provided or exposed to the active compound by inhalation, injection, catheterization, maceration, lavage, perfusion, topical application, absorption, adsorption, or oral administration. Furthermore, intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intrathecal, intravitreal, intravaginal, intrarectal, surface , intratumoral, intramuscular, intraperitoneal, intraocular, subcutaneous, subconjunctival, intracapsular, transmucosal, intrapericardial, intraumbilical, intraocular, oral, topical, topical, by inhalation, by injection, by infusion, The biological substance is provided or exposed to the active compound by continuous infusion, by local perfusion, by administration to the biological substance via a catheter, or via irrigation.

The methods and devices of the present invention involve protective agents, which in some embodiments are oxygen antagonists. In still further embodiments, the oxygen antagonist is a reducing agent. Furthermore, oxygen antagonists can be characterized as chalcogenide compounds. It should be understood that the active compound may also be a protectant. Moreover, any chalcogenide compound can be considered as an active compound, regardless of whether it is an oxygen antagonist or not, as long as it achieves the object of the present invention.

In certain embodiments, the chalcogenide compound includes sulfur, while in other embodiments it includes selenium, tellurium, or polonium. In certain embodiments, a chalcogenide compound contains one or more exposed sulfide groups. It is contemplated that such chalcogenide compounds contain 1, 2, 3, 4, 5, 6 or more exposed sulfide groups, or may be derived from any range therein. In a particular embodiment, this sulfur-containing compound is _CS2 (carbon disulfide).

Also, in some methods of the invention, arrest is induced in the cell by exposing the cell to a reducing agent having the following chemical structure (referred to as Formula I):

Wherein X is N, O, Po, S, Se or Te;

where Y is N or O;

Wherein _R is H, C, lower alkyl, lower alcohol or CN;

Wherein R ₂ is H, C, lower alkyl, lower alcohol or CN;

where n is 0 or 1;

where m is 0 or 1;

where k is 0, 1, 2, 3 or 4; and,

where p is 1 or 2.

The terms "lower alkyl" and "lower alcohol" are used according to their ordinary meanings, and the symbols are those used to refer to chemical elements. Such a chemical structure will be referred to as a "reductant structure", and any compound having such a structure will be referred to as a reductant structure compound. In additional embodiments, k is 0 in the reducing agent structure. Also, in other embodiments, the _R1 and/or _R2 groups can be amines or lower alkylamines. In other embodiments, R ₁ and/or R ₂ may be short chain alcohols or short chain ketones. Also, _R1 and _R2 may be linear or branched bridges and/or the compound may be a cyclic compound. In still further embodiments, X can also be halogen. The term "lower" means 1, 2, 3, 4, 5 or 6 carbon atoms or any range derivable therein. Also, _R1 and/or _R2 can be other small organic groups including _C2 - _C5 esters, amides, aldehydes, ketones, carboxylic acids, ethers, nitriles, anhydrides, halides, acid halides, sulfides, sulfones , sulfonic acids, sulfoxides and/or mercaptans. With respect to R ₁ and/or R ₂ , these substitutions are explicitly contemplated. In certain other embodiments, R ₁ and/or R ₂ may be small organic groups discussed above in short chain form. "Short chain" means 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 carbon molecules, or any range derivable therein.

Consider that in some cases, the reducing agent compound may be a chalcogenide compound. In certain embodiments, the chalcogenide compound has an alkyl chain that contains exposed chalcogenides. In additional embodiments, the chalcogenide compound has a chalcogenide that becomes exposed upon uptake by the biomass. In this respect, the chalcogenide compounds are analogous to prodrugs that act as oxygen antagonists. Thus, upon exposure of biological matter to the chalcogenide compound, one or more molecules of sulfur, selenium, oxygen, tellurium, polonium, or ununhexium on the compound become available. In this case, "available" means that sulfur, selenium, oxygen, tellurium, polonium, or element 116 will retain a negative charge.

In certain embodiments, the chalcogenide is a salt, preferably a salt wherein the chalcogen is in the -2 oxidation state. Sulfide salts encompassed by embodiments of the present invention include, but are not limited to, sodium sulfide ( _Na2S ), sodium hydrosulfide (NaHS), potassium sulfide ( _K2S ), potassium hydrosulfide (KHS), lithium sulfide ( _Li2 S), rubidium sulfide (Rb ₂ S), cesium sulfide (Cs ₂ S), ammonium sulfide ((NH ₄ ) ₂ S), ammonium bisulfide ((NH ₄ )HS), beryllium sulfide (BeS), magnesium sulfide ( MgS), calcium sulfide (CaS), strontium sulfide (SrS), barium sulfide (BaS), etc. Similarly, embodiments of the present invention include, but are not limited to, corresponding selenide and telluride salts. In particular, the invention is contemplated to include compositions containing chalcogenide salts (chalcogenide compounds that are salts) having a pharmaceutically acceptable carrier or prepared as a pharmaceutically acceptable formulation. In a further embodiment, the reducing agent structure compound is selected from _H2S , _H2Se , _H2Te and _H2Po . In some cases, the reducing agent structure of formula (I) has X which is S. In other cases, X is Se, or X is Te, or X is Po, or X is O. Furthermore, in some embodiments, k in the reducing agent structure is 0 or 1. In certain embodiments, the reducing agent structural compound is dimethyl sulfoxide (DMSO), dimethyl sulfide (DMS), carbon monoxide, methyl mercaptan ( _CH3SH ), mercaptoethanol, thiocyanate, hydrogen cyanide , methyl mercaptan (MeSH) or CS ₂ . In particular embodiments, the oxygen antagonist is _H2S , _H2Se , _CS2 , MeSH or DMS. Compounds on the order of the size of these molecules are specifically contemplated (ie, within 50% of their molecular weight average).

In certain embodiments, selenium-containing compounds such as _H2Se are employed. In some embodiments of the invention, the amount of _H2Se may be in the range of 1-1000 parts per billion. It is further contemplated that any of the embodiments discussed in the context of sulfur-containing compounds may be practiced with selenium-containing compounds. This involves replacing one or more sulfur atoms in the sulfur-containing molecule with the corresponding selenium atom.

A further aspect of the invention comprises compounds represented by formula IV:

in:

X is N, O, P, Po, S, Se, Te, O-O, Po-Po, S-S, Se-Se or Te-Te;

n and m are independently 0 or 1; and

Wherein R ^{and R} are independently hydrogen, halogen, cyano, phosphate, thio, alkyl, alkenyl, alkynyl, alkoxy, aminoalkyl, cyanoalkyl, hydroxyalkyl, haloalkane radical, hydroxyhaloalkyl, alkylsulfonic acid, thiosulfonic acid, alkylthiosulfonic acid, thioalkyl, alkylthio, alkylthioalkyl, alkylaryl, carbonyl, alkylcarbonyl, Haloalkylcarbonyl, alkylthiocarbonyl, aminocarbonyl, aminothiocarbonyl, alkylaminothiocarbonyl, haloalkylcarbonyl, alkoxycarbonyl, aminoalkylthio, hydroxyalkylthio, cycloalkyl, cycloalkene radical, aryl, aryloxy, heteroaryloxy, heterocyclyl, heteroepoxy, sulfonic acid, alkyl sulfonate, thiosulfate, or sulfonylamino; and

Y is cyano, isocyano, amino, alkylamino, aminocarbonyl, aminocarbonylalkyl, alkylcarbonylamino, amidino, guanidine, hydrazino, hydrazide, hydroxyl, alkoxy, aryloxy, hetero Aryloxy, cycloalkoxy, carbonyloxy, alkylcarbonyloxy, haloalkylcarbonyloxy, arylcarbonyloxy, carbonylperoxy, alkylcarbonylperoxy, arylcarbonylperoxy, Phosphate, alkyl phosphate, sulfonic acid, alkyl sulfonate, thiosulfate, thiosulfenyl, sulfonamide, -R ²³ R ²⁴ , where R ²³ is S, SS, Po, Po-Po , Se, Se-Se, Te or Te-Te, and R ²⁴ is as defined herein for R ²¹ , or Y is

wherein X, R ²¹ and R ²² are as defined herein.

Furthermore, it is contemplated that in some embodiments of the present invention, the biological material is provided with precursor compounds which become active forms of compounds of formula I or IV upon exposure to the biological material, eg, by chemical or enzymatic means. Furthermore, the compound may be provided to the biological material as a salt of the compound, in free radical form or in a negatively, positively or multiple charged form. Some compounds conform to both Formula I and Formula IV structures, and in this case, use of the phrase "Formula I or Formula IV" is not intended to imply the exclusion of such compounds.

In certain embodiments, compounds defined by the structure of Formula I or Formula IV are also characterized as oxygen antagonists, protective metabolizers, or precursors, prodrugs, or salts thereof. It is further contemplated that said compound need not itself be characterized or certified as a compound for use in the invention, so long as it performs a particular method of the invention. In some other embodiments, the compound may be considered a chalcogenide compound. It is particularly contemplated that any compound determined by the structure of Formula I or Formula IV or proposed in this disclosure may be used in place of or in combination with an oxygen antagonist in the methods, compositions and apparatus of the invention; similarly , any of the embodiments discussed with respect to any structure having Formula I or Formula IV or otherwise suggested in this disclosure may be used in place of or in combination with an oxygen antagonist. Furthermore, any compound identified by the structure of Formula I or Formula IV or set forth in this disclosure may be combined with any oxygen antagonist or any other active compound described herein. It is also contemplated that in the methods, compositions, and other articles of manufacture of the present invention, any combination of these compounds may be used together, sequentially (overlapping or non-overlapping), and/or in an overlapping sequential manner (initiating administration of one compound and prior to commencing administration of another compound) provided or formulated to achieve the desired effects set forth herein.

In certain embodiments, more than one compound having the structure of Formula I or Formula IV is provided. In certain embodiments, multiple different compounds are employed that have the structure of the same formula (ie, Formula I or Formula IV), while in other embodiments, when multiple different compounds are employed, they are from different formulas.

In a particular embodiment, the use of multiple active compounds is contemplated, one of which is carbon dioxide (CO ₂ ). It is contemplated that in some embodiments at least one other compound is also a compound of formula I and/or IV. In some cases, carbon dioxide is provided to the biomass in combination with _H2S or a _H2S precursor (together, sequentially, or in overlapping sequential fashion).

The amount of carbon dioxide to which the biomass can be exposed is about, at least about, or at most about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30% or more, or any range derivable therein. In certain embodiments, the amount is expressed in ppm, such as about, at least about, or at most about 350, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600 , 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100 , 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 2 , 22000, 23000, 24000, 25000, 26000, 27000, 28000, 29000, 30000, 31000, 32000, 33000, 34000, 36000, 37000, 39000, 40000, 41000, 43000, 44000, 45000, 466000 、47000、48000、49000、50000、60000、70000、80000、90000、100000、110000、120000、130000、140000、150000、160000、170000、180000、190000、200000、210000、220000、230000、240000、250000、260000 , 270,000, 280,000, 290,000, 300,000 or more ppm, or any range derivable therein, and expressed in molar equivalents. It is contemplated that these concentrations are applicable to any other active compound in gaseous form.

In other embodiments, it is specifically contemplated that the active compound is sodium sulfide, sodium thiomethoxide, cysteamine, sodium thiocyanate, cysteamine-S-phosphate sodium salt or tetrahydrothiopyran-4-ol. In other embodiments, the active compound is dimethylsulfoxide, thioacetic acid, selenourea, 2-(3-aminopropyl)-aminoethanethiol-dihydrogen-phosphate, 2-mercapto-ethanol, mercapto Diethyl ether (thioglycolicether), sodium selenide, sodium methanesulfinate, thiourea, or dimethyl sulfide. It is particularly contemplated that these compounds, or any other compounds discussed herein, including any compound of formula I, II, III, or IV, may be present in amounts of about, at least about, or at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800, 5900, 6000, 6100, 6200, 6300, 6400, 6500, 6600, 6700, 6800, 6900, 7000, 7100, 7200, 7300, 7400, 7500, 7600, 7700, 7800, 7900, 8000, 8100, 8200, 8300, 8400, 8500, 8600, 8700, 8800, 8900, 9000, 9100, 9200, 9300, 9400, 9500, 9600, 9700, 9800, 9900, 10000mM or mmol/kg (biological matter) or source An amount within any range therein is provided or applied to a biological substance.

It is specifically contemplated that any subset of the active compounds identified by name or structure may be used in the methods, compositions and articles of manufacture. It is also specifically contemplated that any subset of these compounds may be disclaimed and do not constitute embodiments of the invention. The present invention also relates to pharmaceutical compositions containing a therapeutically effective amount of one or more active compounds. Such pharmaceutical compositions will, of course, be formulated as pharmaceutically acceptable compositions. For example, the composition can include a pharmaceutically acceptable diluent.

In certain embodiments, the pharmaceutical compositions contain an effective amount of an active compound to provide a Cmax or steady state plasma concentration of the active compound when administered to a patient to produce a therapeutically effective benefit. In certain embodiments, the Cmax or steady state plasma concentration to be achieved is about, at least about, or at most about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000 (M or more , or may be derived from any range therein. In certain embodiments, for example for _H2S , the desired Cmax or steady state plasma concentration is between about 10 μM to about 10 mM, between about 100 μM to about 1 mM, or about 200 μM to about 800 [mu]M. Suitable means may be considered and the level of compounds already in the blood, such as sulfur, may be assessed.

In certain embodiments, the pharmaceutical composition provides an effective amount of _H2S to provide between about 10 μM to about 10 mM, between about 100 μM to about 1 mM, or between about 200 μM to about 800 μM when administered to a patient. Cmax or steady state plasma concentration. In relation to the administration of hydrogen sulfide in relation to the administration of a sulfide salt, in typical embodiments, the administration of the salt is based on the administration of approximately the same sulfur equivalents as the administration of _H2S . Suitable means will be considered and used to assess the level of sulfur already in the blood.

In certain embodiments, compositions contain one or more active compounds specified above in gaseous form. In another embodiment, the composition comprises salts of one or more of these compounds. In a particular embodiment, the pharmaceutical composition comprises formula I or IV or a salt of formula I or IV in gaseous form. The gaseous form or salt of _H2S is specifically contemplated in some aspects of the invention. The amount of gas considered to be provided to the biomass is about, at least about or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120 ,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370 ,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620 ,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870 , 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200 . , 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000, 16000, 17000, 18000, 19000, 20000, 21000, 22000, 23000, 250000, 27000, 27000 , 28000, 29000, 300, 31000, 32000, 33000, 34000, 35000, 36000, 37000, 39000, 40000, 41000, 42000, 43000, 45000, 47000, 47000, 49000, 50000, 70000, 70000, 70000 、80000、90000、100000、110000、120000、130000、140000、150000、160000、170000、180000、190000、200000、210000、220000、230000、240000、250000、260000、270000、280000、290000、300000、310000、320000 , 330000, 340000, 350000, 360000, 370000, 380000, 390000, 400000 or more ppm, or any range that may be derived therefrom. Alternatively, an effective amount of gas may be expressed as about, at least about, or at most about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any range derivable therein. Also, considering that for some embodiments, the amount of gas provided to the biomass is about, at least about, or at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60 parts per billion ,70,80,90,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310 ,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560 ,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810 , 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000, 1100, 1200, 1300, 1400, 1500, 1600 , 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4000, 4100 , 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5000, 6000, 7000, 8000, 9000, 10000 parts (ppb) or any range that may be derived therefrom. In particular embodiments, the amount of hydrogen selenide provided to the biomass is on this order of magnitude.

In some embodiments of the invention, the pharmaceutical composition is a liquid. As discussed elsewhere, the composition may be a liquid with the compound of interest dissolved or bubbled into the composition. In some instances, the pharmaceutical composition is a medical gas. According to the U.S. Food and Drug Administration, a "medical gas" is a gas that is regulated by §201(g)(1) of the Federal Food, Drug, and Cosmetic Act ("the Act") (21 U.S.C. §321(g)). ) within the meaning of ), and require a prescription to be dispensed under §503(b)(1)(A) of the act (21 U.S.C. §353(b)(1)(A)). As such, these medicinal gases require proper FDA labeling. A medical gas includes at least one reactive compound.

The invention further encompasses apparatus and articles of manufacture comprising packaging material and an activity arresting compound contained within the packaging material, wherein the packaging material comprises a label indicating that it can be used to induce arrest in biological material in vivo.

In some embodiments, the apparatus or article of manufacture further comprises a pharmaceutically acceptable diluent. In particular other embodiments, the instrument or article of manufacture has a buffer. The active compound is provided in a first sealed container and the pharmaceutically acceptable diluent is provided in a second sealed container. In other embodiments, the device or article further has instructions for mixing the active compound and diluent. In addition, active compounds can be reconstituted to perform any of the methods of the invention, for example for inducing stasis in biological material in vivo. It is contemplated that any labeling will specify the result to be obtained and the use of the compound for the patient in need of that result.

The present invention also relates to an article of manufacture comprising, packaged together, an active compound, instructions for using said activity-stabilizing compound, said instructions comprising: (a) identifying the body tissue in need of arresting treatment; and (b) administering an effective amount of active compound to the biological substances in the body.

In a further embodiment of the invention there is a medicinal gas containing article of manufacture comprising an active compound and a label comprising details or usage and administration for inducing stagnation in biological material or any other method of the invention.

The invention also relates to kits and methods of using these kits. In some embodiments, there is a kit for delivering an active compound to a tissue site in need of arrest treatment, or any other treatment of the claimed invention, comprising: a drape, suitable for tissue The site forms a sealed envelope; a container containing the oxygen antagonist; and an inlet in the covering, wherein the container containing the active compound communicates with the inlet. In certain embodiments, the kit includes an outlet in the cover, wherein the outlet is in communication with a source of negative pressure. In some cases, the covering comprises an elastic material and/or has a pressure sensitive adhesive covering the edges of the covering. The outlet may be placed in fluid communication with a source of negative pressure, which may or may not be a vacuum pump. There may also be flexible conduit communication between the outlet and the source of negative pressure. In some embodiments, the kit includes a canister, which may or may not be removable, in fluid communication between an outlet and a source of negative pressure. Consider a container containing an active compound in gas communication with an inlet. In certain embodiments, the container includes the active compound that is a gas or liquefied gas. The kit may also comprise a vaporizer in communication between the vessel containing the oxygen antagonist and the inlet. Furthermore, it may have a reflux outlet in communication with the container containing the active compound.

In particular embodiments, the active compound in the kit is carbon monoxide, carbon dioxide, _H2Se and/or _H2S . In certain embodiments, the tissue site to which the kit or method is applied is injured.

Moreover, it is generally understood that any compound discussed herein as an oxygen antagonist may be provided to a biological substance in the form of a prodrug, which means that the biological substance or other substances in the environment of the biological substance convert the prodrug into its active form, i.e. Oxygen antagonist. The term "precursor" is considered to cover compounds considered to be "prodrugs".

The oxygen antagonist or other active compound may be or may be provided as a gas, a semi-solid liquid (such as a gel or paste), a liquid or a solid. It is contemplated that a biological substance may be exposed to more than one such active compound and/or more than one state of the active compound. Furthermore, the active compounds can be formulated for a particular mode of administration, as discussed herein. In certain embodiments, the active compound is in a pharmaceutically acceptable formulation for intravenous delivery.

In certain embodiments, the active compound is a gas. In particular embodiments, the gas active compound comprises carbon monoxide, carbon dioxide, nitrogen, sulfur, selenium, tellurium or polonium, or mixtures thereof. Furthermore, it is particularly contemplated that the active compound is a chalcogenide compound in gaseous form. In some embodiments, the active compound is in a gas mixture containing more than one gas. In some embodiments, the other gas is a non-toxic and/or non-reactive gas. In some embodiments, the other gas is an inert gas (helium, neon, argon, krypton, xenon, radon, or ununoctium), nitrogen, nitrous oxide, hydrogen, or mixtures thereof. For example, the non-reactive gas may simply be the mixture that makes up "room air", which is a mixture of nitrogen, oxygen, argon, and carbon dioxide, with trace amounts of other molecules such as neon, helium, methane, krypton, and hydrogen. Although a typical sample might contain about 78 percent nitrogen, 21 percent oxygen, 0.9 percent argon, and 0.04 percent carbon dioxide, the precise amounts of each gas vary. Contemplated in the context of the present invention, a "house atmosphere" is one containing from about 75% to about 81% nitrogen, from about 18% to about 24% oxygen, from about 0.7% to about 1.1% argon, and from about 0.02% to about 0.06% carbon dioxide mixture. The gas active compound may be first diluted with a non-toxic and/or non-reactive gas prior to application or exposure to biological matter. Additionally or alternatively, any gas active compound may be mixed with room air prior to application or exposure to the biological material, or the compound may be applied or exposed to the biological material in the room air.

In some cases, the gas mixture also contains oxygen. In other embodiments of the invention, the reactive compound gas is mixed with oxygen to form an oxygen ( _O2 ) mixture. Particularly contemplated are oxygen mixtures wherein the amount of oxygen in said oxygen mixture is less than the sum of all other gases in the mixture.

In some embodiments, the active compound is carbon monoxide, and the amount of carbon monoxide approximately equals or exceeds any amount of oxygen in the oxygen mixture. In a particular embodiment, carbon monoxide is used for bloodless biological material. The term "blood-free biological material" refers to cells and organs whose oxygenation is independent, or no longer dependent, on the vasculature, such as organs for transplantation. Preferably the atmosphere will be 100% CO, but it will be apparent to those skilled in the art that the amount of CO can be equilibrated with a gas other than oxygen so that the amount of available oxygen is reduced to a level that prevents cellular respiration. In this aspect, the ratio of carbon monoxide to oxygen is preferably 85:15 or higher, 199:1 or higher or 399:1 or higher. In certain embodiments, the ratio is about, at least about, or at most about 1:1, 2:1, 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8 :1, 9:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, 55:1, 60:1 , 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 110:1, 120:1, 130:1, 140:1, 150 :1, 160:1, 170:1, 180:1, 190:1, 200:1, 210:1, 220:1, 230:1, 240:1, 250:1, 260:1, 270:1 , 280:1, 290:1, 300:1, 310:1, 320:1, 330:1, 340:1, 350:1, 360:1, 370:1, 380:1, 390:1, 400 :1, 410:1, 420:1, 430:1, 440:1, 450:1, 460:1, 470:1, 480:1, 490:1, 500:1 or more, or may be derived from any range in it.

In still further embodiments, the above figures apply to the ratio of carbon monoxide to a mixture of oxygen and one or more other gases. In some cases, it is contemplated that the other gas is a non-reactive gas, such as nitrogen ( _N2 ). Thus, in other embodiments of the invention, the above numbers apply to the ratio of carbon monoxide to a combination of oxygen and nitrogen ( _O2 / _N2 ) useful in the methods and apparatus of the invention. Accordingly, it should be understood that other gases may or may not be present. In some embodiments, the CO:oxygen ratio is balanced with one or more other gases (non-carbon monoxide and non-oxygen). In a particular embodiment, the CO:oxygen ratio is balanced with nitrogen. In a still further embodiment, the amount of CO is the ratio of CO to room air, as described by the numbers above.

In some cases, the amount of carbon monoxide is relative to the amount of oxygen, while in other cases it is an absolute amount. For example, in some embodiments of the invention, the amount of oxygen is in "parts per million (ppm)", which is one million parts of air at a standard temperature and pressure of 20°C and one atmosphere. Measurement of the volume fraction of oxygen in a medium in which the balance of the gas volume is made up with carbon monoxide. In this respect, carbon monoxide is related to the amount of oxygen in parts per million of oxygen balanced with carbon monoxide. The atmosphere contemplated for exposure or incubation of biological material can be at least 0, 50, 100, 200, 300, 400, 500, 1000, or 2000 parts per million (ppm) of oxygen balanced with carbon monoxide, and in some cases with Non-toxic and/or non-reactive gas mixed with carbon monoxide balanced with oxygen. The term "environment" refers to the immediate environment of a biological material, ie the environment it is in direct contact with. Therefore, the biomass must be directly exposed to carbon monoxide, and a sealed tank of carbon monoxide in the same chamber as the biomass is considered insufficient to incubate in the "environment" according to the invention. Alternatively, the atmosphere may be expressed in kPa. It is generally considered that 1 million parts = 101 kPa at 1 atmospheric pressure. In an embodiment of the invention, the environment in which the biological material is incubated or exposed is about, at least about or at most about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11 . , 0.65, 0.70, 0.75, 0.80, 0.5, 0.90, 0.95, 1.0 kPa or more O ₂ , or any range derivable therein. As described above, these levels can be balanced with carbon monoxide and/or other non-toxic and/or non-reactive gases. Also, the atmosphere can be defined in terms of CO levels in kPa. In certain embodiments, the atmosphere is about, at least about, or at most about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 101, 101.3 kPaCO or any range that can be derived therefrom. In particular embodiments, the partial pressure is about or at least about 85, 90, 95, 101, 101.3 kPa CO or any range derivable therein.

In embodiments of the invention, the amount of time the sample is incubated or exposed to carbon monoxide may also vary. In some embodiments, the sample is incubated or exposed to carbon monoxide for about, at least about, or at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more minutes and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more days.

In some embodiments, the present invention is directed to compositions and articles of manufacture containing one or more active compounds. In certain embodiments, the composition has one or more of these active compounds as a gas that is bubbled through the composition such that the composition provides the compound to the composition when biological matter is exposed to the composition. biological matter. These compounds may be gels, liquids or other semi-solid substances. In certain embodiments, the solution has an oxygen antagonist as a gas bubbled through it. Considering the amount of bubbling in the gas will provide the appropriate amount of compound to the biological material exposed to the solution. In certain embodiments, the amount of gas bubbled into the solution is about, at least about, or at most about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 times or more, or any range that may be derived therefrom.

In some embodiments of the invention, biological matter is exposed to gas in a closed container. In some cases, a closed container can maintain a specific environment or adjust the environment as desired. The environment refers to the amount of oxygen antagonist to which the biological substance is exposed and/or the temperature, gas composition or pressure of the environment. In some cases, the biological material is placed under vacuum before, during, or after exposure to the oxygen antagonist or other active compound. Also, in other cases, the biological material is exposed to a normoxic environment after exposure to an oxygen antagonist or other active compound. In certain embodiments, the present invention includes a method for inducing arrest or protecting a biological substance from damage or from disease comprising providing an active compound to the biological substance in combination with another arrest-inducing active compound or environmental conditions to the biological substance. Such joint processing occurs in any order, eg simultaneously or sequentially. In certain embodiments, an active compound is provided to the biological material and the biological material is subsequently subjected to anoxic conditions, e.g., 5% O ₂ , or is subsequently exposed to progressively hypoxic conditions , such as 5% O ₂ , followed by 4% O ₂ , 3% O ₂ , 2% O ₂ , 1% O ₂ , or no O ₂ , or any sequenced combination of these conditions.

Also, in other embodiments, the environment containing the biological material is cycled at least once to a different amount or concentration of the oxygen antagonist or other active compound, wherein the difference in the amount or concentration is at least a percent difference. The environment may cycle back and forth between one or more amounts or concentrations of the oxygen antagonist or other active compound, or it may gradually increase or decrease the amount or concentration of that compound. In some cases, the different amount or concentration is between about 0 and 99.9% of the amount or concentration of the oxygen antagonist or other active compound to which the biological material was initially exposed. Contemplated, the difference in amount and/or concentration is about, at least about or at most about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 , 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, or any range derivable therein.

The methods of the invention may also include the step of subjecting the biomass to a controlled temperature environment. In certain embodiments, exposing the biological material to a temperature is a "non-physiological temperature environment", which refers to a temperature in which the biological material cannot survive for more than 96 hours. The controlled temperature environment can have about, at least about, or at most about -210, -200, -190, -180, -170, -160, -150, -140, -130, -120, -110, -100, - 90, -80, -70, -60, -50, -40, -30, -20, -10, -5, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 , 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 ,111,112,113,114,115,116,117,118,119,120,121,122,123,124,125,126,127,128,129,130,131,132,133,134,135 ,136,137,138,139,140,141,142,143,144,145,146,147,148,149,150,151,152,153,154,155,156,157,158,159,160 ,161,162,163,164,165,166,167,168,169,170,171,172,173,174,175,176,177,178,179,180,181,182,183,184,185 , 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200° C. or higher, or any range of temperatures derivable therein. The biological material may also be exposed to the oxygen antagonist or other reactive compound at room temperature, which means a temperature between about 20°C and about 25°C. Furthermore, the core temperature at which the biomass reaches any of the amounts or ranges of amounts discussed is contemplated.

It is contemplated that the biological material may be subjected to a non-physiological temperature environment or a controlled temperature environment before, during or after exposure to the oxygen antagonist or other active compound. Additionally, in some embodiments, the biomass is subjected to a non-physiological temperature environment or a controlled temperature environment for a period of time between about one minute and about one year. The amount of time can be about, at least about, or at most about 30 seconds, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, December, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years and any combination or range derivable therein. Furthermore, there may also be a step of increasing the ambient temperature relative to the reduced temperature.

Also, it is contemplated that the temperature may be varied or cycled during a process in which the temperature is controlled. In some embodiments, the temperature of the biological material can be lowered first before subjecting it to an environment with an oxygen antagonist or other active compound, while in other embodiments, the temperature of the biological material can be reduced by placing the biological material below its The biomass is cooled in an environment with active compounds at high temperature. The biomass and/or environment may be gradually cooled or heated such that the temperature of the biomass or environment is initially at one temperature but then reaches another temperature.

The methods of the invention may also include the step of subjecting the biological material to a controlled pressure environment. In certain embodiments, the organism is exposed to a pressure that is lower than the pressure to which the biological material is normally subjected. In certain embodiments, the biological material is subjected to a "non-physiological stress environment," which refers to a stress in which the biological material cannot survive for more than 96 hours. The controlled pressure environment may have about, at least about, or at most about 10 ⁻¹⁴ , 10 ⁻¹³ , 10 ⁻¹² , 10 ⁻¹¹ , 10 ⁻¹⁰ , 10 ⁻⁹ , 10 ⁻⁸ , 10 ⁻⁷ , 10 ⁻⁶ , 10 ^-5 , ^10-4 , ^10-3 , 10-2, ^10-1 , ^0.2 , 0.3, 0.4, or 0.5 atmospheres or greater, or any range of pressures derivable therein.

It is contemplated that the biological material may be subjected to a non-physiological stressful environment or a controlled stressful environment before, during or after exposure to the active compound. Additionally, in some embodiments, the biological material is subjected to a non-physiological stressful environment or a controlled stressful environment for a period of time between about one minute and about one year. The amount of time can be about, at least about, or at most about 30 seconds, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, December, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years, and any combination or range derivable therein.

Also, it is contemplated that the pressure may be varied or cycled during a process in which the pressure is controlled. In some embodiments, the biological material may first be exposed to reduced pressure prior to placing it in an environment with the active compound, while in other embodiments, the biological material is placed under pressure after exposure to the active compound. The pressure can be gradually lowered so that the pressure of the environment is initially at one pressure, but then at 10, 20, 30, 40, 50, 60 seconds, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 , 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, Another pressure is reached within 20, 21, 22, 23, 24 hours and/or 1, 2, 3, 4, 5, 6, 7 days or more, or any combination or range that may result therefrom. In certain embodiments, the methods include adjusting the oxygen level of the environment or removing biological material from the environment with oxygen. Operatively, exposing the biological material to an environment in which oxygen is reduced or absent mimics the exposure of the biological material to the oxygen antagonist. It is contemplated that in some embodiments of the invention, biological material is exposed to or provided with an active compound under conditions in which the environment of the biological material is anoxic or anaerobic, as described in further detail below. This can be intentional or unintentional. Thus, in some embodiments of the invention, biological material is intentionally placed in an oxygen-free or low-oxygen environment or placed in an environment that results in an oxygen-free or low-oxygen environment. In other embodiments, the biological substance is in such a condition caused by unintentional circumstances, for example, if the biological substance is in an ischemic or potentially ischemic condition. Therefore, it is contemplated that in some cases hypoxic or anaerobic conditions will damage the material in the absence of the active compound.

In certain methods of the invention there is also the step of assessing the level of oxygen antagonists and/or oxidative phosphorylation in the biological material in which arrest is induced. Also, in some embodiments of the invention, there is a step of assessing the level of cellular metabolism normally occurring in the biological material. In some cases, the amount of active compound in the biomass is measured and/or the reduction in temperature in the biomass is assessed. Also, in some methods of the invention, the extent of one or more therapeutic effects is assessed.

In certain other embodiments, any toxic effects of active compounds and/or environmental changes (temperature, pressure) on biological substances are monitored or controlled. It is contemplated that toxicity can be controlled by altering the level, amount, duration or frequency of the active compound and/or changes in the environment to which the biological substance is exposed. In certain embodiments, the change is a decrease, while in certain other embodiments, the change is an increase. Consider that the skilled artisan knows many methods of assessing toxic effects in biological substances.

Other optional steps of the methods of the invention include identifying a suitable active compound; diagnosing the patient; considering the patient's history and/or performing one or more tests on the patient before administering or prescribing the active compound to the patient.

The compositions, methods and articles of manufacture of the invention are useful on biological material to be transferred back to a biological material source (autologous) donor organism or to a different recipient (heterologous) subject. In some embodiments, biological material is obtained directly from a donor organism. In other embodiments, the biological material is placed in culture prior to exposure to the oxygen antagonist or other active compound. In some cases, biological material is obtained from donor tissue that has undergone extracorporeal membrane oxygenation, a technique performed to help preserve biological material, prior to removal of the biological material. Furthermore, the method comprises administering or transplanting a biological substance in which stasis is induced to a living recipient organism.

The methods of the invention also involve inducing arrest in biological material in vivo, comprising incubating the biological material with an oxygen antagonist or other active compound that produces hypoxic conditions for an effective amount of time for bringing the tissue into arrest.

Additionally, other embodiments of the invention include methods of reducing the oxygen demand of biological materials in vivo comprising contacting the biological materials with an effective amount of an oxygen antagonist or other reactive compound to reduce their oxygen demand. Considering that the oxygen demand is reduced by about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100%, or source within any range.

Other aspects of the invention relate to methods for preserving biological material in vivo comprising exposing biological material in vivo to an effective amount of an oxygen antagonist or other active compound to preserve biological material in vivo.

The present invention also relates to a method of delaying or reducing the effects of trauma on or in an organism comprising exposing a biological substance at risk of trauma to an effective amount of an oxygen antagonist or other active compound.

In other aspects of the invention there are methods for treating or preventing hemorrhagic shock in a patient comprising exposing the patient to an effective amount of an oxygen antagonist or other active compound. Alternatively, in some embodiments, the method prevents lethality in patients caused by hemorrhage and/or hemorrhagic shock. In such methods of preventing bleeding death in a patient or preventing mortality in a bleeding patient, the step includes exposing the patient to an effective amount of an oxygen antagonist or other active compound. In certain embodiments, it is specifically contemplated that the oxygen antagonist is a chalcogenide compound such as _H2S .

Also included as part of the invention are methods for reducing heart rate in an organism. Such methods involve contacting a biological sample or organism with an effective amount of an oxygen antagonist or other active compound.

One embodiment of the present invention is directed to a method of inducing hibernation in a mammal comprising contacting the mammal with an effective amount of an oxygen antagonist or other active compound.

In another embodiment, there is a method of anesthetizing an organism comprising exposing a biological substance in which anesthesia is desired to an effective amount of an oxygen antagonist or other active compound. It is contemplated that the anesthesia may be similar to local or general anesthesia.

The invention further includes a method of protecting a mammal from radiation therapy or chemotherapy comprising contacting said mammal with an effective amount of an oxygen antagonist or other active compound prior to or during radiation therapy or chemotherapy. With regard to local application of cancer therapy, it is especially contemplated that oxygen antagonists or other active compounds may also be administered locally to the affected organs, tissues and/or cells. In certain embodiments, the methods are useful for preventing or reducing hair loss in chemotherapy patients. Consider that such patients may have already received chemotherapy or are candidates for chemotherapy. In particular instances, it is contemplated that the active compound may be provided to the patient as a topical gel to be applied where hair loss is expected or present.

The present invention also covers reducing the oxygen demand of the biomass, which means that the amount of oxygen required by the biomass to survive is reduced. This can be achieved by providing an effective amount of one or more active compounds. It is generally known how much oxygen a particular biological substance needs to survive, which also depends on time, pressure and temperature. In certain embodiments of the invention, the oxygen requirement of the biomass is reduced by about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any range derivable therein.

In additional embodiments, there is a method of treating a hyperproliferative disease (eg, cancer) in a patient comprising contacting a mammal with an effective amount of an oxygen antagonist or other active compound and subjecting the mammal to hyperthermia.

While the methods of the invention may be applied to preserving organs for transplantation, other aspects of the invention relate to the recipient organism. In some embodiments, there is a method of inhibiting rejection of an organ transplant in a mammal, the method comprising providing the mammal with an effective amount of an oxygen antagonist or other active compound.

Temperature regulation can be accomplished in organisms through the use of oxygen antagonists or other reactive compounds. In some embodiments, there is a method of treating a subject with hypothermia comprising (a) contacting the subject with an effective amount of an oxygen antagonist, and then (b) subjecting the subject to the ambient temperature of the user. In other embodiments, the present invention includes methods of treating a subject with hyperthermia comprising (a) contacting the subject with an effective amount of an oxygen antagonist or other active compound. In some instances, treating hyperthermia also includes (b) subjecting the subject to an ambient temperature that is at least about 20°C lower than the temperature of the subject. As discussed above, exposing a subject to a non-physiological or controlled temperature environment can be used in additional embodiments. It is contemplated that the method can generally be performed with active compounds.

In some instances, the invention relates to methods for inducing cardioplegia in a patient undergoing bypass surgery, the method comprising administering to the patient an effective amount of an oxygen antagonist or other active compound. It is considered that the application may be localized to the heart in order to protect it.

Other aspects of the invention relate to methods for preventing hemorrhagic shock in a patient comprising administering to the patient an effective amount of an oxygen antagonist or other active compound.

Furthermore, there are methods for promoting wound healing in an organism comprising administering to the organism or wound an effective amount of an oxygen antagonist or other active compound.

Furthermore, the present invention covers a method for preventing or treating neurodegeneration in a mammal comprising administering to the mammal an effective amount of an oxygen antagonist or other active compound.

The present invention also encompasses reducing the oxygen demand of the biomass, which means that the amount of oxygen the biomass needs to survive is reduced. This can be achieved by providing an effective amount of one or more active compounds. It is generally known how much oxygen a particular biological substance needs to survive, which also depends on time, pressure and temperature. In certain embodiments of the invention, the oxygen requirement of the biomass is reduced by about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any range derivable therein.

Additional embodiments of the present invention relate to methods for preventing alopecia, eg, alopecia induced by chemotherapy, by administering to a patient who has or will undergo chemotherapy an effective amount of at least one active compound.

In the case where biological material is protected from damage or further damage, it is contemplated that it may be about, at least about or at most about 30 seconds, 1, 2, 3, 4, 5, 10 seconds after the initial injury (wound or trauma or degeneration) occurs. , 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5, 6, 7 days, 1, 2, 3, 4, 5 weeks, 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20 or more years, and any combination or range that may result therefrom, exposing the biological material to the oxygen antagonist. Thus in further embodiments of the invention, the method includes an initial assessment of any injury, trauma, trauma or degeneration.

In certain embodiments of the invention there are methods for treating a patient affected by a hematological disorder, by which is meant a disease, disorder or condition affecting any hematopoietic cell or tissue. Examples include sickle cell disease and thalassemia. Thus, in some embodiments, there are methods of treating a patient with sickle cell disease and thalassemia with an effective amount of an active compound. In other embodiments, there is a method for enhancing the viability of a patient with cystic fibrosis (CF) by administering or providing an effective amount of an active compound. In other methods of the invention there are methods for treating cyanide poisoning in a subject comprising administering an effective amount of an active compound. In certain embodiments, the compound is _H2S .

Other aspects of the invention relate to methods for preserving one or more cells separated from an organism, the method comprising contacting the cells with an effective amount of an oxygen antagonist or other reactive compound to preserve the one or more cells. In addition to the cells and cell types discussed above and elsewhere in this application, shrimp embryos are specifically contemplated for use in the present invention.

Also, in some embodiments of the invention, there are methods for preserving platelets. Using the techniques of the present disclosure, the disadvantages of the prior art are reduced or eliminated. Embodiments involving platelets and oxygen reduction have wide application including, but not limited to, any application that would benefit from long-term preservation of platelets.

In one embodiment, oxygen reduction technology can be embodied in a kit. For example, a kit currently sold under Product No. 261215 provided by Becton Dickinson utilizes the selection technique described herein. The kit includes an anaerobic generator (such as a hydrogen generator), a palladium catalyst, an anaerobic indicator, and an airtight, sealable "BioBag" in which the above components (along with the platelets in an airtight bag) are placed inside and sealed.

In other embodiments of the invention there is a method of reversibly inhibiting the metabolism of a cell and/or organism by providing an effective amount of an active compound. It is specifically contemplated that rotenone is not a compound employed in this method or possibly other methods of the invention. Furthermore, it is also contemplated that in some embodiments, rotenone is excluded as an active compound. Similarly, consideration may exclude nitric oxide as an active compound.

In other embodiments of the invention, methods are provided for protecting the biological material from damage or injury by providing an effective amount of an active compound to enhance the ability of the biological material to enter into arrest in response to injury or disease, thereby enhancing the survival of the biological material. Related embodiments include methods of preparing or eliciting a biological substance into stasis in response to injury or disease by providing an effective amount of an active compound. Other related embodiments include methods of inducing biological material into pre-stagnation, thereby protecting the biological material from damage or damage. For example, treatment with the active compound at a dose or for less time than required to induce arrest allows the biological substance to more readily or more completely achieve the favorable state of arrest in response to injury or disease, Instead of treatment with the active compound, the biological material will die or suffer damage or injury before it reaches a protective level of stagnation, eg a level sufficient to render the biological material resistant to lethal hypoxia.

Certain injury and disease states cause biological substances to reduce their metabolism and/or temperature to a point where stagnation may not be achieved. For example, hypoxia, ischemia, and blood loss all reduce the amount of oxygen available and provided to oxygen-utilizing biomass, thereby reducing oxygen utilization in the cells of the biomass, reducing energy production from oxidative phosphorylation, And thus reduces heat production, leading to hypothermia. Depending on the severity or time elapsed after the onset or progression of harmful injury or disease injury, "stasis" may or may not have been achieved. Treatment with an active compound reduces the threshold for inducing arrest (i.e., the severity or duration of damage required to achieve arrest), or it may increase or potentiate a nociceptive or disease stimulus in biological matter under deleterious conditions. Induction of arrest, wherein said deleterious conditions would not have resulted in arrest had not been applied to the active compound treatment. This activity of the active compound is achieved by comparing the effects (magnitude, kinetics) of noxious or disease stimuli alone inducing arrest with those in which the biological substance was exposed to the active compound before, at the same time, after exposure, or any combination thereof to measure. For example, as described in Example 11 of this patent application, pre-exposing mice to 150 ppm _H2S in air prior to exposure to hypoxia (5% _O2 ) caused an approximately 2-fold decrease in _CO2 production. Subsequently, _CO2 production in the pretreated mice was reduced approximately 50-fold during hypoxia. In contrast, although _CO2 production was also decreased in control, non- _H2S -treated mice, hypoxic viability of the mice was not achieved, probably because the mice died before reaching stasis.

In other aspects of the invention there is a method for inducing sleep in an organism comprising exposing the organism to an effective amount of an active compound, wherein said effective amount is less than an amount that induces sleep in the organism. The term "sleep" is used according to its usual and ordinary meaning in medical terms. Sleep is distinguished from other states of unconsciousness, which is also considered a state obtainable by the method of the present invention.

The invention also relates to a method for anesthetizing a biological substance, the method comprising exposing the substance to an effective amount of an active compound, wherein said effective amount is less than that which would induce stagnation in the biological substance.

In the methods discussed above, the duration and/or amount of less than an effective amount that can induce stagnation in the biomass can be reduced. Said reduction may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 percent of the amount to induce arrest. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or any range of reductions in amounts derivable therein. The reduction can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, 1, 2, 3, 4, 5 , 6, 7 days, 1, 2, 3, 4, 5 weeks and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or may be derived therefrom A reduction in the duration of any range. Alternatively, said reduction may be with respect to the total effective amount provided to the biological material, which may be 1, 2, percent, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or any range reduction.

It is particularly contemplated that the invention may be used to preserve organisms for consumption or laboratory research, such as flies, frogs, fish, mice, rats, dogs, shrimp and their embryos.

The methods of the invention may involve the use of apparatus or systems that maintain the environment in which the biological material is placed or exposed. The invention comprises an apparatus in which an oxygen antagonist or other active compound is provided, in particular as a gas. In some embodiments, the apparatus comprises a container having a sample chamber containing biological material, wherein the container is connected to a gas supply comprising an oxygen antagonist. It is particularly contemplated that the container may be strong or it may be flexible, such as a bag.

In some embodiments, the invention is an apparatus for preserving cells, the apparatus comprising: a container having a sample chamber having a volume no greater than 775 liters; a first gas supply in fluid communication with the sample chamber, a first gas Supply includes carbon monoxide. In a further embodiment, the instrument also includes a cooling unit to adjust the temperature of the sample chamber, and/or to adjust the amount of oxygen antagonist or other reactive compound in the sample chamber or the amount of oxygen antagonist or other reactive compound in solution in the sample chamber. amount of gas regulator.

It is contemplated that there may be a gas supply for a second or additional gas or a second or additional gas supply for an oxygen antagonist or other active compound. The second gas supply can be connected to the sample chamber or it can be connected to the first gas supply. As discussed above, the additional gas may be a non-toxic and/or non-reactive gas.

In some embodiments of the invention a gas regulator is part of the apparatus. One, two, three or more gas regulators may be used. In some cases, the gas regulator regulates the gas provided to the sample chamber from the first gas supply. Alternatively, it regulates the gas supplied to the sample chamber or the first gas supply from the second gas supply, or there is a regulator for both the first and the second gas supply. It is further contemplated that any gas regulator may be programmed to control the amount of gas supplied to the sample chamber and/or other gas supplies. The adjustment may or may not be for a specified period of time. There may be a gas regulator, which may or may not be programmable for any gas supply connected directly or indirectly to the sample. In some cases, the gas regulator is electronically programmed.

In some cases, the pressure and/or temperature within the sample chamber can be adjusted with a pressure regulator or temperature regulator, respectively. Like gas regulators, these regulators may be electronically programmable. The apparatus of the present invention may also have cooling and/or heating units to achieve the temperatures discussed above. The unit may or may not be electronically programmable.

In other embodiments, the apparatus includes a wheeled cart on which the container is placed, or it may have one or more handles.

It is particularly contemplated that the invention includes an apparatus for cells, tissues, organs, and even whole organisms, wherein the apparatus has: a container having a sample chamber; a first gas supply in fluid communication with the sample chamber, the first gas supply comprising oxygen An antagonist or other active compound; an electronically programmable gas regulator that regulates the gas supplied to the sample chamber from the first gas supply.

In some embodiments, the instrument also has structures configured to provide a vacuum within the sample chamber.

Furthermore, any oxygen antagonist or other active compound described in this application is contemplated for use with the apparatus of the present invention. In a particular embodiment, carbon monoxide may be administered with the apparatus. In other cases, chalcogenide compounds or compounds having a reducing agent structure may be applied. In still further embodiments, the device is used to administer the active compound. In particular embodiments, the invention covers a device or use thereof. In certain embodiments, the device is a single dose delivery device. In other embodiments, the device is an inhaler or nebulizer. In still further embodiments, other devices include, but are not limited to infusion devices, such as pens, pumps such as infusion pumps, or patches. Also, it is contemplated that these devices may or may not be single dose delivery devices.

Furthermore, the present invention relates to screening assays. In some embodiments, candidate substances are screened for their ability to act as oxygen antagonists or active compounds, in particular including protective metabolizers. This can be done with any of the assays described herein, for example by measuring carbon dioxide output. Any substance identified to exhibit properties of an oxygen antagonist or other active compound can be further characterized or tested. Furthermore, it is contemplated that such substances can be applied to biological substances to induce stagnation or subsequent production of the substances.

In certain embodiments, there are screening methods for active compounds, including activity arresting compounds. In addition, screening methods can be used for oxygen antagonists or for any other compound that can carry out the methods discussed herein. In some embodiments, there is a screening method comprising a) exposing a zebrafish embryo to a substance; b) measuring the heart rate of the embryo; c) comparing the heart rate of the embryo in the presence of the substance to the heart rate in the absence of the substance , a reduction in heart rate, for example by 50% or more, identifies the substance as a candidate active compound. In addition to zebrafish embryos, it is contemplated that other non-human organisms, such as fish, frogs, flies, shrimp or embryos thereof, may also be used. In a further embodiment, the embryo's heart rate is measured by counting heartbeats. In some cases, this can be done by viewing the embryos under a dissecting microscope.

Other screening embodiments include: a) exposing nematodes to substances; b) measuring one or more of the following cellular respiration factors: i) core body temperature; ii) oxygen consumption; iii) motility; or iv) carbon dioxide production; c) The cellular respiration factor of nematodes in the presence of the substance is compared to the cellular respiration factor in the absence of the substance, wherein a reduction in said characteristic identifies the substance as a candidate active compound. In some methods of the invention, it is specifically contemplated to measure the motility of nematodes.

In some embodiments, the method first involves identifying suitable screening substances. In certain embodiments, the species will be a chalcogenide compound, a reducing species, or have a structure of Formula I or Formula IV, or any other compound discussed herein.

It is further contemplated that subsequent screens may be performed in organisms considered higher or more complex than those used for the primary or initial screen. Therefore, it is contemplated that one or more factors of cellular respiration will be tested in these other organisms for further evaluation of candidate compounds. In certain embodiments, subsequent screening involves the use of mice, rats, dogs, and the like.

It is contemplated that many different organisms or biological substances (other cells or tissues) can be used and many different factors of cellular respiration can be assayed in the screening methods of the present invention. Furthermore, it is contemplated that in some embodiments of the invention, multiple such screens are performed simultaneously.

It will of course be understood that in order for a substance to be considered a candidate active compound (or an oxygen antagonist, or a stasis inducer or a protective metabolizer, etc.), the substance cannot kill organisms or cells in the assay and the effect must be reversible ( That is, the altered characteristics need to return to their pre-exposure levels).

It will of course be understood that any method of manipulation may be used in the manufacture of a medicament for the treatment or combating of a particular disease or condition. This includes, but is not limited to, the preparation of medicaments for the treatment of hemorrhagic or hematological shock, wound and tissue injury, hyperthermia, hypothermia, neurodegeneration, sepsis, cancer and trauma. Furthermore, the present invention includes, but is not limited to, the preparation of a medicament for the treatment of preventing death, shock, trauma, organ or tissue rejection, damage caused by cancer therapy, neurodegeneration, and wound or tissue damage.

As discussed above, biological stasis is not any of the following states: sleep, coma, death, anesthesia, or grand mal seizures. However, it is contemplated that in some embodiments of the invention, such states are desirable objects for use in the methods, compositions and articles of manufacture of the invention. Any embodiments discussed in relation to one aspect of the invention also apply to the other aspects of the invention. Also, embodiments may be combined.

Any of the embodiments involving "exposing" a biological substance to an active compound can also be practiced such that the active compound is provided to the biological substance or the active compound is administered to the biological substance. The term "provide" is used according to its usual and ordinary meaning. "Provide or provide for use" (Oxford English Dictionary), in relation to a patient, may refer to the act of a physician or other medical professional who prescribes a particular active compound or administers it directly to the patient.

The embodiments in the Examples section are to be understood as embodiments of the invention applicable to all aspects of the invention.

The use of the term "or" in the claims is used to mean that "and/or" unless expressly stated refers only to the alternative or the alternatives are mutually exclusive, although this disclosure supports only referring to the alternative and "and/or "Definition.

Throughout this application, the term 'about' is used to denote a value including the standard deviation of error of the device or method used to determine the value. In any embodiment discussed in the context of a numerical value used in conjunction with the term 'about' , especially considering that the term approximately can be omitted.

According to long-standing patent law, the words "a" and "an", when used in conjunction with the word "comprising" in a claim or specification, mean one or more unless specifically stated otherwise.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications will be seen from the detailed description to be within the spirit and scope of the invention. Modifications will become apparent to those skilled in the art.

Brief description of the drawings

The following drawings form part of this specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments provided herein.

Figure 1 Human keratinocytes survived exposure to 100% CO. Cells were observed with the naked eye using an inverted phase-contrast microscope. Quantification of the number of viable keratinocytes as judged by trypan blue staining, an indicator of cell death.

Figure 2. Discontinuities in viability in hypoxia. In wild-type embryos, the adult stage was assessed after 24 hours of exposure to anaerobic (pure N ₂ ), moderate hypoxia ((0.01 kPa O ₂ , 0.05 kPa O ₂ , or 0.1 kPa O ₂ ), or mild hypoxia Viability. All data points are the result of at least 3 independent experiments and unexplained worms were removed from the total.

Figure 3. Carbon monoxide protects against hypoxia. Viability to adulthood was assessed after 24 hours of exposure to pure carbon monoxide, 0.05 kPa _O2 / _N2 or 0.05 kPa _O2 /CO in wild-type embryos. All data points are the result of at least 3 independent experiments and unexplained worms were removed from the total.

Figure 4A When mice are exposed to hydrogen sulfide, metabolic rate decreases in front of body core temperature. Exposure of mice to 80 ppm (at 0 min on the X-axis) resulted in an approximately 3-fold decrease in _CO2 production (black line) in less than 5 min. This is before the animal's core temperature decreases towards ambient temperature (gray line).

Figure 4B Temperature of mice exposed to hydrogen sulfide. Each line represents a serial measurement of the core body temperature of an individual mouse exposed to 80 ppm of _H2S or room air. Numbers on the vertical axis are degrees Celsius. On the horizontal axis, numbers reflect time in hours. Recovery was recorded after the test was run for 6 hours. The starting point was at 1:00 and the end of the 6 hour treatment was approximately 7:00.

Figure 5 Exposure to 80 ppm hydrogen sulfide causes the core body temperature of mice to approach ambient temperature. Starting at time 0:00, the gas is turned on and the temperature is lowered. At time 6:00, the atmosphere was switched back to room air. Triangles indicate the core body temperature of mice measured by radiotelemetry. This is approximately 39°C at time 0:00. The diamonds represent the ambient temperature, which dropped from 23°C to 13°C in the first 3 hours of the test, and then rose again from 6:00 hours close to 23°C, stabilizing at about 9:00 hours.

Figure 6. The rate at which body core temperature decreases depends on the concentration of hydrogen sulfide administered to mice. All lines represent the core body temperature of individual mice as determined by radiotelemetry. Mice receiving 20 ppm and 40 ppm _H2S showed a small decrease in core temperature. Exposure to 60 ppm induced a large drop in temperature starting around 4:00. Mice exposed to 80 ppm showed a large drop in temperature starting at about 2:00.

Figure 7 Minimum core body temperature. The lowest core body temperature recorded for mice exposed to 80 ppm hydrogen sulfide was 10.7°C. Triangles indicate the core body temperature of the mice as determined by radiotelemetry, starting at approximately 39°C at time 0. The diamonds represent the ambient temperature which started at about 23°C and dropped below 10°C at the midpoint of the experiment before it then rose again towards room temperature.

Figure 8A Mild temperature acclimated mice have increased endogenous hydrogen sulfide levels. The gray histograms (two left bars) represent the endogenous H ₂ S concentrations of two individual mice adapted to 4°C; the black histograms (two right bars) represent the concentrations of two individual mice adapted to 30°C Endogenous H ₂ S concentration. Hydrogen sulfide concentration was determined by GC/MS.

Figure 8B Effect of ambient temperature on H2S-dependent temperature drop. The rate of decrease in core temperature due to exposure to hydrogen sulfide depends on the acclimatization temperature. Mice were exposed to gas at 1:00. Triangles indicate core temperature of mice acclimatized to 12°C as determined by radiotelemetry. Squares indicate the core body temperature of animals acclimated to 30 °C.

9 is a block diagram illustrating a breathing gas delivery system according to an embodiment of the present invention.

10 is a schematic diagram illustrating a breathing gas delivery system according to an embodiment of the present invention.

Figure 11 is a schematic diagram illustrating a breathing gas delivery system according to a further embodiment of the present invention.

Figure 12 is a flowchart illustrating operation in accordance with an embodiment of the present invention.

13 is a schematic diagram illustrating a tissue treatment gas delivery system according to an embodiment of the present invention.

Figure 14 is a flowchart illustrating operation in accordance with an embodiment of the present invention.

Figure 15 Metabolic inhibition protects nematodes from hypothermia-induced death. Nematodes exposed to cold temperature (4°C) did not survive after 24 hours. However, if anaerobic conditions were maintained during hypothermia (and for 1 hour before and after it), a large proportion of nematodes survived.

Figure 16 Short _CO2 pretreatment results in longest anaerobic survival. Adult flies were exposed to 100% _CO2 for the indicated time, the atmosphere was made anoxic by flushing with _N2 , and the tubes were then sealed. After 22 hours, the tubes were opened to room air. Flies were allowed to recover for 24 hours before scoring for viability.

Figure 17 CO ₂ enhances hypoxic survival to varying degrees. Adult flies were hypoxic in a low-flow test, either directly in room air (without preconditioning), or after 10 min exposure to 100% _CO2 . After the indicated time, the tubes were opened to room air. Flies were allowed to recover for 24 hours before scoring for viability.

Figure 18 Addition of 50 ppm _H2S to CO increases the proportion of flies that survive hypoxia. Adult flies were hypoxic in a low-flow test, either directly in room air (without pretreatment), or after exposure to 50 ppm _H2S equilibrated with CO.

19 is a schematic diagram of an exemplary system for removing oxygen from platelets and a scheme according to embodiments of the present disclosure.

Figures 20A-B show changes in core temperature in rats exposed to hydrogen sulfide (A) and mice exposed to carbon dioxide (B).

Figure 21 shows the gas matrix of the step-by-step test plan used to determine the concentration of active compound.

22A-B show negative pressure devices that can be used to deliver or administer active compounds.

Figure 23 Survival of mice in 5% oxygen. Mice were either exposed to room air for 30 min before exposure to 5% _O2 (control; black line; n=9) or to room air for 10 min before exposure to 5% _O2 and then exposed to 20 min 150 ppm _H2S (experiment; red line; n=20) and the length of time they survived was measured. At 60 minutes the test was terminated and if the animals were still alive (all in the experiment and none in the control group) they were returned to their cages.

Figure _24H2S increases survival under lethal oxygen tension. The graph shows the results of the experiments described in Figure 23. The x-axis shows the time in minutes that the mice survived the low oxygen tension. The black histogram shows the survival time in the absence of _H2S , while the light histogram shows the survival time in the presence of _H2S . In the latter group, mice were exposed to 150 ppm _H2S before the partial pressure of oxygen decreased to between 5% and 2.5%. Survival time was measured and was at least 60 minutes in all _H2S treated groups.

Figure 25. Metabolic rate of mice in 5% oxygen. Mice were exposed to room air for 10 min followed by 150 ppm _H2S for 20 min before exposure to 5% _O2 . Metabolic rate is measured by _CO2 output. The _CO2 output was about 2500ppm before exposure, the metabolic rate was reduced by about 2 times after 20 minutes of _H2S exposure, and after several hours of exposure to 5% _O2 , the _CO2 output was already reduced by about 50 times, from pre-exposure levels to approximately 50 ppm. At 6 hours, mice were returned to room air and allowed to recover. This data is from one of the mice included in Figure 23 (experimental group).

Figure 26 Mice exposed to 100 ppb _H2Se . The graph shows exposure to _H2Se in minutes (x-axis) with a decrease in core body temperature (Celsius shown on the right is drawn with a line showing a gradual decrease) and with a decrease in respiration (at The ppm _CO2 shown on the left is drawn with a jagged line showing the reduction).

Figure 27 Mice exposed to 10 ppb _H2Se . The graph shows exposure to _H2Se in minutes (x-axis) with a decrease in core body temperature (Celsius shown on the right is drawn with a line showing a gradual decrease) and with a decrease in respiration (at The ppm _CO2 shown on the left is plotted with a zigzag line showing a decrease, with a nadir occurring at 5 minutes of exposure).

Figure 28 H ₂ S pretreatment enhances survival of mice under hypoxic conditions. Mice were exposed to 5% O ₂ (5%), 4% O ₂ (4%), 5% O ₂ for 1 hour and then exposed to 4% O ₂ (4% + 5% for 1 hour) or 5% O 2 ₂ 1 hour followed by 3% O ₂ exposure (3% + 1 hour 5%) followed by 30 minutes exposure to room air (no PT) or 10 minutes exposure to room air followed by 20 minutes exposure to 150ppm H ₂ S (PT) and measure their survival time. At 60 minutes the test was terminated and the animals were returned to their cages if they were still alive.

Figure 29 _CO2 production during transition to lethal hypoxia. Changes in _CO2 production upon switching to 5% _O2 or 4% _O2 were measured in mice exposed to room air for 30 minutes (no PT) or exposed to room air for 10 minutes followed by exposure to 150ppm _H2S for 20 minutes (PT). Also, the change in _CO2 production was measured for a stepwise switch to 5% _O2 for 1 hour followed by a switch to 4% _O2 . The percent change in _CO2 production is plotted, along with the standard error.

Figure 30 Survival of human keratinocytes upon exposure to 100% carbon monoxide (CO). Cells were inspected visually with an inverted phase-contrast microscope. Quantification of the number of viable keratinocytes as judged by trypan blue staining, an indicator of cell death.

Figure 31 Chronic exposure to low levels of _H2S results in heat resistance in C. elegans. Nematodes adapted to environments containing approximately 50 ppm _H2S in room air were significantly more resistant to the lethal effects of raising the ambient temperature to 35°C compared to siblings reared in room air alone.

Figure 32 Chronic exposure to low levels of _H2S increases lifespan in C. elegans. Nematodes adapted to an environment containing about 50 ppm _H2S in the room air had a longer lifespan than untreated controls.

Figure 33 Example of transient core temperature reduction in Sprague-Dawley rats. Core temperature measurements in rats exposed to 0.03% hydrogen sulfide mixed with room air (grey/dashed line) or 15% carbon dioxide/8% oxygen/77% helium (black/solid line). In this test, the temperature of the environmental chamber was 10°C during the treatment phase. When the gas returned to room air, the temperature of the environmental chamber was returned to room temperature (22°C). In each case, this is the point in time when the core temperature starts to rise (approximately 2 hours for the black/solid line and approximately 7.4 hours for the gray/dashed line).

Figure 34 Mouse core body temperature during exposure to 1.2 ppm hydrogen selenide in room air at 5°C ambient temperature during 2 hours and 10 minutes.

Figure 35 Core body temperature of rats during exposure to room air in an environmental chamber at an ambient temperature of 10°C. The black line depicts the core body temperature of the rat. The gray line depicts the ambient temperature.

Figure 36 Core body temperature of rats exposed to 80% helium 20% oxygen at an ambient temperature of 7°C. Time is depicted in hours on the x-axis. The total exposure time was approximately 5 hours (from 9:15AM to 2:15PM). No significant decrease in core body temperature was seen.

Figure 37 Core body temperature of rats during exposure to 15% carbon dioxide, 20% oxygen and 75% helium at an ambient temperature of 7°C. The exposure time is about 2 hours. Rats were exposed to room air starting at the time point when the temperature started to rise (shortly after the point marked 38512.6). During exposure of the rats to room air, the ambient temperature was returned to room temperature.

Figure 38 Core body temperature of rats exposed to 15% carbon dioxide, 8% oxygen and 77% helium at an ambient temperature of 7°C. The exposure time is approximately 4 hours. The gray line depicts the ambient temperature. The black line depicts the core temperature. The point at which ambient and core temperatures rise is when the gas transitions to room air.

Figure 39 Core temperature of dogs exposed to carbon dioxide/helium/oxygen. The dashed line is the core temperature when the gas is turned on (about 24 minutes) and off (about 55 minutes). Figure 40. Core temperature of dogs exposed to increasing concentrations of carbon dioxide. Dashed lines indicate when changes are made to the gas. At approximately 63 minutes, the gas was changed from room air to 9% carbon dioxide in room air. At approximately 85 minutes, the atmosphere was changed from 9% carbon dioxide in room air to 12% carbon dioxide in room air. At approximately 115 minutes, the atmosphere was changed from 12% carbon dioxide in room air to 15% carbon dioxide in room air. The test was terminated at approximately 135 minutes.

Figure 41. Instrumentation employed in the screening method.

Figure 42 Oxygen consumption (gray histogram) and carbon dioxide production (black histogram) of animals exposed to hydrogen sulfide for 4 hours per day for at least 1 week and control animals exposed to the same conditions lacking hydrogen sulfide.

Figure 43 Respiratory quotients of animals exposed to hydrogen sulfide for 4 hours per day for at least 1 week ( _H2S 2900 and _H2S 2865) and control animals exposed to the same conditions lacking hydrogen sulfide (2893 and 2894).

Description of Illustrative Embodiments

I. stagnation

In "arrest" or "arrest," cells, tissues, or organs or organisms (collectively "biomass") are alive, but cell division, cellular functions, and/or metabolic states necessary for developmental progression are slowed or even stopped . This state is desirable in many situations. Arrest itself can be used as a preservation method, or it can be induced as part of a cryopreservation protocol. Biological material can be preserved for example for research use, for transport, for transplantation, for therapeutic treatment (eg ex vivo therapy) and for prophylaxis of traumatic episodes. Stasis with respect to the whole organism serves a similar purpose. For example, if the creature has entered stasis, it can facilitate the transport of the creature. This may reduce physical and physiological damage to the organism by reducing or eliminating stress or physical damage. These embodiments are discussed in further detail below. Stasis can be beneficial by reducing the oxygen requirement of biological matter and thus blood flow. This can extend the period of time over which biological material can be separated from a life-sustaining environment and exposed to a death-inducing environment.

Although recovery from relatively prolonged periods of unexpected hypothermia has been reported (Gilbert et al., 2000), there has recently been interest in intentionally inducing stasis in organisms. (Discussion of any reference should not be construed as an admission that such reference constitutes prior art. In fact, some of the references discussed herein are not prior art with respect to the priority application). Controlled hyperthermia therapy has been studied, as well as administration of cold solution flow into the aorta (Tisherman, 2004), induction of asystole (Behringer et al., 2003) or nitric oxide-induced arrest (Teodoro et al., 2004).

A stagnant creature is different from a creature under general anesthesia. For example, a mildly stagnant creature (between 2x and 5x reduction in cellular respiration) exposed to room air will begin to tremble, whereas an anesthetized creature will not. Also, the mildly immobilized organisms were expected to respond to the toe squeeze, whereas the anesthetized organisms did not. So stasis is not the same thing as being in a commonly practiced state of anesthesia.

_CO2 production is a direct marker of cellular respiration associated with the metabolism of an organism. This _may be different from " _CO2 out," which refers to the amount of _CO2 exhaled by the lungs. Certain reactive compounds, for example, hydrogen sulfide, can inhibit carbonic anhydrase activity in the lungs, which inhibits the conversion of carbonic acid to _CO2 and its release from the blood in the lungs, thus exhibiting a concomitant decrease in _CO2 evolution without Corresponding decrease in cellular _CO2 production.

The present invention is based on the observation that certain types of compounds are effective at inducing reversible arrest in biological matter. Other patent applications discuss induction of arrest, including the following applications: U.S. Patent Application 10/971,576, 10/972,063, and 10/971,575; U.S. Patent Application 10/971,576; U.S. Patent Application 10/972,063; and U.S. Patent Application 10/971,575 , all of which applications are hereby incorporated by reference.

A. to regulate body temperature

Stasis in warm-blooded animals will affect thermoregulation. Thermoregulation is a property of so-called "warm-blooded" animals that allows organisms to maintain a relatively constant core body temperature, even when exposed to significantly changing (cold or hot) ambient temperatures. The ability to control thermoregulation by inducing stasis is an aspect of the invention and allows for uses similar to those discussed above.

Thermoregulation can be facilitated by placing an organism, limb, or isolated organ or tissue within a chamber/apparatus whose temperature can be controlled. For example, a greenhouse or chamber-type device similar to an autoclave can house a whole organism and can be connected to a device that can regulate temperature. Smaller devices such as blankets, sleeves, cuffs, or gloves are also contemplated (eg, AVAcore Technologies, Palo Alto, CA's CORE CONTROL Cooling System, US Patent 6,602,277). This chamber/device can be used to increase or decrease the ambient temperature.

B. Biomass

Biological materials contemplated for use in the present invention include materials derived from invertebrates and vertebrates (including mammals); biological materials include organisms. In addition to humans, the present invention is applicable to mammals of veterinary or agricultural importance, including those from the following classes: canines, felines, equines, bovines, ovines, murines , suids, goats, rodents, lagomorphs, wolves and bears. The invention also extends to fish and birds. Other examples are disclosed below.

Also, there are different types of biological matter. It can be cells, tissues or organs as well as different compositions, methods and apparatus related organisms. Nonprovisional US Patent Applications 10/971,576, 10/972,063, and 10/971,575 are hereby incorporated by reference in their entirety.

In some embodiments, the biological material is or comprises cells. Consider that a cell can be any oxygen-utilizing cell. Cells can be eukaryotic or prokaryotic. In certain embodiments, the cells are eukaryotic. More specifically, in some embodiments, the cells are mammalian cells. Mammalian cells contemplated for use in the present invention include, but are not limited to, those from humans, monkeys, mice, rats, rabbits, hamsters, goats, pigs, dogs, cats, ferrets, cows, sheep and horses.

Also, the cells of the invention may be diploid, but in some cases the cells are haploid (sex cells). Furthermore, the cells may be polyploid, aneuploid, or anucleated. Cells can be derived from specific tissues or organs, such as cells from the following tissues or organs: heart, lung, kidney, liver, bone marrow, pancreas, skin, bone, vein, artery, cornea, blood, small intestine, large intestine, brain, spinal cord, Smooth muscle, skeletal muscle, ovaries, testes, uterus and umbilical cord. Furthermore, cells can also be characterized as one of the following cell types: platelets, myeloid cells, erythrocytes, lymphocytes, adipocytes, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, skeletal muscle cells, endocrine cells, glia cells, neurons, secretory cells, barrier function cells, contractile cells, absorptive cells, mucosal cells, limbal cells (from the cornea), stem cells (totipotent, pluripotent, or pluripotent), unfertilized or fertilized oocytes cells or sperm cells.

1. Different sources

The following are examples of sources from which biomass can be obtained: Embodiments of the invention include, but are not limited to, these examples.

a. Mammals

In certain aspects of the invention, the mammal is of the orders Monotremes, Marsupiales, Insectivora, Macroscelidia, Dermoptera, Chiroptera, Scandentia, Primates, Odontia .

科(Microbiotheriidae)(例如Monito del Monte)、新袋鼠科(例如新袋鼠类)、袋鼬科(例如袋鼬类)、袋食蚁兽科(例如袋食蚁兽)、袋狼科(例如袋狼)、袋狸科(例如袋狸类)、兔袋狸科(例如兔袋狸类)、袋鼹科(例如袋鼹类)、袋貂科(例如袋貂类)、袋鼯科(例如浣熊类、袋鼯类)、鼯科(例如矮小负子袋鼠(Pygmy Possums))、袋鼠科(例如袋鼠类、小袋鼠类)、蜜貂科(例如蜂蜜负子袋鼠(Honey Possum))、袋熊科(例如袋熊类)和树袋熊科(例如树袋熊类)。Examples of Monotremes include Echidnas (eg, Echidnas) and Platypuses (eg, Platypus). Examples of Marsupiales include Possumidae (e.g. Opossums), small

Microbiotheriidae (e.g. Monito del Monte), Neokangarooidae (e.g. Neokangaroos), Quollidae (e.g. quolls), Numbatidae (e.g. Numbats), Thylacidae (e.g. Numbats Wolves), Bandicoots (e.g. Bandicoots), Cymbalidae (eg Bandicoots), Concuspidae (eg Bandicoots), Possumidae (eg Possums), Possumidae (eg Procionidae, Pygmy Possums), Glididae (e.g. Pygmy Possums), Kangaroosidae (e.g. Kangaroos, Wallabies), Mineridae (e.g. Honey Possums), Pygmy Possums Ursidae (eg wombats) and Koalas (eg koalas).

The order Insectivora includes, for example, the family Sloodonidae (e.g., Scutellaria), Falkidae (e.g., Analhog, Otter Shrew), Golden Moleidae (e.g., Golden Mole), Hedgehogidae (e.g., Hedgehog, Hedgehogs), Shrewidae (e.g. Shrews) and Moleidae (e.g. Moles, Musk Moles). The order Rock mice includes the family Rock mice (eg elephant shrews). The order Climotheria includes the family Treeshrews (eg Treeshrews). The order Dermoptera includes the family of flying foxes (such as spotted flying monkeys). The order Chiroptera includes the following families: Pteropidae (e.g., fruit bats, flying foxes), Pterobatidae (e.g., sage-tailed foxes), Pteropidae (e.g., hog-nosed or hornet bats), Sheath-tailed bats (e.g., tailed bat), night-faced bats (e.g. pit-faced bats), giant-eared bats (e.g. false vampire bats), horseshoe bats (e.g. horseshoe bats), chrysoplastids (e.g. pitbull dog bats, murlocs FishermanBats), Meadowattidae, Leaf-Nosed Bats (such as New World Leaf-Nosed Bats (New World Leaf-NosedBats)), Long-legged Bats, Smoky Bats, Orchididae, Squirrel-Nosed Bats, Bats (eg, Common (Common) bats), Brachyptidae (eg, Short-tailed bats), and Wrinkled-nosed bats (eg, Wrinkled-nosed bats).

The order Primates includes the following families: Lemuridae (e.g., lemurs), Mouse Lemuridae (e.g., small-billed lemurs), Macrophuridae (e.g., giant lemurs, woolly lemurs), Ayepiidae (e.g., Lemurs), Lorises (e.g. slow lorises, baby monkeys, bush monkeys), tarsiers (e.g. tarsiers), capuchins (e.g. New World monkeys, marmosets, tamarins), gibbonidae (eg, gibbons), orangutans (eg, apes), and hominids (eg, humans).

Examples of the order Antodontidae include Anteatidae (e.g., Anteater), Slothidae (e.g., Three-toed Sloth), Two-toed Slothidae (e.g., Two-toed Sloth), and Armadilloidae (e.g., Armadillo armadillo). Examples of Lepidoptera include Pangolinidae (eg, pangolins). Examples of the order Aardvaridae include the Aardvaridae (eg, Aardvarks). Examples of Lagomorpha include pikas (eg, pikas) and leporidae (eg, hares and rabbits).

The order Rodentia includes the following families: Mountain Beaveridae (e.g., Mountain Beavers), Sciuridae (e.g., squirrels, marmots, chipmunks), Gochurididae (e.g., Gochudidae), Xenomuridae (e.g., Pocket Mice, Kangaroo Rats), Beaveridae (e.g., beavers), Lepidopteridae (e.g., scaly-tailed squirrels), Leptosciuridae (e.g., Derboa), Dormotae (e.g., rats and mice), Dormotae (e.g., Dormouse), Dormotae (e.g., Desert Dormouse), Dormotae (e.g., Jumping Mice), Dormotae (e.g., Jumping Mice), Dormotae Porcupines (e.g., Jerboa), Porcupines (e.g., Old World Porcupines), American Porcupines (e.g., New World Porcupines), Caviaceae (e.g., guinea pigs, Patagonian hares (Maras)) , Capybaridae (eg, Capybara), Chinchidae (eg, Cavy), Camelidae (eg, Thornidae), Agoutiidae (eg, Agouti), Chinchidae ( For example, Chinchilla, Lagomus), Coryciidae (e.g., Coypus), Coypuidae (e.g., Coypus), Combs (e.g., Tuco-Tucos), Deguidae (e.g., For example, Octododonts, degus), Abrocomidae (e.g. chinchillas), Echimyidae (e.g. Spiny Rat), Thryonomyidae ( For example, Cane Rat), Petromyidae (e.g., African Rock Rat), Bathyergidae (e.g., Mole Rat), and Peptotoyd porcupines (e.g., , porcupine).

The order Cetacea includes the following families: Iniidae (such as Amazon Popoise), Lipotidae, Platanistidae, Pontoporiidae, beaked whales family Ziphiidae (e.g., Beaked Whales), Physeteridae (e.g., Sperm Whales), Monodontidae (e.g., Beluga Whale, Beluga Whale ( Narwhal), Delphinidae (e.g., Marine Dolphin, Killer Whale), Phocoenidae (e.g., Porpoises), Balenopteridae (e.g., Whale ), Balaenidae (eg, Right Whale), and Eschrichtiidae (eg, Gray Whale).

The order Carnivora includes the following families: Canidae (e.g., dogs, foxes, wolves, jackals, coyotes), Ursidae (e.g., bears), Procyonidae (e.g., raccoons, Proboscis, honey bear, red panda), Ailuropodidae (e.g., giant panda), Mustelidae (e.g., ferret, skunk, badger, otter), Viverridae (e.g., Civet, Civet), Herpestidae (e.g. mongoose), Protelidae (e.g. hyena), Hyaenidae (e.g. hyena), Felidae (e.g. , cats), Otariidae (e.g., Eared Seals, sea lions), Odobenidae (e.g., walruses), and Phocidae (e.g., Earless Seals ).

The order Proboscidea includes the family: Elephantidae (eg, elephants). The order Hyracoidea includes the family Procaviidae (eg, hyraxes). The order Sirenia includes the families Dugongidae (eg, dugongs) and Trichechidae (eg, manatees). The order Perissodactyla includes Equidae (eg, horses, donkeys, zebras), Tapiridae (eg, tapirs), and Rhinocerotidae (eg, rhinos). The order Artiodactyla includes the following families: Suidae (e.g., pigs, wild boars), Tayassuidae (e.g., peccaries), Hippopotamidae (e.g., hippopotamuses), Camelidae (e.g., ) (e.g., camel, llama, vicuna), Tragulidae (e.g., hevrotains), Moschidae (e.g., musk deer), Cervidae (e.g., deer, elk , moose), Giraffidae (e.g., giraffe, okapi), Antilocapridae (e.g., pronghorn), and Bovidae (e.g., cattle, sheep, antelope, goat ).

b. Reptiles

In some embodiments, the biological material is or is derived from a reptile. The reptiles may be of the order Turtles, Sidenectes, Squamates, Rhinocephala, or Crocodiles. The reptiles of the order Turtles can be, for example, Dichonidae, Snapping Turtles (e.g., soft-shelled turtles), Turtleidae (e.g., loggerhead turtles, sea turtles), mud turtles (e.g., leatherbacks), Turtleidae (e.g., Painted turtles, yellow-bellied slider turtles, water turtles, snail turtles, box turtles), kinetothoracids (e.g. musk turtles), Saurotypidae, tortoises (e.g. elephant tortoises, desert gopher tortoises, Arda Bulla tortoise, European tortoise, Hermann's tortoise), soft-shelled turtles (eg, Chinese soft-shelled turtle, spiny soft-shelled turtle), or ratites. The reptile of the suborder Side-necked Turtles may be, for example, of the family Ophiocididae (eg, Snake-necked turtles) or the family of Osicilidae (eg, Marsh side-necked turtles).

The scaly reptiles may be, for example, Iguanidae (e.g. Crested Tree Lizard, Chow Lion, Indian Bloodsucker, Syrian Spinytail), Chamaeleontdidae (e.g. Chameleon), American Iguana (e.g., anole lizard, crowned iguana, common collared lizard, iguana, crested horned lizard, black tapped lizard, slender edged lizard, male flank-spotted lizard), gecko (e.g., gecko), scale Lizardidae, Lizardidae (e.g., whiptail lizards, arboreal lizards), Lizardidae (e.g., lizards, monocular lizards, viviparous lizards, geckos, long-tailed lizards), yellow lizards, skinks ( e.g., Skinks), African lizards (e.g., sun lizards), Amphiridae, Alligatoridae, snake lizards (e.g., scale-footed lizards, Alligator lizards, brown snake lizards , brittle lizards), Gilaidae (e.g., Venomous lizards), Borneusidae, Varanidae (e.g., Varanus lizards), Lepidopteridae, Blindidae, Hesperididae, Cylindidae (e.g. , pipe snakes), Uropeitidae, Scrophiidae, Anacondae (e.g., anaconda, anaconda, rock python), Scrofulidae (e.g., Javan Scrofula), Scropididae (e.g., mangrove snakes, emerald Forest snake, slippery snake, egg-eating snake, South African tree snake, rat snake, Esculapu snake, four-striped snake, eastern elapid snake, snout snake, hognose snake, king snake, (Montpelier) snake, Grass snakes, venomous snakes, black snakes, vine snakes, keelback snakes), cobras (e.g., lethal vipers, krait snakes, bonnet-headed cobras, coral snakes, cobras, copperheads, drum-bellied vipers), vipers Family (e.g., Venomous Vipers, Right Adders, Rattlesnakes, Rattlesnakes, Vipers), Hyphiridae (e.g., Sea Snakes (Sea Brait)), Phlomatidae (e.g., Basilidae), Burmidae or Brachycephalic lizards (for example, Burrowing lizards).

The beaked reptile may be, for example, a lizard (eg, Lizard). The reptile of the order Crocodiles can be, for example, of the family Crocodidae (eg, crocodile, Caiman), Crocodiidae (eg, crocodilians), or Gharialidae (eg, crocodilians).

c. Amphibians

The biological material of the present invention may be amphibian or derived from amphibians. An amphibian can be, for example, a frog or a toad. The frogs or toads can be, for example, of the family Pleurocaridae (e.g., Reicheria), Cryopharidae (e.g., P. , true (true) toads), Pleurocaridae (e.g., glass frogs and leaf frogs), poison dart frogs (e.g., poison dart frogs), Aphididae (e.g., Bombinae), Bomberidae (e.g., , ghost (ghost) frogs), shovel-nosed frogs (e.g., shovel-nosed frogs), hyla family (e.g., New World tree frogs), African tree frogs (e.g., African tree frogs), smoothfoot frogs (e.g., New Zealand Frogs), Lepidopteridae (e.g., Neotropical frogs), Ceratoporidae (e.g., South Asian frogs), Aphididae (e.g., microhylid frogs), Turtlebudidae (e.g., Australian frogs) . Frogs), Ranaidae (e.g., Shore frogs and its (true) frogs), Ranaidae (e.g., New World tree frogs), Snorriidae (e.g., Darwin's (burrowing toads), Ranaidae (eg, Seychelle frogs), Cauura (eg, salamanders) or Caecilians (eg, caecilians).

The amphibian may be a salamander. The salamanders can be, for example, of the family Axolotidae (e.g., Buramosauridae), Amphisaltidae (e.g., Amphiloxidae), Cryptogillidae (e.g., Giant Salamander and Cryptobranchidae), Macrosaltidae (e.g., , Gigaphyllidae), Microsaltidae (e.g., Asian salamanders), Pneumonidae (e.g., Pneumonidae), Olmidae (e.g., American salamanders, (torrent salamanders), salamanders (eg, oriental salamanders and newts) or eeloids (eg, sirens). Alternatively, the amphibian may be a caecilian. The caecilian can be, for example, of the family Euclididae (e.g., Eucalyptus), Ichthyophthidae (e.g., Asian caecilian), Rhizomatidae (e.g., Neotropical caecilian), Vermectidae (e.g., African Caecilians), Cecilidae (e.g., Aquatic Caecilians), or Caecilidae (e.g., Indian Caecilians).

d.birds

The biological material of the invention may be or may be derived from birds. The birds can be, for example, of the order Anseriformes (e.g., waterbirds), Swifts (e.g., Kyohos and swifts), Nighthawks (e.g., nocturnal birds), Phasianidae (e.g., shorebirds), storks Orders (e.g., storks), Mousebirds (e.g., mousebirds), Columbiformes (e.g., pigeons and doves), Buddhist monks (e.g., kingfishers), Pheasants (e.g., juvenile pheasants, crested pheasants pheasants, crested pheasants, pheasants), cuckooformes (for example, rhododendrons, musk pheasants, turacos), falconiformes (for example, gray diurnal birds), galliformes (for example, chicken-like birds), loons ( For example, loons), Craneformes (e.g., roosters, cranes, crakes), passerines (e.g., perching birds), Pelicans (e.g., pelicans), flamingos (e.g. e.g., flamingos), Woodpeckers (e.g., woodpeckers), Grebes (e.g., grebes), albatrosses (e.g., tube-nosed seabirds), psittaciformes (e.g., parrots), penguins (e.g., penguins), ostriches (e.g., owls), ostriches (e.g., cassowaires, emus, kiwi, ostriches, rheas), ostriches (e.g., For example, Quetto) or the order of the three-toed quail (for example, the three-toed quail).

e. Fish

The biological material of the invention may be fish or may be derived from fish. The fish can be, for example, of the order Acipenseriformes (e.g., sturgeons, oonfishes and sturgeons), polyfins (e.g., bichirs, birchers, lobed-finned fishes, finned pike) and reed fishes), chrysalis (e.g., green gilt and silver hanfish), chinfish (e.g., fish and needlefish), goldeneyes, milkfish order, Odontocyps (e.g., killifishes), leopard breams (e.g., leopard breams), spinyfishes (e.g., sea dragons and sticklebacks), mullets (e.g., mullets), sea moths ( For example, megamouths and sea moths), Perciformes (e.g., perch-like fish), Plaiceiformes (e.g., flounder, flounder, and tongue sole), Scorpioformes (e.g., scorpion fish and sculpins), Chineyes, Synbranchs (e.g., eels), Pufferiformes (e.g., boxfish, porcupine fish, scalefish, puffer fish, triggerfish, and boxfish), Dories (e.g. , pike-nosed fishes, dories, and breams), cocklefish superorders, herringiformes (e.g., anchovies and herrings), nymphae, northern pike, eels (e.g., eels), tarponiformes (e.g., Arpons), Acanthians (e.g., spiny eels and sticklebacks), Pharynchoichthyes, Lunar fishes (e.g., moonfishes and hairtails), Porphyrides (e.g., harefish (leporins and piranhas), Cypriniformes (e.g., minnows, mullets, zebrafish), Muratas (e.g., milkfish and shellears), Cyprinidae, Siluriformes (e.g., catfish), Aphredoderiforme (e.g., cavefish and perch), Chrysalis (e.g., cod and cod), Throatfishes, Angleriforme (e.g., anglerfish), Orders of the Weasel-blennles, Salmonidae (e.g., Salmonidae), Pleobreams (e.g., Barbreamia), Cetaceans, Ctenopterygia, Pikefishes (e.g., Shawn and Pike), Smeltfishes ( e.g., smelts and smelts), Salmoniformes (e.g., salmon), Lanternfishes (e.g., Latern Fishes), Braidfishes, Megastomas, Bowfinches (e.g., Bowfishes) finfishes), Hemisprayfishes (e.g., gars), Seasaurs (e.g., sea dragons and seahorses), Lungfishes (e.g., barramundi), Bactrophytes (e.g., American lungfishes and African Lungfish) or of the order Coelacanths (eg, Coelacanths).

f. Invertebrates

Biological material can be invertebrate or derived from an invertebrate. The invertebrate can be, for example, the phylum Polyporia (e.g., sponges), Cnidaria (e.g., jellyfish, hydra, sea anemones, man-of-war, and corals), platyhelminths (e.g., mollusca phyllodes, including planarians, trematodes, and tapeworms), nematodes (e.g. roundworms, including rotifers and nematodes), molluscs (e.g. molluscs, snails, slugs, octopuses, squids), annelids (e.g. knotted worms, including earthworms, leeches, and marine worms), Echinodermata (e.g., starfish, sea cucumbers, sand dollars, sea urchins), Sculptozoa (e.g., Scutellaria), Tardigrades (e.g., tardigrades genus), Acanthocephala (e.g., Acanthocephala), Ctenophora (e.g., Ctenophora), or Arthropods (e.g., spiders, crustaceans, myriapods, lipopods, insects).

Arthropods can be, for example, Coleoptera (e.g., beetles), Diptera (e.g., flies), Hymenoptera (e.g., ants, bees, wasps), Lepidoptera (e.g., butterflies, moths), Mecoptera (e.g., scorpionflies), Pleuroptera, Neuroptera (e.g., lacewings and related animals), Plephales (e.g., fleas), Twisteroptera (e.g., parasitic insects and twister beetles), Trichoptera Orders (e.g., caddis moths), Licides (e.g., sucking lice), Hemiptera (e.g., Hemiptera longworms and their related animals), Trichophaera (e.g., biting lice), Rodentia (e.g., gnats), Thysanoptera (e.g., pasture insects), Orthoptera (e.g., locusts, cicadas), Dermatoptera (e.g., earwigs), Dictoptera, Spinopods (e.g., silk ants), Glyceroptera, Mantidia (for example, gladiators), Pteroptera (for example, stoneflies), Anoptera (for example, zorapterans), Ephemeroptera (for example, mayflies), dragonflies Orders (e.g., dragonflies and damselflies), Phytophthora (e.g., stick insects), Thysanoptera (e.g., borers), Lycognathia, Collembola (e.g., snow flies and springtails) , Chilopoda (for example, lipopods), diplopoda (for example, myriapods), sauropods (for example, pauropodas, classes (pauropodans) and prophylloids), syntheses (for example , pseudocentipedes (pseudocentipedes and symphylans)), molluscs (e.g., crabs, krill, ball shrimps, river prawns), jawopodas, branchiopods (e.g., branchlets), cephalopods Shrimps, Ostracodas (e.g., Ostracodas), Palloropods, Branchura, Cirripedes (e.g., Barnacles), Arachnida (e.g., Arachnoides, including whip spiders, Spiders, blind spiders, blind spiders, microscorpions, book scorpions, pseudoscorpions, pseudoscorpions, scorpions, sun avoiders, sun spiders and uropygids), acrostomes (for example, Limulus ) or sea arachnids (eg, sea spiders).

g. Fungus

The biological material of the invention may be a fungus or may be derived from a fungus. The fungus may be, for example, Ascomycota (Ascomycetes), Basidiomycota (Coral Fungi), Chytridiomycota (chytrids), Deuteromycota or Zygomycota. The fungi may be Rhizopus, Pilobolus, Arthrobotrys, Aspergillus, Allomyces, Chytridium, Mushrooms ( Agaricus, Amanita, Cortinarius, Neurospora, Morchella, Saccharomyces, Pichia, Candida Candida, Schizosaccharomyces or Ergot. In particular embodiments, the fungus may be Saccharomyces cerevisiae, Schizosaccharomycespombe, Candida albicans or Pichia pastoris.

h. plants

The biological material of the invention may be a plant or may be derived from a plant. The plant can be a bryophyte (e.g., mosses, liverworts, hornworts), a lycophyte (e.g., a lycopodium, a lycopodium), an equiseta (e.g., an equiseta), a fern (e.g., a fern genus), cycads (for example, cycads), vines (for example, genus Ephedra, Ephedra, orchid), coniferous plants (for example, conifers), ginkgo plants (for example, Ginkgo biloba) or flowering plants (eg, flowering plants). Flowering plants can be monocots or dicots. Non-limiting examples of monocots include wheat, corn, rye, rice, turfgrass, sorghum, millet, sugar cane, lily, iris, agave, aloe, orchids, bromeliads, and palms. Non-limiting examples of dicots include tobacco, tomato, potato, soybean, sunflower, alfalfa, brassica, rose, Arabidopsis, coffee tree, citrus fruit, bean, alfalfa, and cotton.

i. Protists

The biological material of the invention may be a protist or may be derived from a protist. Protists can be Rhodophyta (e.g., red algae), Phaeophyta (e.g., brown algae, seaweed), Chlorophyta (e.g., green algae), Euglephyta (e.g., Euglena), Myxophyta (e.g., Myxomycetes), Oomycetes (eg Saprolegnia, Downy Mildew, Potato Blight) or Diatoms (eg Diatoms).

j. Prokaryotes

In certain aspects of the invention, the biological material is or is derived from a prokaryote. In certain embodiments, the prokaryote is an archaebacteria. The archaea can be, for example, Euryarchaeota, Euryarchaeota, Korarchaeota or Nanoarchaeota. In certain aspects, the Cranearchaeota are Halobacteria, Methanobacteria, Methanococci, Methanomicrobia, Methanosarcinae, Methanosarcinae (Methanopyri), Archeoglobi, Thermoplasmata or Thermococci. Specific non-limiting examples of archaea include: Aeropyrum pernix, Methanococcus jannaschii, Halobacterium marismortui, and Thermoplasma acidophilum.

In certain embodiments, the prokaryote is Eubacteria. Eubacteria can be, for example, Actinobacteria, Aquificae, Bacteroidetes, Green sulfur bacteria, Chlamydiae, Verrucomicrobia ), Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyobacteria ( Dictyoglomi, Fibrobacteres/Acidobacteria, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Omnivore Omnibacteria, Planctomycetes, Proteobacteria, Spirochaetes, Thermodesulfobacteria or Thermotogae. Non-limiting examples of Actinomycetes include Actinomyces, Arthrobacter, Corynebacterium, Frankia, Micrococcus, Monospora Bacteria of the genera Micromonospora, Mycobacterium, Propionibacterium and Streptomyces. Specific examples of actinomycetes include Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium avium, Corynebacterium glutamicum, Propionibacterium acnes and Rhodococcus equi.

Non-limiting examples of the Hydrogenous phylum include Aquifex, Hydrogenivirga, Hydrogenobacter, Hydrogenobaculum, Thermocrinis, Hydrogenothermus, Persephonella, Sulfurihydrogenibium, Balnearium, Except for Desulfurobacterium and Thermovibrio. Non-limiting examples of Firmicutes include bacteria of the genera Bacilli, Clostridia, and Molecutes. Specific examples of Firmicutes include: Listeria innocua, Listeria monocytogenes, Bacillus subtilis, Bacillus anthracis, Bacillus thuringiensis, Staphylococcus aureus, Clostridium acetobutylicum, Clostridium difficile, Clostridium perfringens, genitalia Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus mutans, Lactococcus lactis ) and Enterococcus faecalis.

Non-limiting examples of Chlamydia/Vrucomicrobia include bacteria such as Chlamydia trachomatis, Chlamydia pneumoniae, Chlamydia psittaci. Non-limiting examples of the Deinococcus-Thermus phylum include Deinococcus and Thermus.

Proteobacteria are Gram-negative bacteria. Non-limiting examples of Proteobacteria include Escherichia, Salmonella, Vibrio, Rickettsia, Agrobacterium, Brucella Brucella, Rhizobium, Neisseria, Bordetella, Burkholderia, Buchnera, Yersinia, Klebsiella, Proteus, Shigella, Haemophilus, Pasteurella, Actinobacillus , Legionella, Mannheimia, Coxiella, Aeromonas, Francisella, Moraxella, Pseudomonas ( Pseudomonas), Campylobacter and Helicobacter. Specific examples of the Proteobacteria phylum include: Rickettsia conorii, Rickettsia prowazekii, Rickettsia typhi endemic, Rickettsia typhi Ehrlichiabovis, Agrobacterium tumefaciens, Brucella melitensis, Rhizobium rhizogenes, Neisseria meningitides, Bordetella parapertussis (rod) Bordetella parapertussis, Bordetella pertussis, Burkholderi mallei, Burkholderi pseudomallei, Neisseria gonorrhoeae, Escherichia coli Escherichia coli, Salmonella enterica, Salmonella typhimurium, Yersinia pestis, Klebsiella pneumoniae, Yersinia enterocolitica Yersinia enterocolitica, Proteus vulgaris, Shgella flexneri, Shigella sonnei, Shigella dysenterica, Influenza (Haemophilus) (Haemophilusinfluenzae), Pasteurella multocida, Actinobacillus actinomycetemcomitans, Actinobacillus pleuropneumoniae, Haemophilus somnus, pneumophilia Legionellapneumophila, Mannheimia haemolytica, Vibrio cholerae, Vibrio parahaemolyticus, Coxiella burnetii, hydrophile Aeromonas hydrophila, Aeromonas salmonicida, Francisella tularesis, Moraxella catarrhalis, Pseudomonas aeruginosa, Pseudomonas putida, Campylobacter jejuni, and Helicobacter pylori.

Non-limiting examples of the spirochete phylum include bacteria of the families Brachyspiraceae, Leptospiraceae, and Spirochaetaceae. Specific examples of the spirochete phylum include Borrelia burgdorferi and Treponema pallidum.

2. Different types of biological matter

The method and apparatus of the present invention can be applied to living things. Stasis of an organism may be induced or stasis within cells, tissues and/or organs of an organism may be induced. The biological matter in which stasis can be induced is contemplated for use in the methods and apparatus of the present invention only to the extent that it comprises cells that utilize oxygen for energy production.

Can be found in organs including the heart, lungs, kidneys, liver, bone marrow, pancreas, skin, bone, veins, arteries, cornea, blood, small intestine, large intestine, brain, spinal cord, smooth muscle, skeletal muscle, ovaries, testes, uterus, and umbilical cord Induction of arrest in cells, tissues or organs.

Furthermore, arrest can be induced in the following cell types: platelets, myeloid cells, erythrocytes, lymphocytes, adipocytes, fibroblasts, epithelial cells, endothelial cells, smooth muscle cells, skeletal muscle cells, endocrine cells, glial cells, Nerve cells, secretory cells, barrier function cells, contractile cells, absorptive cells, mucosal cells, limbal cells (from the cornea), stem cells (totipotent, pluripotent, or pluripotent), unfertilized or fertilized oocytes or sperm cells.

Furthermore, stagnation can be induced in plants or plant parts, including fruits, flowers, leaves, stems, seeds, cuttings. Plants may be agricultural, medicinal or ornamental. Induction of arrest in plants can enhance shelf life or pathogen resistance of whole plants or plant parts.

The methods and apparatus of the invention can be used to induce stagnation of biological material in vivo. This can be used to protect and/or preserve biological substances or the organism itself or to prevent damage or injury to them or to the whole organism.

3. Assay

Arrest can be measured in a number of ways, including by quantifying the amount of oxygen consumed by a biological sample, the amount of carbon dioxide produced by the sample (an indirect measure of cellular respiration), or by characterizing motility.

To determine the rate of oxygen consumption or carbon dioxide production, the biomass was placed in a sealed chamber with two openings for gas input and output. Gas (room air or other gas) is passed into the chamber at a given flow rate and out the outlet to maintain approximately 1 atmosphere in the chamber. The amount of each compound in the gas mixture is measured (per second) by passing the gas through a carbon dioxide detector and or an oxygen detector before and after exposure to the chamber. Comparing these values over time yields the rate of oxygen consumption or carbon dioxide production.

II. Oxygen antagonists and other active compounds

The present invention relates to methods, compositions and articles of manufacture involving one or more agents that act on biological substances to produce a number of effects including, but not limited to, Induces stasis, enhances or increases viability, reversibly inhibits metabolism, induces metabolism and activity of cells or organisms, reduces oxygen demand, reduces or prevents injury, prevents ischemic type injury, prevents aging or senescence, and/or obtains a variety of Therapeutic applications of this discussion. In certain embodiments, the agent qualifies as an "active compound."

In some embodiments, the agent is an oxygen antagonist, which can act directly or indirectly. Oxygen metabolism is a fundamental requirement of aerobic metazoan life Aerobic respiration is responsible for the vast majority of energy production in most animals and is also used to maintain the redox potential necessary for important cellular reactions. At low oxygen, reduced oxygen availability leads to ineffective transfer of electrons to molecular oxygen in the final step of the electron transport chain. This ineffectiveness results in both a decrease in aerobic energy production and an increase in the production of damaging free radicals, mainly due to the premature release of electrons at complex III and the formation of O ₂ ^- by cytochrome oxidase (Semenza, 1999). Limited energy supply and free radical damage can interfere with fundamental cellular processes, such as protein synthesis and maintenance of membrane polarity (Hochachka et al., 1996), and will eventually lead to cell death.

In other embodiments, the agent is a protective metabolic agent. Metabolism is generally understood to refer to the chemical processes (in cells or organisms) required for life; they involve a variety of reactions to sustain energy production and to synthesize (anabolism) and break down (catabolism) complex molecules.

In certain embodiments of the invention, the active compound has a chemical structure as set forth in Formula I or IV as described herein, or is a precursor of Formula I or IV.

Various chemical structures and compounds are described herein. The following definitions apply to the terms used to describe the structures and compounds discussed herein:

"Alkyl", when used alone or within other terms such as "arylalkyl", "aminoalkyl", "thioalkyl", "cyanoalkyl" and "hydroxyalkyl", refers to Straight-chain or branched groups having from one to about 20 carbon atoms. The term "lower alkyl" refers to a C ₁ -C ₆ alkyl group. As used herein, the term alkyl includes hydroxy, halo (eg, F, Cl, Br, I), haloalkyl, alkoxy, haloalkoxy, alkylthio, cyano, isocyano, carboxy (- COOH), alkoxycarbonyl (-COOR), acyl, acyloxy, amino, alkylamino, urea (--NHCONHR), mercapto, alkylthio, sulfoxy, sulfonyl, arylsulfonyl , alkylsulfonyl, sulfonylamino, arylsulfonylamino, heteroaryl, heterocyclyl, heterocycloalkyl, amidyl, alkyliminocarbonyl, amidino, guanidono, Those groups substituted by hydrazine, hydrazide, sodium sulfonyl (—SO ₃ Na), sodium sulfonylalkyl (—RSO ₃ Na) and the like. Examples of such groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, etc. .

"Hydroxyalkyl" means an alkyl group, as defined herein, substituted with one or more hydroxy groups. Examples of hydroxyalkyl groups include, but are not limited to, hydroxymethyl, 2-hydroxyethyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxybutyl, 3-hydroxybutyl, 4-hydroxybutyl , 2,3-dihydroxypropyl, 1-(hydroxymethyl)-2-hydroxyethyl, 2,3-dihydroxybutyl, 3,4-dihydroxybutyl and 2-(hydroxymethyl)- 3-hydroxypropyl etc.

"Arylalkyl" refers to the group R'R- wherein an alkyl group "R" is replaced with an aryl group "R'". Examples of arylalkyl groups include, but are not limited to, benzyl, phenethyl, 3-phenylpropyl, and the like.

"Aminoalkyl" refers to the group _H2NR'- in which an alkyl group is substituted with an amino group. Examples of such groups include aminomethyl, aminoethyl, and the like. "Alkylaminoalkyl" refers to an alkyl group substituted with an alkylamino group.

"Alkylsulfonylamino" refers to a sulfonylamino group (-S(O) ₂ -NRR') appended to an alkyl group as defined herein.

"Sulfuryl" refers to an alkyl group in which one or more mercapto groups are substituted. "Alkylsulfanyl" means an alkyl group in which one or more alkylthio groups are substituted. Examples include, but are not limited to, methylthiomethyl, ethylthioisopropyl, and the like. "Arylsulfanyl" means wherein an alkyl group as defined herein is substituted with one or more arylthio groups.

"Carboxyalkyl" refers to the group _-RCO2H in which an alkyl group is substituted with a carboxyl group. Examples include, but are not limited to, carboxymethyl, carboxyethyl, carboxypropyl, and the like.

"Alkylene" means a bridged alkyl group.

The term "alkenyl" refers to an unsaturated, acyclic hydrocarbon group in that it contains at least one double bond. Such alkenyl groups contain from about 2 to about 20 carbon atoms. The term "lower alkenyl" refers to a C ₁ -C ₆ alkenyl group. As used herein, the term alkenyl group includes those groups substituted with alkyl groups. Examples of suitable alkenyl groups include propenyl, 2-chloropropenyl, buten-1-yl, isobutenyl, pent-1-en-1-yl, 2-2-methyl-1-butene- 1-yl, 3-methyl-1-buten-1-yl, hex-2-en-1-yl, 3-hydroxyhex-1-en-1-yl, hept-1-en-1-yl and oct-1-en-1-yl, etc.

The term "alkynyl" refers to an unsaturated, acyclic hydrocarbon group in that it contains one or more triple bonds, such groups containing from about 2 to about 20 carbon atoms. The term "lower alkynyl" refers to a C ₁ -C ₆ alkynyl group. As used herein, the term alkynyl includes those groups substituted with alkyl groups. Examples of suitable alkynyl groups include ethynyl, propynyl, hydroxypropynyl, but-1-yn-1-yl, but-1-yn-2-yl, pent-1-yn-1-yl , Pent-1-yn-2-yl, 4-methoxypent-1-yn-2-yl, 3-methylbut-1-yn-1-yl, hex-1-yn-1-yl, Hex-1-yn-2-yl, hex-1-yn-3-yl, 3,3-dimethyl-1-butyn-1-yl, etc.

"Alkoxy" refers to the group R'O-, wherein R' is an alkyl group as defined herein. Examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, isopropoxy, t-butoxy, and the like. "Alkoxyalkyl" refers to an alkyl group substituted with one or more alkoxy groups. Examples include, but are not limited to, methoxymethyl, ethoxyethyl, methoxyethyl, isopropoxyethyl, and the like.

"Alkoxycarbonyl" refers to the group R-O-C(O)-, wherein R is an alkyl group as defined herein. Examples of alkoxycarbonyl groups include, but are not limited to, methoxycarbonyl, ethoxycarbonyl, sec-butoxycarbonyl, isopropoxycarbonyl, and the like. Alkoxythiocarbonyl refers to R-O-C(S)-.

"Aryl" means a monovalent aromatic carbocyclic group consisting of a single ring or of one or more fused rings, at least one of which is aromatic in nature, which may optionally be surrounded by one or more Multiple, preferably one or two of the following substituents, unless otherwise indicated, for example: hydroxy, halogen (eg F, Cl, Br, I), haloalkyl, alkoxy, haloalkoxy, alkylthio, cyano , carboxyl (-COOH), alkoxycarbonyl (-COOR), acyl, acyloxy, amino, alkylamino, urea (--NHCONHR), mercapto, alkylthio, sulfoxyl, sulfonyl, arylsulfonyl Acyl, alkylsulfonyl, sulfonylamino, arylsulfonylamino, heteroaryl, heterocyclyl, heterocycloalkyl, amido, alkylimino, amidino, guanidino, hydrazino, hydrazide, Sodium sulfonyl (-SO ₃ Na), Sodium sulfonyl alkyl (-RSO ₃ Na). Alternatively, two adjacent atoms of the aromatic ring may be substituted with methylenedioxy or ethylenedioxy. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, indanyl, diphenylbutanthracenyl, tert-butylphenyl, 1,3-benzodioxa Cyclopentenyl, etc.

"Arylsulfonylamino" means a sulfonylamino group, as defined herein, appended to an aryl group, as defined herein.

"Thiaryl" refers to an aryl group substituted with one or more mercapto groups.

"Alkylamino" refers to an amino group substituted with one or two alkyl groups. Examples include monosubstituted N-alkylamino groups and N,N-dialkylamino groups. Examples include N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino, N-methyl, N-ethyl-amino, and the like.

"Aminocarbonyl" refers to the group _H2NCO- . "Aminocarbonylalkyl" means an alkyl group as defined herein substituted with one or more aminocarbonyl groups.

"Acylamino" means RCO-NH-, where R is H or alkyl, aryl or heteroaryl as defined herein.

"Imidocarbonyl" refers to a carbon group that shares two of the four covalent bond sites with an imino group. Examples of such iminocarbonyl groups include, for example, C=NH, C= _NCH3 , C=NOH and C= _NOCH3 . The term "alkyliminocarbonyl" refers to an imino group substituted with an alkyl group. The term "amidino" refers to a substituted or unsubstituted amino group bonded to one of the two available bonds of an iminocarbonyl group. Examples of such amidino groups include, for example, _NH2 -C=NH, _NH2 -C= _NCH3 , NH-C= _NOCH3 and NH( _CH3 )-C=NOH. The term "guanidino" refers to an amidino group as defined above bonded to an amino group which may be bonded to a third group. Examples of such guanidino groups include, for example, NH ₂ -C(NH)-NH-, NH ₂ -C(NCH ₃ )-NH-, NH ₂ -C(NOCH ₃ )-NH- and CH ₃ NH- C(NOH)-NH-. The term "hydrazino" refers to -NH-NRR', where R and R' are independently hydrogen, alkyl, and the like. "Hydrazide" refers to -C(=O)-NH-NRR'.

The term "heterocyclyl" refers to saturated and partially saturated heteroatom-containing ring groups having 4 to 15 ring members, referred to herein as " _C4 - _{C15heterocyclyl} ", the ring members being selected from carbon , nitrogen, sulfur and oxygen, wherein at least one ring atom is a heteroatom. A heterocyclyl group may contain one, two or three rings, wherein such rings may be attached in a pendant fashion, or may be fused. Examples of saturated heterocyclic groups include saturated 3- to 6-membered heteromonocyclic groups [such as pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl, etc.] containing 1 to 4 nitrogen atoms; A saturated 3- to 6-membered heteromonocyclic group [e.g. morpholinyl] containing 2 oxygen atoms and 1 to 3 nitrogen atoms; a saturated 3- to 6-membered heterocyclic group containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms membered heteromonocyclic groups [eg thiazolidinyl, etc.]; examples of partially saturated heterocyclic groups include dihydrothiophene, dihydropyran, dihydrofuran and dihydrothiazole. Non-limiting examples of heterocyclic groups include 2-pyrrolinyl, 3-pyrrolinyl, pyrrolindinyl, 1,3-dioxolanyl, 2H-pyranyl, 4H-pyranyl , piperidinyl, 1,4-di-alkyl, morpholinyl, 1,4-dithianyl, thiomorpholinyl, etc. Such heterocyclic groups may optionally be substituted with groups such as, for example, hydroxyl, halogen (eg F, Cl, Br, I), haloalkyl, alkoxy, haloalkoxy, alkylthio, cyano, carboxy (-COOH), alkoxycarbonyl (-COOR), acyl, acyloxy, amino, alkylamino, urea (--NHCONHR), mercapto, alkylthio, thiooxy, sulfonyl, arylsulfonyl, Alkylsulfonyl, sulfonylamino, arylsulfonylamino, heteroaryl, heterocyclyl, heterocycloalkyl, amido, alkyliminocarbonyl, amidino, guanidino, hydrazino, hydrazide, sulfonyl Acyl sodium (-SO ₃ Na), sulfonyl alkyl sodium (-RSO ₃ Na) substitution.

"Heteroaryl" means a monovalent aromatic ring group having one or more rings, preferably one to three rings, each ring having 4 to 8 atoms, incorporating one or more heteroatoms, preferably one or two (selected from nitrogen, oxygen or sulfur), said heteroaryl may be optionally substituted with one or more, preferably one or two, substituents selected from the following substituents, unless otherwise indicated, for example: hydroxyl, halogen ( For example F, Cl, Br, I), haloalkyl, alkoxy, haloalkoxy, alkylthio, cyano, carboxyl (-COOH), alkoxycarbonyl (-COOR), acyl, acyloxy, amino, Alkylamino, urea (--NHCONHR), mercapto, alkylthio, thiooxy, sulfonyl, arylsulfonyl, alkylsulfonyl, sulfonylamino, arylsulfonylamino, heteroaryl, heterocycle group, heterocycloalkyl, amido, alkyliminocarbonyl, amidino, guanidino, hydrazino, hydrazide, sodium sulfonyl (-SO ₃ Na), sodium sulfonylalkyl (-RSO ₃ Na). Examples of heteroaryl groups include, but are not limited to, imidazolyl, oxazolyl, thiazolyl, pyrazinyl, thienyl, furyl, pyridyl, quinolinyl, isoquinolyl, benzofuryl, benzothienyl , Benzothiopyryl, Benzimidazolyl, Benzopropazolyl, Benzothiazolyl, Benzopyranyl, Indazolyl, Indolyl, Isoindolyl, Quinolinyl, Isoquinolyl , Naphthyridine, benzenesulfonyl-thienyl, etc.

"Heteroaryloxy" refers to a heteroaryl group attached to an oxy group. Examples of such groups include, but are not limited to, 2-thiophenoxy, 2-pyrimidinyloxy, 2-pyridyloxy, 3-pyridyloxy, 4-pyridyloxy, and the like.

"Heteroaryloxyalkyl" refers to an alkyl group substituted with one or more heteroaryloxy groups. Examples of such groups include 2-pyridyloxymethyl, 3-pyridyloxyethyl, 4-pyridyloxymethyl and the like.

"Cycloalkyl" means a monovalent saturated carbocyclic group consisting of one or more rings, typically one or two rings, each ring having from 3 to 8 carbons, said cycloalkyl group usually being represented by one or a plurality of the following substituents, unless otherwise indicated: hydroxy, halogen (e.g. F, Cl, Br, I), haloalkyl, alkoxy, haloalkoxy, alkylthio, cyano, carboxy (-COOH), Alkoxycarbonyl (-COOR), acyl, acyloxy, amino, alkylamino, urea (--NHCONHR), mercapto, alkylthio, sulfoxy, sulfonyl, arylsulfonyl, alkylsulfonyl, Sulfonylamino, arylsulfonylamino, heteroaryl, heterocyclyl, heterocycloalkyl, amido, alkyliminocarbonyl, amidino, guanidino, hydrazino, hydrazide, sodium sulfonyl (-SO ₃ Na), sodium sulfonylalkyl (-RSO ₃ Na). Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, 3-ethylcyclobutyl, cyclopentyl, cycloheptyl, and the like. "Cycloalkenyl" refers to a group having 3 to 10 carbon atoms and one or more carbon-carbon double bonds. Typical cycloalkenyl groups have 3 to 7 carbon atoms. Examples include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, and the like. "Cycloalkenylalkyl" means a group wherein an alkyl group as defined herein is substituted with one or more cycloalkenyl groups.

"Cycloalkoxy" refers to a cycloalkyl group attached to an oxy group. Examples include, but are not limited to, cyclohexyloxy, cyclopentyloxy, and the like.

"Cycloalkoxyalkyl" refers to an alkyl group substituted with one or more cycloalkoxy groups. Examples include cyclohexyloxyethyl, cyclopentyloxymethyl, and the like.

"Sulfinyl" means -S(O)-.

"Sulfonyl" refers to -S(O) _2- , wherein "alkylsulfonyl" refers to a sulfonyl RSO2- substituted with an alkyl group, and arylsulfonyl refers to an aryl group attached to a sulfonyl group _. "Sulfonylamino" refers to -S(O) ₂ -NRR'. "Sulphonic acid" refers to -S(O) _2OH . "Sulfonate" refers to -S(O) _2OR , where R is alkyl in a group such as alkylsulfonate.

"Thio" means -S-. "Alkylthio" refers to RS- wherein a mercapto group is substituted with an alkyl R group. Examples include methylthio, ethylthio, butylthio, and the like. "Arylthio" means R'S- wherein the thio group is substituted with an aryl group as defined herein. Examples include, but are not limited to, phenylthio and the like. Examples include, but are not limited to, thiophenylmethyl, and the like. "Alkylthiosulfonic acid" refers to the group _HO3SR'S- wherein the alkylthio group is substituted with a sulfonic acid group.

"Thiosulfenyl" means -S-SH.

"Acyl", alone or in combination, means a carbonyl or thiocarbonyl group bonded to a group selected from, for example, hydrido, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, alk Oxyalkyl, Haloalkoxy, Aryl, Heterocyclyl, Heteroaryl, Alkylsulfinylalkyl, Alkylsulfonylalkyl, Aralkyl, Cycloalkyl, Cycloalkylalkyl, Cycloalkyl Alkenyl, alkylthio, arylthio, amino, alkylamino, dialkylamino, aralkoxy, arylthio and alkylthioalkyl. Examples of "acyl" are formyl, acetyl, benzoyl, trifluoroacetyl, phthaloyl, malonyl, nicotinoyl, and the like.

The terms "acylthio" and "acyldithio" refer to RCOS- and RCOSS-groups, respectively.

The term "thiocarbonyl" refers to compounds and moieties containing a carbon double bonded to a sulfur atom, -C(=S)-. "Alkylthiocarbonyl" means a thiocarbonyl group in which a thiocarbonyl group is substituted with an alkyl group R as defined herein to form a monovalent group RC(=S)-. "Aminothiocarbonyl" refers to thiocarbonyl substituted with amino, _NH2C (=S)-.

"Carbonyloxy" refers to -OCOR.

"Alkoxycarbonyl" refers to -COOR.

"Carboxy" refers to -COOH.

For those compounds having stereoisomers, all stereoisomers thereof, including cis/trans geometric isomers, diastereomers and individual enantiomers, are contemplated.

A) carbon monoxide

Carbon monoxide (CO) is a colorless, odorless and tasteless gas that can be toxic to animals, including humans. According to the Centers for Disease Control, more than 450 people die unexpectedly from carbon monoxide every year.

It is toxic to organisms whose blood carries oxygen to sustain them. It can become toxic by entering the lungs through normal breathing and displacing oxygen in the bloodstream. Interruption of normal oxygen supply compromises the function of the heart, brain, and other vital functions of the body. However, carbon monoxide is being investigated for medical applications (Ryter et al., 2004).

At levels of 50 parts per million (ppm), carbon monoxide exhibits no symptoms to those exposed to it. However, at 200 ppm, carbon monoxide can cause a mild headache within 2 to 3 hours; at 400 ppm, within 1 to 2 hours, it can cause frontal pain, which can become widespread within 3 hours, respectively; and At 800 ppm, it can cause dizziness, nausea, and/or convulsions within 45 minutes and render subjects numb within 2 hours. At levels of about 1000 ppm, organisms can die after exposure for more than about 1-2 minutes.

Due to the well-known and well-documented toxic effects of carbon monoxide on organisms, it was surprising and unexpected that carbon monoxide could be used to induce stasis and/or aid in the preservation of living biological samples. It is thus contemplated that carbon monoxide could be used to induce stagnation in isolated biological matter, such as blood-free biomatter (which is a different pathway than that involved in inducing stasis due to the effect carbon monoxide has on hemoglobin).

In addition to exposure to carbon monoxide to induce stasis or to limit or prevent any damage caused by stasis-inducing agents, the present invention contemplates the use of carbon monoxide in combination with agents or methods that facilitate the preservation and/or transplantation/grafting process of biological material.

B. Chalcogenide compounds

Compounds (but not oxides) containing chalcogens (those in Group 6 of the Periodic Table) are commonly referred to as "chalcogenides" or "chalcogenide compounds" (used interchangeably herein). These elements are sulfur (S), selenium (Se), tellurium (Te) and polonium (Po). Ordinary chalcogenides contain one or more of S, Se and Te in addition to other elements. Chalcogenides include micronized and/or nanonized particles of elemental forms such as S and Se. Chalcogenide compounds can be employed as reducing agents.

The inventors, while not being bound by the following theory, believe that the ability of chalcogenides to induce stasis in cells, and allow regulation of core body temperature in animals, stems from the binding of these molecules to cytochrome oxidase. Thus, chalcogenides inhibit or reduce the activity of oxidative phosphorylation. Chalcogenides block spontaneous thermoregulation, the ability to allow the core body temperature of "warm-blooded" animals to be manipulated by controlling the temperature of the environment, is thought to arise from the same mechanism as listed above - binding to cytochrome oxidase, And block or reduce the activity of oxidative phosphorylation. Chalcogenides are available in liquid as well as gaseous form.

Chalcogenides can be toxic to mammals and at some levels lethal. According to the present invention, it is expected that levels of chalcogenides should not exceed lethal levels in suitable environments. Lethal levels of chalcogenides can be found, for example, in the Material Safety Data Sheets for each chalcogenide, or from information sheets available from the US government's Occupational Safety and Health Administration (OSHA).

Although both carbon monoxide and chalcogenide compounds can induce arrest by acting as oxygen antagonists, they have different toxic effects that are dissociated from their ability to induce arrest. Also, because of the different affinities of cytochrome oxidases, the concentration required to mediate the arresting effect is different. Cytochrome oxidase has an affinity for oxygen compared to carbon monoxide of about 1:1, while its affinity for _H2S to oxygen is on the order of about 300:1. This affects what toxic effects would be observed using the arrest-inducing concentration. Therefore, chalcogenide compounds are considered to be particularly suitable for inducing stagnation of biomass in whole organisms and for inducing stasis of whole organisms.

It may also prove useful to provide an additional stimulus to the biological material prior to removal of the chalcogenides. In particular, it is envisioned that animals may be subjected to increased ambient temperatures prior to removal of the chalcogenide source.

1.H ₂ S and other sulfur-containing compounds

Hydrogen sulfide ( _H2S ) is a potentially toxic gas commonly associated with petrochemical and natural gas, sewage, pulp, leather tanning and food processing. The main effect at the cellular level is the inhibition of cytochrome oxidase or other oxidases, leading to cellular hypoxia. Exposure to extreme levels (500ppm) resulted in outbursts and unconsciousness (the so-called "knockout" effect), followed by recovery. Post-exposure effects may last for years and include coordination inability, memory loss, motor dysfunction, personality changes, hallucinations, and insomnia.

However, most _H2S exposures occur well below this level of acute toxicity. However, long-term exposure at the subacute level is generally of concern. There are several reports that persistent impairment of balance and memory, as well as altered sensorimotor function, may occur in humans following chronic low-level _H2S exposure. Kilburn and Warshaw (1995); Kilburn (1999). Others have reported that daily exposure of rats to low (20 or 50 ppm) _H2S for 7 hours during the perinatal period from gestation to 21 days postpartum resulted in reduced aborization of Purkinje cells in the brain. ) of longer tree-like branches. Other neurological deficits associated with relatively low levels of _H2S include altered brain neurotransmitter concentrations and altered neural responses, such as increased hippocampal и EEG activity.

Behavioral toxicity was studied in rats exposed to moderate levels of _H2S . It was shown that _H2S inhibited the discrimination-avoidance response immediately after the exposure ended (Higuchi and Fukamachi, 1997) and also interfered with the ability of rats to learn a baited radial arm maze task (Partlo et al. , 2001). In another perinatal study using 80 ppm _H2S , no neuropathological effects or altered locomotor activity, passive avoidance or acoustic startle responses were found in exposed rat pups. Dorman et al. (2000). Finally, Struve et al., (2001 ) exposed rats to _H2S for 3 hours daily via various levels of gas for 5 consecutive days. Significant reductions in locomotor activity, water maze performance and body temperature were observed after exposure to 80 ppm or higher of _H2S . Taken together, these reports suggest that _H2S can have various effects on the biochemistry of mammalian tissues, but no clear response pattern with respect to behavior.

Once dissolved in plasma, _H2S will participate in a series of chemical reactions. The chemical reactions described are: (1) dissociation of molecular H ₂ S to form bisulfide ions, (2) dissociation of sulfide ions to form sulfide ions, and (3) self-ionization of water. The response is given below:

Using the equilibrium constants K ₁ = 1.039E ^-07 , K ₂ = 6.43E ^-16 and K _w = 1.019E ^-14 at pH 7.4, the calculated amount of the different species relative to the total S concentration is approximately 23% H ₂ S and 77% of HS ^- , while the amount of S ^2- tends to 0.

The present inventors utilized an extractive alkylation technique coupled with gas chromatography and mass-specific detection to quantify hydrogen sulfide (adapted from Hyspler et al., 2002). This method involves first adding 50 μL of a blood, serum or tissue extract sample in saturated borate buffer, which has been diluted in Nitrogen-purified deoxygenated water to a concentration of 1 mg/mL. First, 100 μL of 15 μM 4-chloro-benzylmethyl sulfide (4CBMS) solution in ethyl acetate was added, followed by 100 μL of 20 mM pentafluorobenzyl bromide (PFBBr) solution in toluene. This solution was then sealed and incubated for 2 hours at 55°C with rotation or shaking. After this incubation period, 200 μL of saturated KH ₂ PO ₄ solution was then added and the organic phase was removed and analyzed by gas chromatography with mass-specific detection according to the method described in Hyspler et al., 2002. These measurements were then compared to a standard curve generated using the same method as described above, starting with known standard concentrations ( _Na2S in the range 1 μM-1 mM prepared in nitrogen-purified deoxygenated water) in order to determine Endogenous hydrogen sulfide levels. For analysis of bound and/or oxidized sulfide levels, the same method was applied, except that a denaturing/reducing reaction buffer consisting of 5 mM BZK with 1% tetraethylammonium hydroxide (TEAH) was used. and 1 mM tris(2-carboxyethyl)-phosphine hydrochloride (TCEP) in saturated borate buffer instead of the reaction buffer described above.

Typical hydrogen sulfide levels contemplated for use in accordance with the present invention include oral, intravenous or transdermal doses of about 1 to about 150 ppm, about 10 to about 140 ppm, about 20 to about 130 ppm, and about 40 to about 120 ppm or equivalents thereof. Other relevant ranges include about 10 to about 80 ppm, about 20 to about 80 ppm, about 10 to about 70 ppm, about 20 to about 70 ppm, about 20 to about 60 ppm, and about 30 to about 60 ppm or their equivalent oral, intravenous or transdermal doses. It is also considered that, for a given animal during a given period of time, the chalcogenide atmosphere should be reduced to avoid potentially lethal accumulation of chalcogenides in the subject. For example, an initial ambient concentration of 80 ppm can be reduced to 60 ppm after 30 minutes, followed by further decreases at 1 hour (40 ppm) and 2 hours (20 ppm).

aH ₂ S precursor

The present invention also relates to the use of compounds and agents that can generate _H2S under certain conditions, such as upon or shortly after exposure to biological matter. Consider this precursor generating _H2S when one or more enzymatic or chemical reactions take place.

3. Other chalcogenides

In certain embodiments, the reducing agent structure compound is dimethyl sulfoxide (DMSO), dimethyl sulfide (DMS), methyl mercaptan ( _CH3SH ), mercaptoethanol, thiocyanic acid, hydrogen cyanide, methyl sulfide Alcohol (MeSH) or _CS2 . In particular embodiments, the oxygen antagonist is _CS2 , MeSH or DMS. Compounds on the order of the size of these molecules are specifically contemplated (ie, within about 50% of their molecular weight).

Other compounds envisaged to be useful for inducing arrest include, but are not limited to, the following structures, many of which are readily available and known to those skilled in the art (identified by CAS number): 104376-79-6 (ceftriaxone sodium salt ); 105879-42-3; 1094-08-2 (diethylpromethazine hydrochloride); 1098-60-8 (triflupromazine hydrochloride); 111974-72-2; 113-59-7; 113-98 -4 (penicillin G K ⁺ ); 115-55-9; 1179-69-7; 118292-40-3; 119478-56-7; 120138-50-3; 1229-35-2; 1240-15-9; 1257-78-9 (chlorpromazine ethanedisulfonate); 128345-62-0; 130-61-0 (thioridazine hydrochloride) 132- 98-9 (penicillin V K ⁺ ); 13412-64-1 (dicloxacillin Na ⁺ hydrate); 134678-17-4; 144604-00-2; 146-54-3; 151767-02-1; 159989-65-8; 16960-16-0 (corticotropin fragment 1-24); 1982-37-2; 21462-39-5 (clolindromycin hydrochloride ); 22189-31-7; 22202-75-1; 23288-49-5 (probucol); 23325-78-2; 24356-60-3 (cefepime); 25507-04-4; 26605-69-6; 27164-46-1 (cefazolin Na ⁺ ); 2746-81-8; 29560-58-8; 2975-34-0; 32672-69 -8 (mesoridazine besylate); 32887-01-7; 33286-22-5 (( ⁺ )-cis-diltiazem hydrochloride); 33564-30-6 (cefoxitin sodium); 346-18-9; 3485-14-1; 3511-16-8; 37091-65-9 (benzamicillin sodium); 37661-08-8; 3819-00-9; 41372-02-5; 42540-40-9 (sodium cefamandole formate); 4330-99-8 (isomethazine hemi-( ⁺ )-tartrate); 440-17-5 (two Trifluoperazine hydrochloride); 4697-14-7 (disodium ticarcillin); 4800-94-6 (disodium carbenzylpenicillin); 50-52-2; 50-53-3; 5002-47 -1; 51481-61-9 (cimetidine); 52239-63-1 (6-propyl-2-thiouracil); 53-60-1 (promazine hydrochloride); 5321-32-4; 54965-21-8 (albendazole); 5591-45-7 (thiothixene); 56238-63-2 (cefoxime sodium); 56796-39-5 (cefemetazole sodium); 5714-00 -1; 58-33-3 (promethazine hydrochloride); 58-38-8; 58-39-9 (piperizine); 58-71-9 (cefalotin sodium); 59703-84-3 (piperacillin sodium); 60-99-1 (levomepromazine maleate); 60925-61-3; 61270-78-8; 6130-64-9 (penicillin G procaine salt hydrate ); 61318-91-0 (clobendazole nitrate); 61336-70-7 (amoxicillin trihydrate); 62893-20-3 (cefoperazone sodium); 64485-93-4 (cefotaxime sodium); 64544-07-6; 64872-77-1; 64953-12-4 (Lata cephalosporin sodium); 66104-23-2 (pergolide mesylate); 66309-69-1; 66357-59 -3 (ranitidine hydrochloride); 66592-87-8 (cefodroxil); 68401-82-1; 69-09-0 (chlorpromazine hydrochloride); 69-52-3 (ampicillin sodium); 53-4 (ampicillin); 69-57-8 (penicillin G sodium); 70059-30-2; 70356-03-5; 7081-40-5; 7081-44-9 (cloxacillin sodium H ₂ O ); 7177-50-6 (nafcillin sodium H ₂ O); 7179-49-9; 7240-38-2 (oxacillin sodium H ₂ O); 7246-14-2; 74356-00-6; 74431-23-5; 74849-93-7; 75738-58-8; 76824-35-6 (famotidine); 76963-41-2; 79350-37-1; 81129-83-1; 84- 02-6 (prochlorperazine hydrogen maleate); 87-08-1 (phenoxymethyl penicillic acid); 87239-81-4; 91-33-8 (benzyl thiazine); 91832-40 -5; 94841-17-5; 99294-94-7; 154-42-7 (6-thioguanine); 36735-22-5; 536-33-4 (ethionamide); 52-67 -5 (D-penicillamine); 304-55-2 (meso-2,3-dimercaptosuccinic acid); 59-52-9 (2,3-dimercapto ⁺ propanol) 6112-76 -1 (6-mercaptopurine); 616-91-1 (N-acetyl-L-cysteine); 62571-86-2 (captopril); 52-01-7 (spironolactone) and 80474 - 14-2 (fluticasone propionate). Other compounds considered as potentially useful for arrest include those having the chemical structure of Formula I or IV.

C. Other antagonists or active compounds and relevant environmental conditions

1. Hypoxia and Hypoxia

Hypoxia is a common natural stress and there are several well-conserved responses that promote cellular adaptation to a hypoxic environment. To compensate for the reduced capacity for aerobic energy production in hypoxia, cells must either increase anaerobic energy production or decrease energy demand (Hochachka et al., 1996). Examples of these two reactions are common in metazoans and the particular reaction utilized often depends on the amount of oxygen available to the cell.

In mild hypoxia, oxidative phosphorylation remains partially active, so some aerobic energy production is possible. The cellular response to this condition, mediated in part by the hypoxia-inducible transcription factor HIF-1, is to compensate for reduced aerobic demand by upregulating genes involved in anaerobic energy production, such as glycolytic enzymes and glucose transporters Energy production (Semenza, 2001; Guillemin et al., 1997). This response also promotes the upregulation of antioxidants such as catylase and superoxide dismutase that protect against free radical-induced damage. Thus, cells can maintain near normoxic levels of activity during mild hypoxia.

In extreme forms of hypoxia (termed "anaerobic" - defined herein as <0.001 kPa _O2 ) - oxidative phosphorylation ceases and thus the ability to generate energy is significantly reduced. To survive in this environment, cells must reduce their energy requirements by reducing cellular activity (Hochachka et al., 2001). For example, in oxygen-deprived sea turtle hepatocytes, cell-restricted activities such as protein synthesis, ion channel activity, and directed responses of anabolic pathways resulted in a 94% reduction in ATP demand (Hochachka et al., 1996). In zebrafish (Danio rerio) embryos, exposure to anaerobic results in a complete arrest of heartbeat, locomotion, cell cycle progression and developmental progression (Padilla et al., 2001). Similarly, C. elegans responds to anaerobia by entering stasis, where all observable movement, including cell division and developmental progression, ceases (Padilla et al., 2002; Van Voorhies et al., 2000). C. elegans can remain stagnant for 24 hours or more and will return to high viability once it returns to normoxia. This response allows C. elegans to survive hypoxic stress by reducing the rate of energy-intensive processes and preventing damage, irreversible events such as aneuploidy (Padilla et al., 2002 ; Nystul et al., 2003).

A recently identified response is hypoxia-induced carbon monoxide production by heme oxygenase-1 (Dulak et al., 2003). Endogenously produced CO can activate signaling cascades that attenuate hypoxic injury through anti-apoptotic (Brouard et al., 2003) and anti-inflammatory (Otterbein et al., 2000) activities, and by perfusing exogenous Sexual CO obtained similar cytoprotective effects in transplantation models (Otterbein et al., 2003; Amersi et al., 2002). At higher concentrations, carbon monoxide competes with oxygen for binding to iron-containing proteins, such as mitochondrial cytochromes and hemoglobin (Gorman et al., 2003), although the possible cytoprotective effects of this activity in hypoxia have not been studied.

Despite these complex defense mechanisms against hypoxic injury, hypoxia is often an injurious stress. For example, mammals have both heme oxygenase-1 and HIF-1, and some evidence suggests that stasis is also possible in mammals (Bellamy et al., 1996; Alam et al., 2002). However, hypoxic injury due to trauma such as heart attack, stroke or blood loss is a major cause of death. Understanding the limitations of the two basic strategies for surviving hypoxic stress (remaining active or stagnant) is hampered by the fact that it is based on studies performed in a variety of systems under a variety of conditions.

"Hypoxia" occurs when normal physiological levels of oxygen are not supplied to cells or tissues. "Normoxia" refers to normal physiological levels of oxygen for the particular cell type, cell state or tissue in question. "Hypoxia" is the absence of oxygen. "Hypoxic conditions" are those conditions that result in hypoxic cells. These conditions depend on the cell type, and on the particular structure or location of the cells within the tissue or organ and on the metabolic status of the cells. For the purposes of the present invention, hypoxic conditions include conditions in which the oxygen concentration is at or below normal atmospheric conditions, which is less than 20.8, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0%; alternatively, these numbers may represent the percentage of the atmosphere at 1 atmosphere (101.3 kPa). An oxygen concentration of 0 percent defines anoxic conditions. Thus, hypoxic conditions include hypoxic conditions, although in some embodiments hypoxic conditions of no less than 0.5% are practiced. As used herein, "normoxic conditions" are equivalent to oxygen concentrations of about 20.8% or greater.

Standard methods of achieving hypoxia or hypoxia are well established and include the use of environmental chambers that rely on chemical catalysts to remove oxygen therefrom. Such chambers are commercially available from, for example, BD Diagnostic Systems (Sparks, MD) as GASPAK Disposable Hydrogen+Carbon Dioxide Envelopes or BIO-BAG environmental chambers. Alternatively, oxygen can be depleted by exchanging the air in the chamber with a non-oxygen gas, such as nitrogen. Oxygen concentration can be determined, for example, with a FYRITE oxygen analyzer (Bacharach, Pittsburgh PA).

It is contemplated that the methods of the present invention may use a combination of exposure to an oxygen antagonist or other reactive compound and altering the oxygen concentration compared to room air. Also, the oxygen concentration of the environment containing the biological matter can be about, at least about, or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%, or may be derived from any range therein. Also, it is contemplated that the change in concentration may be any of the above percentages or ranges in terms of a decrease or increase compared to room air or compared to a control environment.

D. Mitochondrial targeting reagents

In some aspects embodiments of the invention contemplate selective targeting of mitochondria for enhanced activity. This selective mitochondrial targeting has been achieved by conjugating reagents to the lipophilic triphenyl_cation, which readily crosses the lipid bis, driven by large potentials (150 to -180 mv) across the inner mitochondrial membrane. layer and accumulate approximately 1000-fold within the mitochondrial matrix. Analogs of both vitamin E and coenzyme Q have been prepared and used to successfully target mitochondria. (Smith et al., 1999; Kelso et al., 2001; Dhanasekaran et al., 2004). The thiol, thibutyltriphosphonium bromide (shown below) has been prepared and used to target mitochondria, where it accumulates hundreds of times (Burns et al., 1995; Burns & Murphey, 1997).

Such conjugates appear to be suitable candidates for active compounds. In addition to free thiol reagents, thiosulfenyl substituted compounds (H-S-S-R) may be useful. It is contemplated that in some embodiments, the reagent has the following structure:

where Z is P or N;

R ¹ , R ² and R ³ are aryl, heteroaryl, alkylaryl, cycloalkyl or alkyl (suitably phenyl, benzyl, tolyl, pyridyl, cyclohexyl, C ₃ -C ₁₀ alkyl, optionally halogenated);

R ⁴ is -R ⁵ SR ⁶ , wherein R ⁵ is C ₁ -C ₁₀ alkyl, and R ⁶ is H or SH, SO ₃ H or PO ₃ H.

III. Detection of stagnation

A variety of compounds useful for inducing arrest can be initially evaluated using a variety of different assays. Arrest can be measured in a number of ways, including by quantifying the amount of oxygen consumed by a biological sample, the amount of carbon dioxide produced by the sample (an indirect measure of cellular respiration), or by characterizing motility.

To determine the rate of oxygen consumption or carbon dioxide production, the biomass was placed in a sealed chamber with two openings for gas input and output. Gas (room air or other gas) is passed into the chamber at a given flow rate and out the outlet to maintain approximately 1 atmosphere in the chamber. The amount of each compound in the gas mixture is measured (per second) by passing the gas through a carbon dioxide detector and or an oxygen detector before and after exposure to the chamber. Comparing these values over time yields the rate of oxygen consumption or carbon dioxide production.

Other screening methods have been established to identify candidate activity arresting compounds. These screening methods and variants thereof may be employed as part of or used to practice aspects of the present invention.

A. Assay Using Zebrafish

Screening assays for arresting inducers were set up with 48-hour-old zebrafish (D. rerio) embryos. These embryos are transparent, allowing one to observe the heartbeat and resulting blood flow into the main vessels along the back and into the tail with a dissecting microscope with a 4-2Ox lens. Heart rate in these animals is an indicator of the metabolic activity of the organism, such that a decrease in heart rate indicates a decrease in metabolism. Embryos were dissected from their egg shells and distributed 5 per well in flat bottom polystyrene tissue culture plates and incubated in 1 mL of standard fish water. The fish water consisted of 1 teaspoon of Instant Ocean (artificial seawater mixture, Aquarium Systems Inc.) per 5 gallons. Calcium chloride was adjusted to 150 ppm and sodium bicarbonate was adjusted to -100 ppm. The conductivity of the water was 900 microSiemens and the pH was about 6.5-7.4. Hydrogen sulfide solution was prepared by bubbling a mixture of hydrogen sulfide equilibrated with room air at a rate of 100 cubic centimeters per minute in a flask containing 150 mL of fish water for 60-90 minutes. It is estimated that this is sufficient to obtain a saturated or nearly saturated or mostly saturated hydrogen sulfide solution. Based on the known solubility of hydrogen sulfide in Ringer's solution at pH 7 at 1 atmosphere pressure and room temperature, it is estimated that fish water contains approximately 0.1 molar hydrogen sulfide. Fish were exposed to hydrogen sulfide solution and their heart rate was monitored by counting beats per minute over the next 24 hours. Control fish (exposed to fish water only) had a heart rate of approximately 160-200 beats per minute, which did not change significantly during the 24 hour observation period. By 2-3 hours after exposure to fish water containing hydrogen sulfide, the cardiac rate had decreased by approximately half to 60-80 beats per minute. By 4 hours, the beat rate further decreased, including some instances of 0 or only a few beats per minute. After 5 h of exposure, replace the hydrogen sulfide solution with normal fish water and allow the embryos to recover overnight at 28 °C. By 24 hours after initial exposure to hydrogen sulfide, treated and flushed animals exhibited a normal heart rate of 160-200 beats per minute. Since hydrogen sulfide induces dormancy, in some cases arrest, and subsequent return to normality of the heartbeat, hydrogen sulfide is considered to have been identified as an arrest inducer or other active compound by the criteria of this screening assay.

B. Assays using nematodes

The screening assay was set up with the nematode (C. elegans). Nematodes do not survive well at 4 degrees Celsius, so after 24 hours at that temperature, they all died. Worms were exposed to an atmosphere containing Y% carbon monoxide for X minutes at room temperature before exposing them to 4 degrees Celsius for 16 hours. Carbon monoxide treated worms survived exposure to low temperatures with high viability compared to control worms pre-exposed to room air which all died. Since carbon monoxide is a known inducer of arrest in nematodes and neonatal foreskin keratinocytes, the nematode assay enables increased exposure to lethal hypothermia when worms are pre-equilibrated in arrest inducers or other active compounds. Ability to identify arrest-inducing compounds for worm viability.

IV. Therapeutic or Prophylactic Use

A. Trauma

In some embodiments, the present invention may be used to treat patients suffering from or susceptible to injury. Trauma can be caused by external injury (such as a burn, wound, excision, gunshot wound, or surgical trauma) internal injury (such as a stroke or heart attack that causes a sharp drop in circulation) or due to non-invasive stress (such as exposure to hypothermia or radiation ) caused by the cycle decline caused. At the cellular level, trauma often results in the exposure of cells, tissues and/or organs to hypoxia, resulting in the induction of programmed cell death, or "apoptosis." Systemically, trauma results in the induction of a cascade of biochemical processes, such as coagulation, inflammation, hypotension, and can cause shock, which, if sustained, can lead to organ dysfunction, irreversible cellular damage, and death. Biological processes are designed to defend the body from traumatic injury; however they can lead to a sequence of events that prove deleterious and, in some cases, fatal.

Thus, the present invention contemplates the immobilization of tissues, organs, limbs and even whole organisms as a means of protecting them from the deleterious effects of trauma. In specific situations where medical care is not readily available, in vivo or ex vivo induction of arrest, alternatively combined with lowering the temperature of a tissue, organ or organism, may "buy time" for the subject to provide medical care to the subject , or transport subjects to medical care. The present invention also contemplates methods for inducing tissue regeneration and wound healing by preventing/delaying biological processes that could lead to delayed wound healing and tissue regeneration. In this regard, in situations where there is a large wound to a limb or organism, in vivo or ex vivo induction of stasis, alternatively, in combination with lowering the temperature of the tissue, organ or organism may inhibit the biological processes of healing and regeneration through management To aid in wound healing and tissue regeneration processes.

In addition to wound healing and hemorrhagic shock discussed below, the methods of the invention can be practiced to prevent or treat trauma such as cardiac arrest or stroke. The present invention is of particular importance for the risk of injury from emergency surgical procedures such as thoracotomy, laparotomy and splenic transection.

1. Wound healing

In many cases, wounds and tissue damage are refractory or require excessive time to heal. Examples are chronic open wounds (diabetic foot ulcers and stage 3 & 4 pressure ulcers), acute and traumatic wounds, flaps and grafts, and subacute wounds (ie, dehiscing incisions). This also applies to other tissue injuries such as burns and lung injuries from smoke/heat inhalation.

Previous experiments have shown that hibernation protects against damage (for example, pin's in the brain), so it may have healing effects. Therefore, this technique could be used to control the wound healing process by bringing tissue into a more metabolically controlled environment. More specifically, the length of time a cell or tissue remains stagnant can vary depending on the injury. In some embodiments of the invention, the biological material is exposed to the oxygen antagonist or other reactive compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24 hours; 1, 2, 3, 4, 5, 6, 7 days; 1, 2, 3, 4, 5 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer.

2. Hematological shock (hemorrhagic shock)

Shock is a life-threatening condition that progresses rapidly when intervention is delayed. Shock is a state in which there is insufficient perfusion to maintain the physiological needs of organ tissues. This is a state of profound hemodynamic and metabolic disturbance characterized by the inability of the circulatory system to maintain adequate perfusion of vital organs. It may be caused by insufficient blood volume (hypovolemic shock), insufficient heart function (cardiogenic shock), or insufficient vasomotor tone, also known as distributive shock (neurogenic shock, septic shock, anaphylactic shock, sexual shock). This often results in the patient's rapid death. Many conditions, including sepsis, blood loss, impaired autoregulation, and loss of autonomic tone can produce shock or a shock-like state. The present invention is expected to prevent the adverse effects of all of the above states of shock and to maintain the life of biological matter undergoing such shock.

In hemorrhagic shock, blood loss exceeds the body's ability to compensate and provide adequate tissue perfusion and oxygenation. This is usually attributed to trauma, but can also result from spontaneous bleeding (eg, gastrointestinal bleeding, childbirth), surgery, and other causes. Most commonly, clinical hemorrhagic shock is caused by acute bleeding events followed by discrete precipitating events. Less commonly, hemorrhagic shock can be seen in chronic conditions with subacute blood loss.

Physiological compensatory mechanisms for bleeding include initial peripheral and mesenteric vasoconstriction to return blood to the central circulation. This is subsequently reinforced by progressive tachycardia. Invasive monitoring can reveal increased cardiac index, increased oxygen delivery (ie, _DO2 ), and increased tissue oxygen consumption (ie, _VO2 ). Lactate levels, acid-base status, and other markers can also provide useful indicators of physiological status. Age, medications, and comorbidities may all affect a patient's response to hemorrhagic shock.

Failure of compensatory mechanisms in hemorrhagic shock can lead to death. In the absence of intervention, a typical trimodal distribution of deaths was observed in severe hemorrhagic shock. The initial peak of death occurs within minutes of bleeding due to immediate exsanguination. Another peak occurs 1 to several hours later due to progressive decompensation. A third peak occurs days to weeks later due to sepsis and organ failure.

In the United States, unintentional injuries are the leading cause of mortality and morbidity among persons between the ages of 1 and 44 steps. In 2001, 157,078 resident deaths occurred due to injuries. Of these, 64.6% were classified as accidental deaths, 19.5% were suicides, 12.9% were murders, 2.7% had undetermined intent, and 0.3% involved legal intervention or acts of war. The leading causes of injury deaths are motor vehicle accidents, firearms and falls. A large proportion of these fatalities result from massive blood loss from trauma, leading to hemorrhagic shock.

In most traumatic injury events, patients arriving in a hospital emergency department are managed by emergency physicians and discharged without surgery or trauma service care. However, patients with severe injuries require stabilization during the "golden time" after injury to improve chances of survival and minimize disability.

Since most shock events are attributed to accident-induced injuries, pre-hosiptal care is critical to patient survival. Prehospital care includes rapid assessment, stabilization, and prompt transport to an appropriate center for evaluation and definitive care. In all patients with shock syndrome, maintaining a patent airway, adequate breathing, and adequate circulation is the primary focus of emergency treatment. Evaluation is required because changes in the patient's condition indicate progression of the shock syndrome. Early intervention is critical to minimize tissue and organ damage and permanent disability, and early identification of the primary clinical cause is critical. Treatment focuses on correcting the cause of shock syndrome and slowing progression. Intravenous infusion and fluid resuscitation (usually IV saline) are standard, however, there is some debate about this. Rapid reversal of hypovolemia may increase bleeding, clot formation in moving parts, and dilution of clotting factors.

Once in the emergency room, focus on optimizing perfusion and oxygenation of vital organs. Diagnosis and management of underlying hemorrhage must be undertaken promptly and concurrently with management of shock. There are two main phases of shock: an early compensatory phase and a progressive phase. It is contemplated that embodiments of the invention may be applied to patients in either or both stages.

When hypovolemic shock is caused by massive bleeding, the alternative fluid of choice is whole blood or packed red blood cells. Crystalloid solutions will temporarily increase circulatory capacity, but patients also need red blood cell substitutes to deliver oxygen to tissues. Management of shock focuses on fluid management, acid-base balance, and enhancing myocardial contractility. The underlying cause of shock should also be addressed in order to minimize the progression of shock syndrome. When whole-body hibernation is induced in mice, there is an immediate drop in overall metabolic state (measured by _CO2 evolution). This was reversible, and the mice appeared to function normally, even after repeated exposures. Thus, the present invention involves the use of _H2S (or other oxygen antagonists or other active compounds) to induce hibernation throughout the body to preserve the vital organs and life of the patient. This would allow transport to a controlled setting (eg, surgery) where the original cause of the shock can be addressed and the patient then return to normal function in a controlled manner. For this indication, the first time after injury known as the "golden time" is critical to a successful outcome. Stabilizing the patient during this time period is the primary goal and transporting the patient to a critical care facility (eg, emergency room, operating room, etc.) where the injury can be appropriately addressed. Therefore, it is ideal to keep the patient in arrest to enable this purpose, and address immediate issues such as the source of the shock, replace blood loss, and reestablish homeostasis. While this will vary widely, in most cases the amount of time that will remain stagnant is between about 6 to about 72 hours after injury. In some embodiments of the invention, the biological material is exposed to the oxygen antagonist or other reactive compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24 hours; 1, 2, 3, 4, 5, 6, 7 days or longer, and any range or combination thereof.

The biology of lethal bleeding and the physiological events leading to shock and ultimately death is not fully understood. However, there are some mechanisms by which _H2S can reduce the lethal effects of ischemic hypoxia. Hydrogen sulfide inhibits cytochrome c oxidase and may reduce oxygen demand by inhibiting this enzyme ³ . Reduced oxygen demand reduces the deleterious effects of low oxygen levels, including reducing metabolic acidosis. Furthermore, tissue sulfhydryl levels decrease during shock (Beck et al., 1954). Exogenous _H2S prevents this hyposulfidic state and maintains the sulfur homeostasis.

Hydrogen sulfide is naturally produced in animals and exhibits potent biological activity (Kamoun, 2004). Most proteins contain disulfide-linked cysteine residues, and the reversible transition from free thiols to disulfides regulates specific enzyme activities (Ziegler, 1985). Furthermore, sulfides are electronegative and exhibit high affinity for transition metals. Proteins containing transition metal atoms, such as cytochrome oxidase, can be strongly affected by _H2S . And finally, the metabolism of _H2S to other molecules containing reduced sulfur increases the number of thiols that can exhibit specific biological activities. In addition to (or perhaps due to) these potential modes of action, _H2S may exert effects on the cardiorespiratory, neuroendocrine, immune and/or hemostatic systems that turn out to be beneficial in injury and disease.

The US provisional application entitled "Methods, compositions and articles of manufacture for treating shock" filed 20 April 2006 by Mark B. Roth, Mike Morrison and Eric Blackstone describes the treatment of shock and is hereby incorporated by reference.

B. Hypothermia

In yet another embodiment, the inventors suggest using the present invention to treat extremely hypothermic patients. The methods and compositions of the invention are useful for inducing hypothermia in a mammal in need of hypothermia. Hypothermia can be mild, moderate or profound. Mild hypothermia involves achieving core body temperature about 0.1-5 degrees Celsius below the normal core body temperature of a mammal. Normal core body temperature in mammals is usually between 35 and 38 degrees Celsius. Moderate hypothermia involves achieving a core body temperature of about 5-15 degrees Celsius below the normal core body temperature of a mammal. Profound hypothermia involves achieving a core body temperature of about 15-37 degrees Celsius below the normal core body temperature of mammals.

Mild hypothermia is known in the art to be therapeutically useful and effective in both non-human mammals and humans. Therapeutic benefits of mild hypothermia have been observed in human clinical trials on out-of-hospital cardiac arrest. Exposure of people to mild hypothermia during cardiac arrest resulted in a survival advantage and improved neurological outcomes compared with the standard of care with normothermia, or the absence of mild hypothermia (Bernard et al., 2002; The Hypothermia After Cardiac Arrest Study Group et al. , 2002).

The methods and compositions of the present invention may have advantages over other methods known in the art for inducing mild, moderate or profound hypothermia in mammals or humans, including, but not limited to Wrap the subject in ice, or in a "cooling veil" that circulates cold air or liquid. In these cases, the subject resists lowering core body temperature below normal body temperature and attempts to generate heat by shivering. Shivering, and the body heat generated therein, can negatively affect the achievement of mild hypothermia by, for example, slowing the rate of core body temperature reduction achieved with standard hypothermia induction methods. Therefore, persons receiving therapeutic levels of hypothermia are also treated with drugs that suppress tremors by blocking neurotransmission at the neuromuscular junction (Bernard et al., 2002).

In preferred embodiments, the methods and compositions of the invention induce therapeutic hypothermia in mammals or humans in combination with invasive procedures or medical devices known in the art. Such invasive methods and devices include, but are not limited to, flexible probes or catheters that can be inserted into the vasculature of a subject requiring hypothermia, where the temperature of the catheter is adjusted below the normal body temperature of the subject, resulting in contact with the catheter Blood on contact cools. The cooled blood then causes the mammal's core body temperature to drop. By incorporating feedback from thermocouple monitoring that monitors the core body temperature of the mammal, the temperature of the catheter can be adjusted to maintain a prespecified core body temperature. Such medical devices for achieving and maintaining mild or moderate hypothermia, known in the art as intravascular thermotherapy, are known in the art and are available, for example, at www.innercool.com and www.radiantmedical.com There is a description.

The method provides for administering or exposing a hypothermic patient to an oxygen antagonist or other active compound, followed by gradual return to normal temperature when the oxygen antagonist or other active compound is withdrawn in a controlled manner. In this way, the oxygen antagonist or other active compound buffers the biological systems in the subject so that they can begin gradually without shocking (or injuring) the subject.

In one embodiment, an oral or intravenous dose of an oxygen antagonist or other active compound will be administered to a subject suffering from hypothermia. Intravenous delivery is preferred due to the potential non-responsiveness of subjects and the ability to provide a controlled dose over a period of time. Alternatively, if available, the oxygen antagonist or other active compound may be provided in a gaseous state, eg, using a mask for inhalation or even a closed chamber that can contain the entire subject.

Ideally, the patient's heart rate, respiration and temperature would be stabilized before any changes are effected. Once stabilized, the ambient temperature will gradually rise again. This is easily accomplished by removing the subject from the cryogenic conditions. A more regulated increase in temperature can be achieved by adding successive layers of clothing or blankets, using thermal wraps with gradually increasing heat, or if possible, placing the subject in a chamber where their temperature can be gradually increased.

The vital signs of the subject are preferably monitored during the temperature increase. Also, in conjunction with increasing the temperature, oxygen antagonists or other active compounds are removed from the subject's environment. Treatment with both heat and oxygen antagonists (or other active compounds) was discontinued at an appropriate endpoint, at the discretion of the medical personnel monitoring the situation, but in any event when the subject's temperature and other vital signs returned to normal range. Continued monitoring for a period of at least 24 hours after cessation of treatment is recommended.

C. High body temperature

In some cases, which can be caused by genetic, infectious, drug, or environmental causes, patients can relax homeostatic thermoregulation, resulting in severe uncontrollable fever (hyperthermia). This can lead to death or long-term morbidity, especially brain damage, if it is not properly controlled.

Mice inhaled 80 ppm _H2S immediately experienced hibernation. This includes the inability to regulate their body temperature when the ambient temperature drops below room temperature. Therefore, this technique can be used to control body temperature in certain hyperthermic states. This would likely include administration of _H2S (or other oxygen antagonists or active compounds) by inhalation or infusion into the blood supply to induce hibernation. It will be useful to keep the patient under arrest for between about 6 and about 24 hours, during which time the source of fever can be resolved. In some embodiments of the invention, the patient is exposed to the oxygen antagonist or other active compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20, 25, 30 seconds , 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours; 1, 2, 3, 4, 5, 6, 7 days or longer, and any range or combination thereof.

This can be combined with some whole body thermoregulation (ice bath/blanket/cooling system).

D. Heart palsy and coronary heart disease

In some embodiments, the present invention is useful as a solution for the treatment of coronary heart disease (CHD), including cardioplegia for cardiac bypass surgery (CABG).

CHD is caused by atherosclerosis, the narrowing and hardening of the arteries that supply oxygen-rich blood to the heart muscle. Arteries harden and narrow due to the buildup of plaque on the inside walls, or lining, of the arteries. Blood flow to the heart is reduced due to plaque narrowing the coronary arteries. This reduces the oxygen supply to the heart muscle. This can manifest as: 1) angina, which is chest pain or discomfort that occurs when the heart does not get enough blood; 2) a heart attack, which can occur when a blood clot suddenly cuts off most or all of the blood supply to part of the heart, And cells in the heart muscle that don't receive enough oxygen-carrying blood begin to die, which can lead to permanent damage to the heart muscle; 3) heart failure, which occurs when the heart can't pump blood effectively to the rest of the body; arrhythmia , which is a change in the normal rhythm of the heartbeat.

Since 1990, more people have died from CHD than from any other cause. CHD kills 3.8 million men and 3.4 million women each year. In 2002, more than 500,000 people died as a direct result of heart disease in the United States alone. Despite improved survival rates, one in four men and one in three women in the United States still die within a year of recognizing their first heart attack.

Medical treatment for CHD includes medications to reduce the risk of heart attack, heart failure, and stroke, as well as important lifestyle changes to prevent further buildup of fatty deposits in the coronary arteries. However, some type of surgical intervention is often required.

About one third of CHD patients will undergo coronary angioplasty and stenting. During balloon angioplasty, a balloon-tipped catheter is used to push plaque back against the artery wall to allow increased blood flow in the artery. Coronary artery stenting is usually accompanied by an angioplasty procedure. Stents are small wire-mesh metal tubes that provide support to the damaged artery wall, which reduces the likelihood of the vessel closing again (restenosis) after angioplasty. In the United States, nearly 1 million balloon angioplasty procedures are performed annually. Not all patients can be treated with this technique; such patients must undergo heart surgery. Michaels et al., 2002.

Approximately 10% of CHD patients will undergo coronary artery bypass grafting (CABG) surgery. Patients with severe narrowing or blockage of the left common coronary artery, or those with disease involving two or three coronary arteries are often considered candidates for bypass surgery. In CABG, the surgeon creates a detour (or bypass) around the blocked portion of the coronary artery using a healthy blood vessel (artery or vein) from another part of the body. Patients typically receive 1 to 5 bypasses during a given procedure. During this procedure, the heart is usually paralyzed, known as cardioplegia (CP), during which a heart-lung machine maintains circulation artificially. The patient is under general paralysis during the procedure, which typically lasts 3-6 hours.

Approximately 13% of all patients will be readmitted to the hospital within 30 days for reasons related to CABG. Hannan et al., 2003; Mehlhorn et al., 2001. One of the leading reasons for readmission is heart failure, possibly due to ischemic damage during surgery. Therefore, much work needs to be done to improve the protection of myocardium during periods when the heart is not properly perfused.

Recent advances in cardiac surgery have focused on optimizing cardioplegic parameters in hopes of preventing postoperative ventricular dysfunction and improving overall outcome. Cohen et al., 1999.

Perfusing the cardioplegia solution through the blood vessels and chambers of the heart causes their intrinsic pulsation arrest while maintaining the viability of the organ. Cardioplegia (paralysis of the heart) is desirable during open heart surgery, during harvesting, transport and preservation of a donor heart for heart transplant surgery.

Early cardioplegia techniques used cold crystalloid solutions to initiate and maintain asystole during surgery. However, it is already clear that hemorrhagic cardioplegia promotes aerobic myocardial metabolism and reduces anaerobic lactate production during cross-clamp. Furthermore, hemorrhagic cardioplegia increases oxygen-carrying capacity, increases myocardial oxygen consumption and maintains myocardial high-energy phosphate reserves. A variety of different cardioplegic solutions are available, and different techniques for using cardioplegic solutions are known in the art. For example, cardioplegia solutions typically have varying amounts of potassium, magnesium, and various other minor components. Sometimes, drugs are added to cardioplegia solutions to help muscles relax and prevent ischemia. Current methods also include blood-only preparations supplemented with appropriate electrolytes, such as glutamate-aspartate. Specific examples of commonly used solutions are St Thomas' Hospital solution, University of Wisconsin solution, Stanford solution, and Bretschneider solution. Examples of other newly developed solutions include the previously mentioned solutions containing adenosine, insulin or L-arginine. Changing the temperature at which the cardioplegia solution is administered also has beneficial effects.

The combination of continuous degenerative cardioplegia with intermittent anterograde cardioplegia reduces myocardial lactate production, preserves ATP reserves, and improves metabolic recovery after cross-clamp release. Warm (29°C) cardioplegia reduces lactate and acid production during cardioplegic arrest and improves postoperative ventricular function. A flow rate of at least 200 mL/min of cardioplegic fluid is required to wash out harmful metabolic end products and improve ventricular function. It is now sufficiently clear that future directions in the management of cardioplegia will involve the use of cardioplegia additives to further improve protection. For example, attempts have been made to fix the beneficial effects of ischemic preconditioning with adenosine. Similarly, insulin cardioplegia has been used to enhance ventricular performance by stimulating early postoperative aerobic metabolism. Finally, L-arginine, a nitric oxide donor, has proven to be beneficial in pilot studies and represents a further option for enhancing myocardial protection during surgery. Future benefits of cardioplegia supplementation are likely to be observed in high-risk groups with poor ventricular function for whom current protective techniques are insufficient. The incidence of current high-risk patients is steadily increasing, and these cases, and subsequent complications, place a disproportionate burden on the health care system. Therefore, improvements in this field hold great promise for the development of nursing in this field.

Despite the protective effect afforded by current methods for inducing cardioplegia, some degree of ischemia-reperfusion injury to the myocardium remains. Ischemia-reperfusion injury during cardiac bypass surgery leads to poor outcomes (both morbidity and mortality), especially due to the already weakened state of the heart. Myocardial ischemia results in anaerobic myocardial metabolism. The end products of anaerobic metabolism rapidly lead to acidosis, mitochondrial dysfunction, and myocyte necrosis. Depletion of high-energy phosphoric acid occurs almost immediately, with 50% of ATP reserves lost within 10 minutes. Contractility reduction occurs within 1 to 2 minutes, and ischemic muscle contracture and irreversible injury occur after 30-40 minutes of normothermic (37°C) ischemia.

Reperfusion injury is a well-known phenomenon following restoration of coronary circulation. Reperfusion injury is characterized by abnormal myocardial oxidative metabolism. In addition to the structural changes produced during ischemia, reperfusion can generate cytotoxic oxygen free radicals. These oxygen free radicals play a major role in the pathogenesis of reperfusion injury by oxidizing the phospholipids of the sarcolemma and thereby disrupting the integrity of the membrane. Oxidized free fatty acids are released into coronary venous blood and are markers of phospholipid peroxidation in the myocardium. Protamine induces complement activation, which activates neutrophils. Activated neutrophils and other leukocytes are an additional source of oxygen free radicals and other cytotoxic substances.

The present invention provides methods and compositions for inducing cardioplegia, which will provide better protection of the heart during bypass surgery. In certain embodiments, the present invention provides a cardioplegia solution comprising _H2S (or another active compound) dissolved in the solution or bubbled through the solution as a gas. In some embodiments, the invention further comprises at least a first device, such as a catheter or cannula, for introducing an appropriate dose of the cardioplegic solution to the heart. In certain aspects, the invention further comprises at least a second device, such as a catheter or cannula, for removing the cardioplegia solution from the heart.

Bypass surgery typically lasts 3-6 hours, however, complications and polyvascular CABG can extend the duration to 12 hours or longer. Consider that the heart should remain stationary during the procedure. Thus, in some embodiments of the invention, the heart is exposed to the oxygen antagonist or other active compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 hours or longer, and any range or combination thereof.

E. Reduce damage caused by cancer treatment

Cancer is the leading cause of mortality in the industrialized countries of the world. The most conventional method of treating cancer is by administering a cytotoxic agent to a cancer patient (or ex vivo treatment of tissue), such that the agent has a greater lethal effect on cancer cells than on normal cells. Higher doses or more lethal agents will be more effective; however, by the same token, such agents are to that extent more toxic (and sometimes lethal) to normal cells. Therefore, chemotherapy and radiation therapy are often characterized by severe side effects, some of which are life-threatening, such as mouth sores, difficulty swallowing, dry mouth, nausea, diarrhea, vomiting, fatigue, bleeding, hair loss and infection, skin irritation, and energy loss (Curran, 1998; Brizel, 1998).

Recent studies have shown that temporarily and reversibly lowering core body temperature, or "hypothermia," can lead to improved resistance to cancer. Hypothermia at 28°C was recently found to reduce radiation, doxorubicin- and cisplatin-induced toxicity in mice. The anticancer activity of these drugs/therapies was not impaired when administered to cooled animals; on the contrary, the activity was enhanced, especially that of cisplatin (Lundgren-Eriksson et al., 2001). Based on this study, as well as other published studies, the inventors propose that further reductions in core temperature would benefit cancer patients. Accordingly, the present invention contemplates the use of oxygen antagonists or other active arresting compounds to induce arrest in normal tissues of cancer patients, thereby reducing the potential effects of chemotherapy or radiation therapy on those tissues. This also allows the use of higher doses of chemotherapy and radiation, enhancing the anti-cancer effects of these treatments.

The treatment of virtually any hyperproliferative disease, including benign and malignant neoplasia, non-neoplastic hyperproliferative disease, preneoplastic conditions, and precancerous lesions is contemplated. Such diseases include restenosis, carcinoma, multidrug-resistant carcinoma, primary psoriasis and metastatic neoplasms, angiogenesis, rheumatoid arthritis, inflammatory bowel disease, psoriasis, eczema, and secondary endogenous as well as oral hairy leukoplakia, bronchial dysplasia, carcinoma in situ, and intraepithelial hyperplasia. In particular, the present invention is intended to treat human cancers including prostate cancer, lung cancer, brain cancer, skin cancer, liver cancer, breast cancer, lymphoid system cancer, stomach cancer, testicular cancer, ovarian cancer, pancreatic cancer, bone cancer, bone marrow cancer, gastric Bowel cancer, head and neck cancer, cervical cancer, esophagus cancer, eye cancer, gallbladder cancer, kidney cancer, adrenal cancer, heart cancer, colon cancer and blood cancer. Cancers involving epithelial and endothelial cells are also contemplated for treatment.

In general, chemotherapy and radiation therapy are designed to reduce tumor size, reduce tumor cell growth, induce tumor cell apoptosis, reduce tumor vasculature, reduce or prevent metastasis, reduce tumor growth rate, accelerate tumor cell death, and kill tumors cell. The object of the present invention is no different. Accordingly, it is contemplated that the oxygen antagonist (or other active compound) compositions of the present invention be combined with a second anti-cancer agent effective in the treatment of hyperproliferative disease (second agent). An "anti-cancer" agent is capable of negatively affecting cancer in a subject, for example by killing cancer cells, inducing cancer cell apoptosis, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size , inhibiting tumor growth, reducing blood supply to a tumor or cancer cell, enhancing an immune response against a cancer cell or tumor, preventing or inhibiting cancer progression, or prolonging the life of a subject with cancer.

Secondary anti-cancer agents include biological agents (biotherapy), chemotherapeutics and radiotherapeutics. More generally, these other compositions are provided in combined dosages effective to kill or inhibit the proliferation of cancer cells or tumor cells, while reducing or minimizing the effects of the second agent on normal cells. The method may involve contacting or exposing the cell simultaneously with the oxygen antagonist (or other active compound) and the second agent. This can be achieved by contacting the cells with a single composition or pharmacological formulation comprising both classes of agents, or by contacting or exposing the cells to two different compositions or formulations at the same time, one of which is One contains an oxygen antagonist and the other composition contains a second agent.

Alternatively, the oxygen antagonist (or other active compound) treatment may precede or follow the second agent treatment by an interval of minutes to weeks. In embodiments where the other agent and the expression construct are administered separately to the cell, it should generally be ensured that the effective period is not terminated between each delivery event so that the agent and expression construct will still be able to exert a beneficial combined effect on the cell. In such cases, it is contemplated that the cells may be contacted with the two modalities within about 12-24 hours of each other, more preferably within about 6-12 hours of each other. In some cases, however, the period of effective treatment is prolonged, with days (2, 3, 4, 5, 6 or 7) to weeks (1, 2, 3, 4, 5, 6, , 7 or 8) intervals may be ideal. In certain embodiments, it is contemplated that the biological material is kept stagnant for between about 2- and about 4 hours while the cancer treatment is being administered. In some embodiments of the invention, the biological material is exposed to the oxygen antagonist or other reactive compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6 hours or longer, and any range or combination thereof.

Various combinations can be used; the active compound is "A" and the second anti-cancer agent, such as radiotherapeutic or chemotherapeutic agent is "B":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/AA/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/AA/B/A/A A/A/B/A

Administration of an oxygen antagonist or other active compound of the invention to a patient will follow the usual methods for administering chemotherapy regimens, taking into account the toxicity, if any, of the compound. It is expected that treatment cycles should be repeated as needed. It is also contemplated that a variety of standard therapies as well as surgical interventions may be used in combination with the anti-cancer therapies described above. It is further contemplated that, for multiple active compounds, any combination therapy contemplated using an active compound and an inactive compound (eg, chemotherapy) may be applied.

1. Chemotherapy

Cancer therapy also includes various combination therapies with chemotherapy and radiation based treatments. Combination chemotherapy includes, for example, cisplatin (CDDP), carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, phenylalanine mustard, chlorambucil, An, nitrosourea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, mitomycin, etoposide (VP16), tamoxifen, ralox phen, estrogen receptor binding agents, paclitaxel, gemcitabine, navelbine, farnesyl-protein transferase inhibitors, transplatinum, 5-fluorouracil, vincristine, vinblastine, and methotrexate), Temazolomide (aqueous form of DTIC), or any analogue or derivative variant of the foregoing. The combination of chemotherapy and biological therapy is called biochemical therapy.

2. Radiation therapy

Other factors that cause DNA damage and have been widely exploited include those commonly referred to as gamma-rays, X-rays, and/or the direct delivery of radioisotopes to tumor cells. Other forms of DNA damaging factors are also considered, such as microwave and ultraviolet radiation. It is likely that all of these factors effect widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. X-ray doses range from daily doses of 50-200 roentgens for prolonged periods (3 to 4 weeks), to single doses of 2000-6000 roentgens. Dosage ranges for radioisotopes vary widely and depend on the half-life of the isotope, the strength and type of radiation emitted, and uptake by neoplastic cells.

The terms "contacting" and "exposing", as they apply to cells, are used herein to describe the delivery of a component of the invention (e.g., a hypoxic antineoplastic compound) or a chemotherapeutic or radiotherapeutic agent to a target cell or to a target cell. The method by which it is placed directly juxtaposed with the target cells. In combination therapy, to achieve cell killing or stasis, the two agents are delivered to the cell in combined doses effective to kill the cell or prevent it from dividing.

3. Immunotherapy

In general, immunotherapeutics relies on the use of immune effector cells and molecules that target and destroy cancer cells. Immune effectors can be, for example, antibodies specific for certain markers on the surface of tumor cells. An antibody alone can act as an effector for a therapy, or it can recruit other cells to actually effect the cell killing. Antibodies can also be conjugated to drugs or toxins (chemotherapeutics, radionuclides, ricin A chain, cholera toxin, pertussis toxin, etc.) and used only as targeting agents. Alternatively, the effectors may be lymphocytes carrying surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

Immunotherapy can also be used as part of a combination therapy. General methods for combination therapy are discussed below. In one aspect of immunotherapy, the tumor cells must have some marker to be targeted, ie absent on most other cells. There are many tumor markers and any of these markers may be suitable for targeting within the scope of the present invention. Common tumor markers include carcinoembryonic antigen, prostate-specific antigen, urinary tumor-associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis antigen, MucA, MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An alternative aspect of immunotherapy is anticancer effects with immunostimulatory effects. Immunostimulatory molecules are also present, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, г-IFN; chemokines such as MIP-1, MCP-1, IL-8; and growth factors For example FLT3 ligand. Combining immunostimulatory molecules as proteins or with gene delivery with tumor suppressors such as mda-7 has been shown to enhance antitumor effects (Ju et al., 2000).

As previously discussed, examples of immunotherapies currently being studied or used are immune adjuvants (e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds) (US Patent 5,801,005, US Patent 5,739,169, Hui and Hashimoto, 1998, Christodoulides et al., 1998), cytokine therapy (eg, interferon alpha, beta, and gamma; IL-1, GM-CSF, and TNF) (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998), gene therapy (e.g., TNF, IL-1, IL-2, p53) (Qin et al., 1998; Austin-Ward and Villaseca, 1998; US Patent 5,830,880 and US Patent 5,846,945) and monotherapy Clonal antibodies (eg anti-ganglioside GM2, anti-HER-2, anti-p185) (Pietras et al., 1998; Hanibuchi et al., 1998). Herceptin (trastuzumab) is a chimeric (mouse-human) monoclonal antibody that blocks the HER2-neu receptor. It has anti-tumor activity and has been approved for the treatment of malignancies (Dillman, 1999). Combination therapy of cancer with Herceptin and chemotherapy has been shown to be more effective than either therapy alone. Accordingly, it is contemplated that one or more anti-cancer therapies may be used in conjunction with the anti-tumor therapies described herein.

F. Neurodegeneration

The present invention is useful in the treatment of neurodegenerative diseases. Neurodegenerative diseases are characterized by degeneration of neuronal tissue and are often accompanied by memory loss, loss of motor function, and dementia. With dementia disease, intelligence and higher general cognitive abilities become increasingly impaired over time. It is estimated that about 15 percent of people aged 65 or older have mild to moderate dementia. Neurodegenerative diseases include Parkinson's disease, primary neurodegenerative diseases, Huntington's disease, stroke and other hypoxic or ischemic processes, neurotrauma, metabolically induced neurological damage, sequelae of cerebral seizures, hemorrhagic Shock, secondary neurodegenerative disease (metabolic or toxic), Alzheimer's disease, other memory impairment or vascular dementia, multi-infarct dementia, dementia with Lewy bodies, or neurodegenerative dementia.

There is evidence that the health of organisms, and especially the health of the nervous system, depends on cycling between oxidative and reducing states, which is closely related to circadian rhythms. That is, oxidative stress on the body during wakefulness is recycled to a reducing environment during sleep. Think this is a big reason why sleep is so important to your health. Certain neurodegenerative disease states, such as Huntington's disease and Alzheimer's disease, as well as the normal aging process, are associated with dissonance in this cyclic pattern. There is also some evidence that brain _H2S levels are reduced in these states (Eto et al., 2002).

The present invention can be used to regulate and control cycling between oxidative and reduced states, for example to prevent or reverse the effects of neurodegenerative diseases and processes. Controlling circadian rhythms may have other applications, for example, for adjusting these cycle patterns after traveling from one time zone to another in order to adapt to the new time zone. Furthermore, a general decrease in metabolic activity has been shown to correlate with health in aging animals and humans. Thus, the present invention will also be useful in inhibiting overall metabolic function to increase longevity and health in the elderly. It is contemplated that this type of treatment will likely be performed for about 6-10 hours each day at night, during sleep. This may require daily processing over extended periods of months to years.

g. aging

Furthermore, in certain stagnant states, including but not limited to, states in which the biological matter is in a stagnant state, senescence itself may be substantially or completely inhibited for the period of time while the biological matter is in that state. Thus, the present invention can inhibit senescence of biological matter with respect to extending the amount of time that biological matter would normally live and/or progress from one developmental stage of life to another.

H. blood disease

A number of blood diseases and disorders can be addressed with the compositions and methods of the present invention. These disorders include, but are not limited to, thalassemia and sickle cell anemia.

1. Thalassemia

Normal hemoglobin contains two b and two beta globin polypeptide (protein) chains, each bound to an iron-containing heme ring. Thalassemias are a group of conditions in which there is an imbalance of б and в chains, resulting in the deposition of unpaired chains on the normally fragile red blood cell membrane, leading to cell destruction. This leads to such anemia that the bone marrow tries to compensate by trying to produce more red blood cells. Unfortunately, this process is very inefficient due to the toxicity of unpaired chains, resulting in massive expansion of the medullary space and the spread of hematopoiesis to other parts of the body. This, along with anemia, leads to major toxicity. Several models exist for why unpaired globin chains are so damaging, but most suggest that increased free radicals generated by iron attached to unpaired globin chains are important for early destruction of red blood cells. Therefore, any intervention that reduces oxidative damage from these free radicals could prolong the lifespan of red blood cells, improve anemia, lead to a reduction in the need to produce red blood cells, and reduce damage from the expansion and spread of the medullary spaces.

It is estimated that more than 30,000 children with thalassemia major are born each year, with the majority estimated to live into their 20s in developed countries and in third world countries (where most patients live) Most of them die in young children. Based on current results in other model systems presented here, it is expected that exposing animals with thalassemia to sulfide will increase the ability of their erythrocytes to undergo oxidative damage, resulting in prolonged erythrocyte survival.

2. Sickle cell disease

Normal hemoglobin (HbA) contains two b and two beta globin polypeptide (protein) chains, each bound to an iron-containing heme ring. Sickle cell disease (SCD; also known as sickle cell anemia) is a class of disorders in which a mutated β chain results in changes in hemoglobin (HbS). Once deoxygenated, HbS can polymerize (crystallize) and precipitate, damaging the normally fragile erythrocyte membrane, leading to cellular destruction and anemia, a reduction in red blood cells (RBC). In addition, cells with aggregated HbS change shape (sickle) and become sticky, and activate mechanisms that lead to coagulation and arrest of blood flow. This can lead to hypoxic damage to surrounding tissues, leading to pain, organ dysfunction and ultimately premature death. A decrease in the stores of sulfur-containing antioxidants was observed in patients. Furthermore, oxidative damage and increased reactive oxygen species (ROS) are associated with crystallization, RBC membrane damage and tissue damage associated with insufficient blood flow. Sulfides are associated with 'recharging' antioxidant stores and potentially minimizing oxidative damage. It is reasonable to think that sulfide may prevent problems in several stages of sickle cell pathology. Furthermore, given the ability of an oxygen antagonist to counteract hypoxia in other systems, it is implied that it should also protect animals and humans suffering from the adverse conditions caused by this disease state.

More than 120,000 children are born with SCD each year. Patients in developed countries are now living into their 40s and 50s, yet have enormous problems with pain and organ damage, including stroke, lung, heart and skin problems. In third world countries (where most patients live), most die as young children. Our hypothesis was that exposing animals with SCD, and eventually humans with SCD, to sulphides would lead to improved health.

IV. Deposit application

The present invention can be used to preserve or preserve a variety of biological substances, including cells, tissues, organs, and whole organisms, for transportation and/or storage purposes. In certain embodiments, biological material is preserved to prevent damage from adverse conditions.

In embodiments of the invention, the biological material may be exposed to the active compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24 hours; 1, 2, 3, 4, 5, 6, 7 days; 1, 2, 3, 4, 5 weeks; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years, and any combination or range that may arise therefrom. It is contemplated that active compounds can be used to induce arrest, and other substances can be used to maintain arrest and preserve them for any meaningful period of time. Alternatively, active compounds are contemplated for inducing and/or maintaining arrest. This can be combined with other factors such as environmental changes in pressure and/or temperature.

1. cells

As discussed above, a variety of cells are contemplated for use in the present invention. It is contemplated that the cells may be preserved in the methods, devices and compositions of the invention.

a. Platelets

In certain embodiments, the invention finds application in the preservation of platelets. Platelets are small cell fragments (~1/3 the size of red blood cells) that play a critical role in clot formation at the site of bleeding. Hemostasis is achieved by adhering to the blood vessel wall, releasing clotting chemicals that form a clot to plug the wall and/or the opening in a narrowed blood vessel. A normal platelet count is between 150,000-400,000 counts/μL. Platelet concentrates are infused for different indications such as: 1) to prevent bleeding due to thrombocytopenia; 2) in bleeding patients to maintain a platelet count greater than 50,000; 3) to address platelet dysfunction, the platelet Dysfunction is congenital or due to medication, sepsis, malignancy, tissue trauma, obstetric complications, cardiopulmonary bypass, or organ failure such as liver or kidney disease.

Each unit of platelets contains an average of 0.8-0.85×10 ¹¹ platelets. Platelet concentrate also contains about 60 mL of plasma (clotting factors) and a small amount of red and white blood cells. Platelet units must be maintained at room temperature (20°C-24°C) and shaken during storage. They can be stored at the Blood Center for up to 5 days. Longer-term storage is currently not possible due to deterioration of the platelets, and the risk of microbial contamination. Two sources of platelets currently exist:

1) Pooled Random Donor Platelet Concentrates are prepared from platelets collected by centrifugation of whole blood units. Up to 8 units of platelets (each from a different donor) may be mixed into a bag for transfusion. Platelets expire 4 hours after mixing. All units are from the same ABO type. If ABO-compatible platelets are not available, ABO-incompatible platelets can be substituted with very little risk. The usual adult dose is 4-6 units of pooled random donor platelets.

2) Apheresis platelets, collected from a single donor, are prepared in standard (corresponding to ~4 pooled units) size and "large" (corresponding to ~6 pooled units) size. Apheresis platelet concentrates contain 200-400 mL of plasma. They can be collected as random units (random apheresis platelets) or can be obtained from family members or voluntary HLA-compatible "targeted" donors for specific recipients. Apheresis platelets expire 4 hours after processing and release from the blood bank.

Platelet storage presents issues not found in storage of whole blood or other components. While whole blood, red blood cells, and white blood cells can be stored at 4°C for several weeks, platelets will aggregate and then settle in refrigeration. Therefore, the standard method of storing platelets is at room temperature, about 20-24°C, with gentle agitation. Even under these conditions, platelets can only be stored for 5 days before they need to be discarded. This overdue problem results in approximately $500 million in lost revenue per year for US hospitals. About 90% of this loss could be avoided if a modest extension of storage life were available.

Another problem with platelet storage is bacterial contamination. Contamination was mainly attributed to staphylococci from the skin during phlebotomy, or to donor bacteremia. Bacterial contamination of platelets represents the greatest risk of infection with any blood transfusion procedure.

An important factor affecting the viability of platelets is the regulation of pH. Almost all platelet units stored according to currently accepted methods show a decrease in pH from their initial value of about 7.0. This reduction was primarily attributed to the production of lactic acid by platelet glycolysis and, to a lesser extent, to the accumulation of _CO2 from oxidative phosphorylation. As the pH drops, the platelets change shape, from discs to spheres. If the pH drops below 6.0, irreversible changes in the morphology and physiology of platelets render them nonviable after infusion. Therefore, an important goal of platelet preservation is to prevent this pH drop. It was previously believed that platelets must be stored in oxygen permeable containers because glycolysis is stimulated when oxygen availability is limited (see, eg, US Patent 5,569,579). However, the present invention demonstrates that the viability of stored platelets can be prolonged by storing them in a hypoxic environment.

The present invention provides methods and compositions for increasing the survival time of stored platelets and reducing bacterial contamination. In one embodiment, the invention provides a sealable, oxygen impermeable container into which platelets are placed. After sealing, an anaerobic generator (eg, a sodium borohydride tablet with a palladium catalyst) converts atmospheric oxygen in the container to water. The container also contains an indicator which indicates the level of oxygen tension. Once under hypoxic conditions, platelets can also be stored at lower temperatures.

Platelets can be suspended and stored in plasma or any platelet storage solution known in the art. For example, US Patent Nos. 4,828,976 and 4,447,415 disclose various commonly used solutions suitable for platelet storage.

Typically, platelets are stored in plasma from a donor and administered in that form.

Generally, the present invention consists of a sealed environment (container, tank, impermeable bag or chamber) in which the oxygen tension can be reduced below 1% (10,000ppm) and more particularly in the range of 10-100ppm , or smaller. Atmospheric oxygen reduction in the environment can be achieved by a number of methods known in the art. For example, reducing oxygen in the atmosphere can be achieved by generating hydrogen (with or without a catalyst) that combines with oxygen to produce water. Other reactions can be catalyzed to combine oxygen with other compounds, such as carbon to produce carbon dioxide, and so on. Likewise, oxygen can be replaced by converting all the air in the chamber to a gas containing any combination of gases that does not include oxygen. Additionally, oxygen can be removed by placing the chamber under vacuum and removing all gases. Alternatively, oxygen can be competed by competing with another gas or compound such as CO. A combination of removing oxygen and competing for the remaining oxygen can also be utilized. The device may also contain means to measure oxygen concentration to ensure proper anaerobic conditions are achieved. For example, oxygen concentration can be measured with an anaerobic indicator based on methylene blue, which turns from blue to colorless in the absence of oxygen. Alternatively, an oxygen meter or other device for measuring oxygen may be used.

The device also includes means to contain the platelets in an airtight environment such that oxygen is removed from the solution containing the platelets, as well as from the platelets themselves. An example of this is having platelets in a gas permeable bag that is placed in a closed environment. Platelets may also be maintained in open containers within a closed environment. Alternatively, the platelets can be placed directly in an impermeable, airtight container/bag.

The Bio-Bag ^™ from Becton Dickinson (Product No. 261215) is an example of a sealable, oxygen-impermeable container that can be used to create an oxygen-deficient environment for the storage of platelets. Bio-Bag, which is a kit sold for isolating anaerobic bacteria, includes a sealable, airtight bag, anaerobic indicator, anaerobic generator (hydrogen generator) and palladium catalyst. The platelets in the air-permeable bag will be sealed in the Bio-Bag for storage.

The anaerobic generator in the Bio-Bag is a device activated by the addition of water, which passes through a series of channels to the filter paper wick. The strip retards and regulates the introduction of water into the sheet chamber, providing a controlled release of hydrogen gas. The gas-generating sheet is composed of sodium borohydride. The hydrogen released from this reaction oxidizes with the atmosphere in the closed vessel to produce water. The reaction is catalyzed by palladium in the vessel.

The Puget Sound Blood Center (PSBC) independently assessed the status of platelets stored under anaerobic conditions on days 0, 5, and 8 using a standardized panel of in vitro assays. The results showed that platelets stored under anaerobic conditions up to 8 days performed as well, or better, than platelets stored under standard conditions. Ongoing research is to repeat the experiment and extend the observation period to 13 days.

Those skilled in the art will be familiar with methods for determining platelet function. For example, as described in U.S. Pat. No. 6,790,603, platelet function can be measured by (1) expression of internal proteins on the cell membrane in response to activation-inducing challenge with an agonist; (2) ability to aggregate when challenged by an agonist and (3) secretion of adenosine triphosphate. Examples of agonists that can cause activation of platelet function include thrombin, epinephrine, ADP and collagen.

Expression of internal proteins can be measured by conjugating molecules to fluorescent dyes followed by sorting in a fluorescent cell sorter. In general, it is preferred to use two monoclonal antibodies, one that binds to a constitutively expressed cell surface molecule and one that binds to a cell surface molecule that is only expressed upon activation. Each monoclonal antibody is conjugated to a different colored dye that can be distinguished by spectrofluorometry. A non-limiting example of a constitutively expressed cell surface molecule is GPIIbIIIa; a non-limiting example of a cell surface molecule expressed upon activation is platelet selectin (P-selectin). The production of monoclonal antibodies to proteins is well known in the art. US Patent No. 5,470,738 is an example of a method of producing monoclonal antibodies against GPIIIa. Another anti-platelet monoclonal antibody is a monoclonal antibody directed against GP IV, as disclosed by U.S. Patent No. 5,231,025. Antibodies are also commercially available from companies such as Becton-Dickinson (Philadelphia).

Another parameter of platelet function is the ability to aggregate upon agonist challenge. The platelet suspension is dense and milky white. Aggregation and subsequent settling of aggregates can be assessed visually, or measured with a densitometer.

Yet another measure of platelet function is ATP secretion. Well-functioning platelets are able to secrete ATP, whereas cells that have been activated or otherwise disabled cannot secrete ATP.

2. Cell culture

The invention can be extended to protect cells in culture that might otherwise die or be induced to undergo apoptosis. In the context of the present invention, the cells are exposed to the active compound before and/or during the culture. Cells that can be cultured according to the invention include those cells that can eventually be placed back into a physiological environment, ie, for subsequent transplantation. These cells include, but are not limited to bone marrow, skin cells and epithelial cells. Also, some transplantable cells would benefit greatly from expansion in culture, thereby increasing the amount of material that can be introduced into the host. Epithelial cells from the gastrointestinal tract are particularly contemplated as cells that may benefit from exposure to active compounds.

Furthermore, the invention extends to the culture of tumor cells. Culturing of tumor cells is known to result in phenotypic changes and, in some cases, death. This makes tissue culture assays of tumor cells very unpredictable.

General cell culture techniques are well known to those skilled in the art. Examples of this knowledge can be found in Shaw (1996) and Davis (1994), both of which are incorporated herein by reference. General information and modifications to conventional cell culture techniques can also be found in US Patent No. 5,580,781, which is incorporated by reference. Additionally, techniques for culturing skin cells are described in US Patent 6,057,148, which is incorporated by reference. It is contemplated that these techniques, as well as others known to those skilled in the art, will be supplemented with media containing one or more active compounds, or perfused with active compounds in fluid and/or gaseous form.

E. Preservation of Cells, Tissues and Organs

In certain embodiments of the invention, it is desirable to preserve biological material so as to prevent damage to the material by death or decomposition as much as possible. Although the first successful kidney transplant was performed in 1954 and the first heart and liver transplant was performed in 1967, thousands of people who need organ transplants die each year. For various reasons, they need hearts, lungs, kidneys and livers. In addition, there are patients for whom pancreas or cornea can be used. While there is a continuing need for organ donors, another significant obstacle to providing organs to those patients in need of organ transplantation is the limitations of current organ preservation techniques. For example, it is generally accepted that a human heart must be shipped within 4 hours for a subsequent transplant to have any chance of being successful. Rager, 2004 (see table below).

The longest cold ischemia time

organ Storage time heart and lungs liver kidneys pancreas small intestine 4-6 hours 12-24 hours 48-72 hours 12-24 hours 12 hours

Furthermore, within the first 30 days, the main cause of organ graft failure in the transplanted heart was ischemia-reperfusion injury.

Organ procurement and preservation, tissue matching, and immunosuppression are major factors in successful solid organ transplantation. The technical aspects of organ procurement operations allow multiple teams to work together to obtain all useful organs from a single donor. On average, 3.6 organs were obtained from a single deceased donor.

Preservation of solid organs depends on rapid intravascular cooling performed in situ, followed by excision, preservation in ice-cold preservation fluid, and rapid transport to the recipient's hospital. Cold ischemia time is the length of time an organ is on ice without blood flow. The longest cold ischemia time limited the amount of time that could pass between organ recovery and organ transplantation (Table 5). 2%-10% of matched and harvested organs are unusable due to prolonged ischemic time, depending on the type of organ. Similarly, approximately 10-20% of harvested organs are unusable due to poor organ function and/or infection (excluding HIV/CMV/hepatitis).

Current preservation techniques involve the use of ice-cold solutions that include electrolytes, antioxidants, hydrogen ion buffers, and sugars. Punch et al., 2001. Appropriate tissue matching depends on blood group matching of all organs (eg, blood group A, B, or O). Immunosuppressive regimens typically include three drugs: a glucocorticoid such as prednisone, an antimetabolite such as azathioprine or mycophenolate mofetil, and a calcineurin inhibitor such as cyclosporine or tacrolimus.

The two most frequently used methods for preserving/shipping hearts for transplantation are cryopreservation and continuous perfusion. In the former method, the heart is stopped, removed from the donor, then rapidly cooled and shipped refrigerated. In the latter approach, the following steps are generally employed: 1) pulsatile flow; 2) hypothermia; 3) membrane oxygenation, and 4) perfusate containing both.

To improve the prospects for successful transplantation, techniques for better preservation of organs for transplantation have been developed. Two general areas of development have emerged, one in the area of preservation solutions and the other in the area of organ containers.

In certain aspects, such as transplantation, adverse outcomes in wound healing can impair or prevent normal engraftment of transplanted tissue. In the context of the present invention, it is contemplated that the donor tissue and the recipient tissue will be treated with oxygen antagonists or other active compounds prior to transplantation, as discussed above in relation to wound healing, aimed at inhibiting biological processes such as inflammation, apoptosis and Other wound healing/post-transplant events that compromise grafted tissue.

F. biology

Such organisms can be used for research purposes, such as testing mice (mouse banking), or for consumption, such as fish. In these cases, consider a standstill that can be maintained indefinitely. Furthermore, stagnation can be induced in plants or parts of plants, including fruits, flowers, leaves, stems, seeds, cuttings. Plants may be agricultural crops, medicinal plants or ornamental plants. Inducing stagnation in plants can increase shelf life or pathogen resistance of whole plants or plant parts. Thus, in an embodiment of the invention, the organism or part thereof is exposed to an oxygen antagonist or other active compound for about, at least about, or at most about 30 seconds; 1, 2, 3, 4, 5, 10, 15, 20 , 25, 30, 35, 40, 45, 50, 55 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours; 1, 2, 3, 4, 5, 6, 7 days; 1, 2, 3, 4, 5 weeks; 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12 months; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more years, and any combination or range derivable therein.

G. Preservative

A variety of preservation solutions have been disclosed, with which the organ is surrounded or perfused during transport. One of the most commonly used solutions is ViaSpan® (Belzer UW), which is used with refrigeration. Other examples of such solutions or components of such solutions include St. Thomas solution (Ledingham et al., J. Thorac. Cardiobasc. Surg. 93:240-246, 1987), Broussais solution, UW solution (Ledingham et al., Circulation 82 (Part 2) IV351-8, 1990), Celsior solution (Menasche etc., Eur.J.Cardio.Thorax.Surg.8:207-213, 1994), Stanford University solution and B20 solution (Bernard etc., J.Thorac.Cardiovasc. Surg.90：235-242，1985)，以及在美国专利6,524,785、6,492,103、6,365,338、6,054,261、5,719,174、5,693,462、5,599,659、5,552,267、5,405,742、5,370,989、5,066,578、4,938,961和4,798,824中描述和/或要求保护的那些。

Besides solutions, other types of materials are known for transporting organs and tissues. These include gel-like or other semi-solid materials such as those described in US Patent 5,736,397.

Some systems and solutions for organ preservation specifically involve perfusing the solution or system with oxygen to expose the organ to oxygen, since maintaining an organ or tissue in an oxygenated environment is believed to enhance viability. See Kuroda et al., (Transplantation 46(3):457-460, 1988) and US Patents 6,490,880, 6,046,046, 5,476,763, 5,285,657, 3,995,444, 3,881,990, and 3,777,507. Isolated hearts deprived of oxygen for longer than 4 hours are considered nonviable and unusable in recipients because of ischemia/reperfusion injury. See US Patent 6,054,261.

Also, most, if not all, of the solutions and containers used for organ preservation and transplantation involve cryogenic temperatures (temperatures below room temperature, usually near but not below 0°C), which have been termed "all useful organs and tissues cornerstone of preservation methods". US Patent 6,492,103.

To improve the prospects for successful transplantation, techniques for better preservation of organs for transplantation have been developed. Two general areas of development have emerged, one in the area of preservation solutions and the other in the area of organ containers.

Also, most, if not all, of the solutions and containers used for organ preservation and transplantation involve cryogenic temperatures (temperatures below room temperature, usually near but not below 0°C), which have been termed "all useful organs and tissues cornerstone of preservation methods". US Patent 6,492,103.

In the field of organ transplantation, certain conditions are believed to correlate with the state of the organ and the prognosis for successful transplantation: 1) Minimizing cellular swelling and edema; 2) Preventing intracellular acidosis; 3) Minimizing ischemic damage and 4) providing a substrate for regeneration of high energy phosphate compounds and ATP during reperfusion. Ischemia/reperfusion injury in organ transplantation is particularly problematic as the harvested organ is removed from the body, separated from its blood source, and thus deprived of oxygen and nutrients over a prolonged period (US Patent 5,912,019). Indeed, one of the most critical problems in transplantation today is the relatively high incidence of delayed graft function (DGF), which is attributed to acute tubular necrosis after surgery. Current methods still experience difficulties in these areas, which highlights the importance of the present invention.

However, the present invention can be used in conjunction with other preservation compositions and methods. As discussed in US Patents 5,952,168, 5,217,860, 4,559,258, and 6,187,529 (which are expressly incorporated by reference), biological material can be preserved, for example, for long-term preservation of transplantable or replaceable organs.

Compounds may be administered to cells, tissues/organs, or cadavers to enhance or maintain the condition of the organ for transplantation. Such methods and compositions include those described in US Patent Nos. 5,752,929 and 5,395,314.

Furthermore, the methods of the invention may include exposing the biological material to a preservation solution, such as those discussed, in addition to exposure to an oxygen antagonist or other active compound.

It is considered that any agent or solution used in biological samples that are living and to be used as living material must be pharmaceutically acceptable or pharmacologically acceptable. The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a human. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to use can also be prepared.

Organs for transplantation can be monitored to assess their status, especially with respect to use as a graft. This method is described in US Patent 5,699,793.

Numerous drugs may be administered to patients after receiving an organ transplant to facilitate the recovery process. These drugs include compounds and agents that reduce or suppress the immune response against the donated organ.

In addition, additional drugs are continuously being investigated and used in organ transplantation, for example in US Patents 6,552,083 (inhibitors containing N-(3,4-dimethoxycinnamoyl)anthranilic acid) and 6,013,256 (binding IL -2 receptor antibodies, such as those described in Humanized Anti-Tax Antibodies).

H. Depositing Instruments and Applications

Systems or containers for transporting organs and tissues have also developed through the years. Any of these embodiments may be combined with the apparatus of the invention which allows the use of oxygen antagonists or other active compounds.

Most involve cooling systems for achieving cooling such as those described in US Patents 4,292,817, 4,473,637 and 4,745,759, which employ active refrigeration with a cooling liquid that is pumped through the system. Several complex devices have been designed containing multiple chambers or dual vessels, such as those described in US Patents 5,434,045 and 4,723,974.

Some constitute systems in which an instrument is designed to perfuse an organ or tissue in a preservation solution, as described in US Patents 6,490,880, 6,100,082, 6,046,046, 5,326,706, 5,285,657, 5,157,930, 4,951,482, 4,502,295, and 4,186,565.

Some systems and solutions for organ preservation specifically involve perfusing the solution or system with oxygen to expose the organ to oxygen, since maintaining an organ or tissue in an oxygenated environment is believed to enhance viability. See Kuroda et al., (Transplantation 46(3):457-460, 1988) and US Patents 6,490,880, 6,046,046, 5,476,763, 5,285,657, 3,995,444, 3,881,990, and 3,777,507. Isolated hearts deprived of oxygen for longer than 4 hours are considered nonviable and unusable in recipients because of ischemia/reperfusion injury. See US Patent 6,054,261.

Also, in some embodiments of the invention, there are methods for preserving platelets, as mentioned above. Using the techniques of the present disclosure, the disadvantages of the prior art are reduced or eliminated. Embodiments involving reduction of platelets and oxygen have wide application including, but not limited to, any application that would benefit from longer term preservation of platelets.

In one embodiment, the oxygen reduction technology can be embodied in a kit. For example, a kit currently sold under Product No. 261215 provided by Becton Dickinson utilizes the selection technique described herein. The kit includes an anaerobic generator (such as a hydrogen generator), a palladium catalyst, an anaerobic indicator, and a gas-tight, sealable "BioBag" in which the above components (along with the platelets in the gas-permeable bag) are placed and seal.

The anaerobic generator in this exemplary kit is activated by the addition of water, which passes through a series of channels to the filter paper strip. The strip retards and regulates the introduction of water into the sheet chamber, providing a controlled release of hydrogen gas. The gas-generating tablet includes sodium borohydride. The hydrogen released from this reaction oxidizes with the atmosphere in the closed vessel to produce water. The reaction is catalyzed by palladium in the vessel.

In a more general aspect, the techniques of this disclosure can be practiced with any number of sealed environments (eg, containers such as tanks, impermeable bags, or chambers) in which oxygen tension can be reduced. In one embodiment, the oxygen level in the container and/or the platelets or associated solution may be reduced to less than about 1% (about 10,000 parts per million). In another embodiment, oxygen may be reduced to a range of about 10-100 parts per million, or less. In yet another embodiment, oxygen may be reduced to any percentage value representing a reduction in oxygen in the container and/or platelets or related solution. In preferred embodiments, the container is airtight and sealable. Those of ordinary skill in the art will appreciate that "breathable" does not necessarily imply an absolute or 100% level of impermeability. Conversely, "airtight" should be interpreted as it is meant in the art, eg capable of maintaining an atmosphere of less than 10 ppm (compared to the gradient of room air, typically 210,000 ppm) for at least 4 days. Typically, commercially available bags are impermeable for 6 weeks or longer.

The container may be sealed with the associated oxygen reducing element placed therein. Atmospheric oxygen reduction in this environment can be achieved by generating hydrogen (with or without a catalyst) and combining oxygen to produce water. Other reactions can be catalyzed to combine oxygen with other compounds, such as carbon, to produce carbon dioxide. Other reactions and combinations will be apparent to those of ordinary skill in the art. Likewise, oxygen can be replaced by switching the gas in the chamber to a gas containing any combination of gases that does not include oxygen. Additionally, oxygen can be removed by placing the container under a vacuum sufficient to remove the gas, and in particular oxygen, to a desired, reduced level. Alternatively, oxygen can be competed by competing with another gas or compound such as CO. A combination of removing oxygen and competing for the remaining oxygen can be used.

In various embodiments, a device may be used to measure oxygen levels to ensure that proper anaerobic conditions have been achieved. Anaerobic indicators based on methylene blue, which changes from blue to colorless in the absence of oxygen, can be used. Alternatively, commercially available oxygen meters (eg, mechanical and/or electronic meters) or other mechanisms for measuring oxygen may be used.

In various embodiments, the platelets are contained in a sealed environment, which removes oxygen from the solution containing the platelets, as well as from the platelets themselves. For example, platelets in a gas-permeable bag can be placed in a sealed environment. Other non-limiting examples may have an open container containing platelets in a sealed environment. Alternatively, platelets can be contained in an impermeable, sealed container (eg, bag) with integrated oxygen scavenging means.

In one embodiment, the invention relates to a method wherein platelets and a solution are introduced into a gas-impermeable container. The container is airtight. Oxygen is removed from the container or from the platelets and solution. Consider placing about, at least about, or at most about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37 in a breathable bag , 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62 , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87 , 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, or any range derivable here, for oxygen removal.

The method may also include indicating the level of oxygen remaining in the container after the oxygen is removed. The oxygen in the container can be reduced to about 10,000 parts per million or less. The oxygen in the container can be reduced to a level between about 10-100 parts per million. Introducing platelets may involve adding a gas-permeable container containing platelets and solution into a gas-impermeable container. Introducing the platelets may involve placing the platelets and solution into a sealable, flexible bag, or a sealable, rigid chamber. Sealing the container may involve the use of an adhesive, and sealing the container may occur at any stage of a given process.

Removing oxygen may involve pumping oxygen out of the container, and such pumping may involve pumping with a roughing pump and/or a turbo pump. Removing oxygen may involve introducing hydrogen gas into the vessel, which combines with oxygen to produce water. Hydrogen can be introduced through chemical reactions. Chemical reactions can be catalyzed. Removing oxygen may involve introducing hydrogen gas into the container using a gas generating chip. Hydrogen gas can be generated by adding water to a gas generating tablet containing sodium borohydride. This water can be added in a delayed and regulated manner. For example, filter paper strips can be used. Water may be introduced to the filter paper strip through one or more channels. Palladium catalyzes the chemical reaction that produces hydrogen gas. Removing oxygen may involve introducing into the vessel one or more reagents that bind oxygen. CO may be introduced into the vessel, where it bonds with oxygen to form _CO2 . Removing oxygen may involve replacing oxygen with one or more gases.

Charting the level of remaining oxygen may involve the use of a methylene blue indicator that changes color in the absence of oxygen. Alternatively, an oxygen meter can be used. Indicating the level of oxygen remaining in the container may involve displaying the level of oxygen remaining in the platelets or solution.

In one embodiment, the invention relates to a method wherein platelets and a solution are introduced into a gas-impermeable container. The container is airtight. Hydrogen gas is produced by the chemical reaction of adding water to sodium borohydride. This chemical reaction removes oxygen from the platelets and solution by combining with hydrogen to form water. Indicates the level of oxygen remaining in the vessel after deoxygenation.

This chemical reaction can be catalyzed by palladium. Addition of water may involve the use of filter paper cartridges.

In one embodiment, the invention relates to a system for removing oxygen from platelets and a solution. The system includes: (a) a sealable, gas-tight container, (b) a generator of reduced oxygen, and (c) an oxygen indicator. The sealable, airtight container is configured and sized to receive platelets and solution. A generator of reducing oxygen is attached to the container and is designed to remove oxygen from the platelets and solution by suction or chemical reaction. An oxygen indicator is attached to the vessel and is designed to indicate the oxygen level in the vessel after deoxygenation.

The container can be a sealable, flexible bag. The generator for reducing oxygen may include a hydrogen generator designed to generate hydrogen for combination with oxygen to produce water. A hydrogen generator may include a gas generating substance that, when combined with a reagent, produces hydrogen gas. Such gas-generating substances may include sodium borohydride flakes, and the reagent may include water. The hydrogen generator may also include a palladium catalyst. The system may also include components designed to delay or regulate a chemical reaction by controlling the introduction of one or more components of the chemical reaction. For example, the member may include a core that retards and regulates chemical reactions.

In one embodiment, the invention is directed to a kit comprising a hydrogen generator, a gas-tight sealable container, and an oxygen indicator.

Hydrogen generators may include gas generating substances that generate hydrogen gas when combined with an agent. Such gas-generating substances may include sodium borohydride flakes, and the reagent may include water. The kit may also contain a palladium catalyst. The kit may also contain a wick designed to retard or modulate a chemical reaction that produces hydrogen gas.

As discussed above, the methods of the invention may involve the use of an apparatus or system that maintains an environment in which biological matter is placed or exposed. The invention includes apparatus in which the active compound is provided, especially as a gas. In some embodiments, the apparatus includes a container having a sample chamber for containing biological material, wherein the container is connected to a supply of a gas containing the active compound. It is particularly contemplated that the container may be a solid container or it may be flexible, such as a bag.

In some embodiments, the invention is an apparatus for preserving cells, the apparatus comprising: a container having a sample chamber having a capacity of no more than 775 liters, and a first gas supply in fluid communication with the sample chamber, the first One gas supply source includes carbon monoxide. In other embodiments, the apparatus further comprises a cooling device for regulating the temperature of the sample chamber and/or a gas regulator which regulates the amount of active compound in the chamber or regulates the amount of active compound in solution within the chamber.

It is contemplated that there may be a second or additional gas supply for the gas, or a second or additional gas supply for the active compound. A second gas supply may be connected to the sample chamber, or it may be connected to the first gas supply. The additional gas (discussed above) may be a non-toxic and/or non-reactive gas.

In some embodiments of the invention, a gas regulator is part of the apparatus. One, two, three or more gas regulators may be used. In some cases, the gas regulator regulates the gas supplied to the sample chamber by the first gas supply. Alternatively, it regulates the gas supplied to the sample chamber or the first gas supply by the second gas supply, or there may be regulators for both the first and second gas supplies. As a further contemplation, any gas regulator may be programmed to control the amount of gas supplied to the sample chamber and/or another gas supply. Conditioning may or may not occur for a specified period of time. There may be a gas regulator, which may or may not be programmable for any gas supply connected directly or indirectly to the sample. In some cases, the gas regulator is electronically programmable.

In some cases, the pressure and/or temperature within the chamber may be adjusted with a pressure regulator or temperature regulator, respectively. Like gas regulators, these regulators can be electronically programmable. The apparatus of the present invention also has cooling and/or heating means to achieve the temperatures discussed above. The device may or may not be electronically programmable.

In other embodiments, the apparatus includes a wheeled cart on which the container rests, or it may have one or more handles.

It is particularly contemplated that the present invention includes an apparatus for cells, wherein the apparatus has: a container having a sample chamber; a first gas supply in fluid communication with the sample chamber, the first gas supply comprising an active compound; and an electronically programmable A gas regulator, which regulates the gas supplied to the sample chamber by the first gas supply source.

In some embodiments, the instrument also has structures configured to provide a vacuum within the sample chamber.

Furthermore, any of the oxygen antagonists described in this application are contemplated for use with the apparatus of the present invention. In a specific embodiment, carbon monoxide can be administered with the device. In other cases, chalcogenide compounds or compounds having a reducing agent structure may be applied.

Figure 19 is a schematic diagram of an exemplary system for removing oxygen from platelets and solution and embodies the concepts discussed above. The air-permeable bag 1920 can be placed in a sealable air-impermeable container 1904 . The gas impermeable container 1904 may be connected to a generator 1906 of reduced oxygen. In one embodiment, the generator of reduced oxygen 1906 may surround the sealable gas-tight container 1904 . In different embodiments, the generator of reduced oxygen 1906 can take different forms. For example, it may be a pump (eg roughing pump and/or turbo pump) or a hydrogen generator. Associated with the generator of reduced oxygen 1906 may be one or more elements such as a wick or other retardation mechanism. Connected to the sealable gas-tight container 1904 is a sensor 1908 and a regulator 1910 . In one embodiment, sensor 1908 may be an oxygen meter, which may take a variety of forms. In other embodiments, sensor 1908 may be a temperature or pressure gauge. Of course, more than one sensor can be used. In one embodiment, regulator 1901 may be a temperature or pressure regulator. For example, regulator 1901 may be a heating or cooling device for regulating the temperature within sealable airtight container 1904 .

v.Diagnostic application

Sulfite is produced by all cells in the body during the normal metabolism of sulfur-containing amino acids. Sulfite oxidase removes, and thus regulates, sulfite levels. Different activities of these enzymes will result in different levels of sulfite being released in a tissue-specific manner. In the example described above, for solid tumors under hypoxic conditions, sulfite can be produced at higher levels to provide a local protective state for tumor cells by reducing the metabolic state and suppressing immune surveillance. Therefore, it would be advantageous to measure sulfite levels and incorporate them as part of the diagnosis of several disease states such as solid tumors. Also, since we recommend the use of sulfites for a variety of applications, it would be useful to follow this recommendation with some type of imaging or other monitoring method.

It is possible to measure sulfite levels in serum to obtain total sulfite levels using current techniques such as HPLC. It is worth investigating the possibility of imaging sulfites. Alternatively, proteomic approaches may enable an understanding of how the regulation of enzymes involved in sulfite metabolism may be altered in certain disease states, enabling the method to be used in diagnosis.

VI. Screening applications

In still further embodiments, the present invention provides methods for identifying oxygen antagonists and molecules that act in a manner similar to the induction of arrest, as well as other active compounds. In some cases, the oxygen antagonist or active compound sought acts like a chalcogenide compound in reducing core body temperature or preserving viability in a hypoxic or hypoxic environment, if the oxygen antagonist or other active compound is not present, The low or anoxic environment would then kill the biological material. These assays may involve random screening of large libraries of candidate substances; alternatively, the assay may be used to focus on particular classes of compounds selected with an eye toward properties thought to make them more likely to act as oxygen antagonists. agent or active compound to provide a candidate active compound;

(a) mixing the candidate active compound with a biological substance;

(b) measuring one or more cellular responses characteristic of oxygen antagonist treatment; and

(c) comparing said one or more responses to the response of the biological material in the absence of the candidate active compound.

Assays can be performed with isolated cells, tissues/organs, or whole organisms.

It will of course be understood that all screening methods of the invention are useful in themselves, despite the fact that no effective candidate may be found. The present invention provides methods for screening such candidates, not just methods for finding them. However, it is also understood that a candidate active compound may be identified as an effective active compound according to one or more assays, meaning that the candidate active compound appears to have some ability to act as an active compound, for example by inducing stagnation in biological matter. In some embodiments, screening comprises identifying modulators using the Examples in this disclosure or assays described elsewhere. Furthermore, in addition to or instead of the methods described in this section, the candidate active compound can be tested as an oxygen antagonist or as another compound with properties of the active compound (e.g. protective metabolizer or therapeutic substance) activity. Some embodiments of screening methods are provided above.

Potential active compounds can be further characterized or determined. In addition, the effective active compounds may be used in vivo in animals or animal models (as discussed below), or may be used in other in vivo animals or animal models, which may involve animals of the same species or different animal species.

Furthermore, it is contemplated that active compounds identified according to embodiments of the present invention may also be produced after screening. Likewise, according to the methods of the present invention, especially with regard to therapeutic or prophylactic embodiments, biological substances may be exposed or contacted with effective active compounds.

A. Active compounds

As used herein, the term "candidate active compound" refers to any molecule that can induce stasis in biological matter by, for example, altering core body temperature. Candidate active compounds can be proteins or fragments thereof, small molecules, or even nucleic acid molecules. Candidate active compounds can also be obtained from various commercial sources, libraries of small molecules considered to meet essential criteria for useful drugs, in an effort to "brute force" identify useful compounds. Screening of these libraries, including combinatorially generated libraries (eg, peptide libraries), is a rapid and efficient method of screening large numbers of related (and unrelated) compounds for activity. The combinatorial approach itself also facilitates the rapid development of potential drugs by generating modeled second, third and fourth generation compounds of active, yet otherwise undesirable compounds.

Candidate active compounds may include fragments or portions of naturally occurring compounds, or are found in active combinations of known compounds that are otherwise inactive. It has been suggested that compounds isolated from natural sources such as animals, bacteria, fungi, plant sources (including leaves and bark) and marine samples could be tested as candidates to detect the presence of potentially useful medicinal substances. It should be understood that the pharmaceutical substances to be screened may also be derived from or synthesized from chemical constituents or man-made compounds. Therefore, it should be understood that the candidate active compounds identified by the present invention may be peptides, polypeptides, polynucleotides, small molecule inhibitors or any other compounds designed by rational drug design starting from known inhibitors or stimulators.

Other suitable active compounds include antisense molecules, siRNA, ribozymes, and antibodies (including single chain antibodies), each of which is specific for the target molecule. Such compounds are described in more detail elsewhere in this document. For example, antisense molecules that bind to translation initiation sites or transcription initiation sites, or splice junctions would be ideal candidate inhibitors.

In addition to the initially identified active compound, the inventors contemplated preparing other structurally similar compounds to mimic key portions of the active compound's structure. Such compounds, which may include peptidomimetics of peptide modulators, may be used in the same manner as the starting active compounds.

B. In vivo assays

In vivo assays involve the use of various animal models. Mice are a preferred embodiment due to their size, ease of handling, and information about their physiological and genetic makeup. However, other animals are also suitable, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, mice, cats, dogs, sheep, goats, pigs, cows, horses, and monkeys (including chimpanzees, gibbons, and baboons) . Fish can also be considered for in vivo assays, as can nematodes. Assays for modulators can be performed using animal models derived from any of these species.

In such assays, one _or more candidate substances that induce arrest, lower core body temperature, or confer The ability to identify regulators of the ability of biological matter to survive under low or anoxic environmental conditions. Treatment of an animal with a test compound will involve administering the compound, in an appropriate form, to the animal. Administration of a candidate compound (gas or liquid) may be by any route available for clinical or non-clinical purposes including, but not limited to, oral, nasal (inhalation or aerosol), buccal or even topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, or intravenous injection. Particularly contemplated routes are systemic intravenous injection, topical administration via the blood or lymphatic supply, or direct administration to the affected site.

VII. Modes of Administration and Pharmaceutical Compositions

An effective amount of a chalcogenide, oxygen antagonist, or pharmaceutical composition of an active compound is generally defined as an amount sufficient to detectably ameliorate, reduce, minimize, or limit the extent of the condition of interest. A more stringent definition could be used, including elimination, eradication or cure of disease.

A. Application

Naturally, the route of administration of the chalcogenide or other active compound will vary with the site and nature of the condition to be treated, and includes, for example, inhalation, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, Subcutaneous, transdermal, intratracheal, intraperitoneal, intratumoral, infusion, lavage, direct injection, and oral administration and formulation. As detailed below, the active compound can be administered as a medical gas by inhalation or intubation, as a liquid for injection by intravascular, intravenous, intraarterial, intracerobroventicular, intraperitoneal, subcutaneous, as a topical Liquid or gel, or solid oral dosage forms are administered.

Furthermore, the amount may vary according to the type of biological material (cell type, tissue type, genus and species of organism, etc.) and/or its size (body weight, surface area, etc.). Usually the larger the organism, the higher the dose. Thus, an effective amount for mice will generally be less than for rats, which will generally be less than for dogs, which will generally be less than for dogs, which will generally be less than for dogs. effective amount for humans. The effective concentration of hydrogen sulfide to achieve stagnation in humans depends on the dosage form and route of administration. For inhalation, in some embodiments, effective concentrations range from 50 ppm to 500 ppm for continuous delivery. For intravenous administration, in some embodiments, effective concentrations range from 0.5 to 50 milligrams per kilogram of body weight delivered continuously.

Similarly, the length of time for administration may vary according to the type of biological material (cell type, tissue type, genus and species of organism, etc.) and/or its size (body weight, surface area, etc.), and will depend in part on the dosage form and route of administration . In particular embodiments, the active compound is provided for about or at least 30 seconds, 1 minute, 2 minutes, 3 minutes, 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 8 hours, 12 hours, 24 hours or more than 24 hours. The active compound may be administered in a single dose or in multiple doses, with varying amounts of time between doses administered.

In the case of transplantation, the present invention can be used pre- and or post-operatively to render the host or graft material dormant. In specific embodiments, a surgical site may be injected or infused with a chalcogenide-containing formulation. The perfusion can be continued postoperatively, such as by having a catheter implanted at the surgical site.

B. Injectable Compositions and Formulations

Preferred methods for delivering the oxygen antagonists or other active compounds of the invention are inhalation, intravenous injection, infusion of specific areas, and oral administration. However, the pharmaceutical compositions disclosed herein may alternatively be administered parenterally, intradermally, intramuscularly as described in US Patent 5,543,158, US Patent 5,641,515, and US Patent 5,399,363 (each of which is expressly incorporated herein by reference in its entirety). , transdermally or even intraperitoneally.

Solutions of the active compounds can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical forms suitable for injectable administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion (US Patent No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and/or vegetable oil. Proper fluidity can be maintained, for example, by coatings such as lecithin, by maintaining the required particle size in the case of dispersions, and by using surfactants. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, intratumoral and intraperitoneal administration. In this regard, the sterile aqueous media that may be used will be known to those skilled in the art in light of the present disclosure. For example, one dose can be dissolved in 1 ml of isotonic NaCl solution and added to 1000 ml of subcutaneous infusion fluid or injected at the suggested infusion site (see, e.g., "Remington's Pharmaceutical Sciences" 15th ed., pp. 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dosage for an individual subject. Furthermore, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA's Office of Biologics standards.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in an appropriate solvent with various other ingredients enumerated above as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying techniques which yield the active substance plus any additional Powder of desired ingredients.

As used herein, "carrier" includes any and all solvents, dispersion media, excipients, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, Suspensions, colloids, etc. The use of such media and agents for pharmaceutically active substances is well known in the art. Their use in therapeutic compositions is contemplated in addition to any conventional media or formulations incompatible with the active ingredient. Supplementary active ingredients can also be incorporated into the compositions.

The phrase "pharmaceutically acceptable" or "pharmacologically acceptable" refers to molecular entities and compositions that do not produce an allergic or similar adverse reaction when administered to a human. The preparation of aqueous compositions containing proteins as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.

C. Intravenous preparations

In one embodiment, the active compounds of the invention may be formulated for parenteral administration (eg, intravenous, intraarterial). In cases where the active compound is a gas at room temperature, solutions containing known and desired concentrations of gas molecules dissolved in a liquid or solution for parenteral administration are contemplated. Solutions of the active compound can be prepared, for example, by contacting (eg, bubbling or injecting) a gas into the solution so that the gas molecules dissolve in the solution. Those skilled in the art will recognize that the amount of gas dissolved in a solution will depend on many variables including, but not limited to, the solubility of the gas in the liquid or solution, the chemical composition of the liquid or solution, its temperature , its pH, its ionic strength, and the gas concentration and exposure (eg, rate and duration of sparging or infusion). The concentration of active compounds in liquids or solutions for parenteral administration can be determined by methods known to those skilled in the art. The stability of an active compound in a liquid or solution can be determined by measuring the concentration of the dissolved oxygen antagonist after various time intervals after preparation or production of the oxygen antagonist solution, wherein the concentration of the oxygen antagonist is compared with the initial concentration. Decrease indicates loss or chemical conversion of active compound.

In some embodiments, a solution containing a chalcogenide compound is produced by dissolving the chalcogenide in its salt form in sterile water or saline (0.9% sodium chloride) to produce a pharmaceutically acceptable intravenous dosage form. Intravenous liquid dosage forms can be buffered to achieve a pH that enhances the solubility of the chalcogenide compound or affects the ionization state of the chalcogenide compound. In the case of hydrogen sulfide or hydrogen selenide, any of a number of salt forms known to those skilled in the art may suffice, including but not limited to sodium, calcium, barium, lithium or potassium. In another preferred embodiment, hydrogen sulfide or hydrogen selenide is dissolved in sterile phosphate-buffered saline and adjusted to pH 7.0 with hydrochloric acid to obtain a known concentration of hydrogen sulfide that can be administered intravenously or intraarterially to a subject. Solution.

It is contemplated that in some embodiments, the pharmaceutical compositions of the present invention are saturated solutions of the active compound. The solution may be any pharmaceutically acceptable formulation, most of which are well known, eg Ringer's solution. In particular embodiments, the concentration of the active compound is about, at least about or at most about 0.001, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0M or larger, may originate from any of these ranges ( at standard temperature and pressure (STP)). For _H2S , for example, in some embodiments the concentration may be from about 0.01 to about 0.5M (at STP). In particular, it is contemplated that the above-mentioned concentrations apply to carbon monoxide and carbon dioxide, either independently or together in solution.

In addition, the following parameters are contemplated for use when administration is intravenous. Flow rate about, at least about or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 gtts/minute or μgtts/minute, or any range derivable therein. In some embodiments, the amount of solution is specified by volume (depending on the concentration of the solution). The amount of time can be about, at least about or at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 minutes; 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours; 1, 2, 3, 4, 5, 6, 7 days; 1, 2 , 3, 4, 5 weeks and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or any range that may be derived therefrom.

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000 ml or liters, or any range thereof in volume may be administered collectively or over a single period of time Administered internally.

In some embodiments, solutions of active compounds for parenteral administration are prepared in a liquid or solution from which oxygen has been removed prior to contacting the liquid or solution with the active compound. Certain oxygen antagonists, especially certain chalcogenide compounds (eg, hydrogen sulfide, hydrogen selenide), are unstable in the presence of oxygen due to their ability to chemically react with oxygen, resulting in their oxidation and chemical transformation. Oxygen can be removed from a liquid or solution using methods known in the art including, but not limited to, applying negative pressure (vacuum degassing) to the liquid or solution, or subjecting the solution or liquid to oxygen-causing or Contact "chelates" the reagent, effectively removing it from solution.

In another embodiment, solutions of oxygen antagonists or other active compounds for parenteral administration can be stored in air-tight containers. This is especially desirable when oxygen has been previously removed from the solution to limit or prevent oxidation of the oxygen antagonist or other active compound. In addition, storage in an airtight container will inhibit volatilization of oxygen antagonist gas or other active compounds from the liquid or solution so that a constant concentration of dissolved oxygen antagonist is maintained. Air-tight containers are known to those skilled in the art and include, but are not limited to, "intravenous bags" containing an air-impermeable construction material, or sealed glass vials. To prevent exposure to air in airtight storage containers, an inert gas, such as nitrogen or argon, can be introduced into the container prior to sealing.

D. Topical formulations and their use

The methods and compositions of the present invention are useful for inducing arrest in the superficial layers of the skin and oral mucosa including, but not limited to, the hair follicle cells, capillary epithelium, and epithelial cells of the mouth and tongue. . Radiation therapy and chemotherapy used to treat cancer can damage hair follicles and normal cells in the oral mucosa, leading to undesirable, debilitating side effects of cancer treatment, hair loss and oral mucositis, respectively. Inducing arrest in hair follicle cells and/or vascular cells that supply blood to the hair follicle slows, limits or prevents damage to hair follicle cells and resulting hair loss that accompanies radiation therapy and chemotherapy, or other alopecia disorders, male pattern baldness , female pattern hair loss, or other loss of hair from the areas of the skin where it normally resides. Inducing arrest in oral epithelial and mesenchymal cells slows, limits or prevents damage to cells lining the mouth, esophagus and tongue, and the resulting painful condition of oral mucositis.

In certain embodiments, the active compounds are administered topically. This is accomplished by formulating the active compound as a cream, gel, paste or mouthwash and applying the formulation directly to the area to be exposed to the active compound (eg, scalp, mouth, tongue, throat).

The topical compositions of the present invention can be formulated as oils, creams, lotions, ointments and the like by choosing appropriate carriers. Suitable carriers include vegetable or mineral oil, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohols (greater than _C12 ). Preferred carriers are those in which the active ingredient can dissolve. Emulsifiers, stabilizers, humectants and antioxidants may also be included, as well as color or fragrance imparting agents, if desired. Additionally, transdermal penetration enhancers can be used in these topical formulations. Examples of such enhancers can be found in US Patent Nos. 3,989,816 and 4,444,762.

Emulsions are preferably formulated with a mixture of mineral oil, self-emulsifying beeswax and water into which is incorporated the active ingredient dissolved in a small amount of oil, such as almond oil. A typical example of such an emulsion is an emulsion containing about 40 parts water, about 20 parts beeswax, about 40 parts mineral oil and about one part almond oil.

An ointment can be formulated by mixing a solution of the active ingredient in a vegetable oil, such as almond oil, with mild soft paraffin, and allowing the mixture to cool. A typical example of such an ointment is one containing about 30% almond oil and about 70% white soft paraffin by weight.

Lotions are conveniently prepared by dissolving the active ingredient in a suitable high molecular weight alcohol such as propylene glycol or polyethylene glycol.

Possible pharmaceutical preparations for rectal use include, for example, suppositories, which consist of one or more active compounds in combination with a suppository base. Suitable suppository bases are, for example, natural or synthetic triglycerides or paraffin hydrocarbons. Furthermore, it is also possible to use gelatin rectal capsules consisting of the active compound in combination with a base. Possible base materials include, for example, liquid triglycerides, polyethylene glycols or paraffin hydrocarbons.

E. Solid Dosage Form

Pharmaceutical compositions include solid dosage forms in which the active compound is entrapped or sequestered within a porous carrier framework capable of attaining a crystalline, solid state. Such solid dosage forms with gas storage capacity are known in the art and can be produced in pharmaceutically acceptable form (eg, Yaghi et al., 2003). A particular advantage of this pharmaceutical composition is associated with chalcogenide compounds (e.g., hydrogen sulfide, carbon monoxide, hydrogen selenide), which in their free form may be toxic to some mammals at certain concentrations . In certain embodiments, the compounds described can be formulated for oral administration.

F. Perfusion system

Perfusion systems for cells can be used to expose tissues or organs to active compounds in liquid or semisolid form. Perfusion refers to the continuous flow of a solution through or over a population of cells. This means that the cells are retained in the culture as opposed to continuous flow culture which uses withdrawn media to wash out the cells (eg chemostat). Perfusion allows better control of the culture environment (pH, _p02 , nutrient levels, active compound levels, etc.) and is a method of significantly increasing the utilization of surface area within the culture for cell attachment.

Perfusion techniques were developed to simulate the in vivo cellular environment in which cells are continuously supplied with blood, lymph or other bodily fluids. Without perfusion with physiological nutrient solutions, cells in culture undergo alternating phases of being fed and starved, thereby limiting the full expression of their growth and metabolic potential. It is also within the scope of the present invention that the perfusion system can also be used to perfuse cells with oxygen antagonists to induce arrest.

Those skilled in the art are familiar with perfusion systems, and there are many commercially available perfusion systems. Any of these perfusion systems may be employed in the present invention. An example of a perfusion system is a perfusion packed bed reactor using a nonwoven bed matrix (CelliGen ^™ , New Brunswick Scientific, Edison, NJ; Wang et al., 1992; Wang et al., 1993; Wang et al., 1994). Briefly, the reactors include modified reactors for culturing both anchorage-dependent and non-anchor-dependent cells. The reactor was designed as a packed bed with means to provide internal recirculation. Preferably, the fibrous matrix support is placed in a basket within the reactor vessel. The basket has holes in the top and bottom to allow media to flow through the basket. A specially designed impeller provides media recirculation through the space occupied by the fibrous matrix to ensure uniform nutrient delivery and waste removal. This simultaneously ensures that a negligible amount of the total cell population is suspended in the medium. The combination of basket and recirculation also provides bubble-free flow of oxygenated medium through the fibrous matrix. The fiber matrix is a non-woven fabric with a "pore" diameter of 10 μm to 100 μm, which provides a large internal volume, and the pore volume is equivalent to 1 to 20 times that of a single cell.

Perfused packed bed reactors have several advantages. Features a fibrous matrix carrier that protects cells from mechanical stress from agitation and foaming. Free medium flow through the basket provides cells with optimally regulated levels of oxygen, pH and nutrients. The product can be continuously removed from the medium, and the harvested product is cell-free and can be produced in low-protein medium, which facilitates subsequent purification steps. This technique is explained in detail in WO 94/17178 (Freedman et al., Aug. 4, 1994), which is hereby incorporated by reference in its entirety.

Cellcube ^™ (Corning-Costar) modules provide a large styrenic surface area for fixation and growth of matrix-attached cells. It is an integrally enclosed sterile single-use device that has a series of parallel culture plates connected to create a thin, sealed laminar flow space between adjacent plates.

The Cellcube ^™ modules have inlets and outlets that are diagonally opposite each other and help regulate the flow of media. During the first few days of growth, the culture is usually saturated with the medium contained in the system after the initial inoculation. The amount of time between initial inoculation and initiation of media perfusion depends on the density of cells in the inoculum being inoculated and the rate of cell growth. Measurement of nutrient concentration in the circulating medium is a good indicator of the state of the culture. When a program is established, it may be necessary to monitor the nutrient composition at various perfusion rates to determine the most economical and productive operating parameters.

Other commercially available perfusion systems include, for example, CellPerf® (Laboratories MABIO International, Tourcoing, France) and Stovall Flow Cell (Stovall Life Science, Inc., Greensboro, NC).

The timing and parameters of the production phase of the culture depend on the type and use of the particular cell line. Many cultures require different media for production than that required for the growth stage of the culture. In traditional culture, switching from one stage to another is likely to require multiple washing steps. However, one of the advantages of perfusion systems is the ability to provide gentle transitions between multiple operating phases. The perfusion system can also facilitate the transition from a growth phase to a static phase induced by oxygen antagonists. Likewise, the perfusion system can facilitate the transition from the stasis phase to the growth phase by replacing the solution containing the oxygen antagonist with, for example, a physiological nutrient medium.

G. Catheter

In certain embodiments, catheters are used to deliver active compounds to organisms. Of particular importance is the administration of such an agent to the heart or vasculature. Typically, catheters are used for this purpose. Yaffe et al., 2004 specifically discuss catheters in the context of stagnation, although the use of catheters was generally known prior to this publication.

H. Gas Delivery

1. Respiratory system

A typical gas delivery system 100 is illustrated in FIG. 9 . Delivery system 100 is adapted to deliver an inhalable gas (including an active agent) to the respiratory system of a subject. Gas delivery system 100 includes one or more gas sources 102 .

Each of the gas sources 102 is connected to a regulator 104 and a flow meter 106 . Gas delivery system 100 also includes active agent source 107 , optional vaporizer 108 , vent controller 110 , scavenger 112 , and alarm/monitoring system 114 . Delivery system 100 may contain certain elements commonly used in anesthesia delivery apparatus. For example, an anesthesia delivery apparatus typically includes a high pressure circuit, a low pressure circuit, a breathing circuit, and a scavenging circuit. As described in FIGS. 10-11, one or more gas sources 102, vaporizer 108, outlet controller 110, purge 112, and/or alarm/monitoring system 114 may be provided as circuit, and these elements may be similar to those commonly used in anesthesia delivery instruments. Anesthesia delivery apparatus are described, for example, in US Patents 4,034,753, 4,266,573, 4,442,856, and 5,568,910, the contents of which are hereby incorporated by reference in their entirety.

The gas source 102 may be provided by a compressed gas tank; however, it should be understood that the gas source 102 may be a gas source or a liquid source that may be converted to a gas. For example, vaporizer 108 may be used to vaporize a source of liquefied gas. Regulators 104 contain valves that reduce the air pressure of each gas source 102 . The depressurized gas then passes through one of the flow meters 106 , which measures and controls the flow of gas from each respective gas source 102 .

Gas source 102 may be a carrier gas for delivering active agent 107 . The carrier gas can be selected to provide a desired environment to the subject to whom the active agent from source 107 is being delivered. For example, if the active agent is delivered to the patient as an inhalable gas, the carrier gas may include sufficient amounts of oxygen, nitrous oxide, or air to meet the needs of the patient. Other inert or reactive gases may be used.

In some embodiments, one of the gas sources 102 includes an active agent source 107 . The active agent from source 107 may be a source of liquefied gas vaporized by vaporizer 108, or the active agent may be a gas source, such as compressed gas at high pressure. The active agent may be mixed with one or more of the gas sources 102 . Vent controller 110 controls the amount of gas mixture provided to the subject.

Scavenger 112 is a device or system that scavengees and/or ventilates the gas provided to the subject. For example, if the active agent from source 107 is provided to the patient as an inhalable gas, scavenger 112 may be used to remove exhaust of the inhaled agent (eg, active agent), unused oxygen, and exhaled carbon dioxide.

Alarm/monitoring system 114 includes sensors that monitor gas flow and/or gas content at one or more locations in delivery system 100 . For example, the flow or flow of oxygen may be monitored as the active agent from source 107 is provided to the patient as an inhalable gas to ensure that the carrier gas contains sufficient oxygen for the patient. The alarm/monitoring system 114 also includes a user interface configured to provide an audible or visual alarm signal or monitor information to the user of the delivery system 100, such as a visual display, light or audible alarm. The alarm/monitoring system 114 may be configured to notify the user and/or provide information regarding gas levels when predetermined conditions are met.

Referring to FIG. 10 , system 100A includes a high pressure circuit 116 , low pressure circuit 118 , breathing circuit 120 and purge circuit 122 .

The high pressure circuit 116 includes a source of compressed gas 102 connected to regulator valves 104b, 104a. Regulator valve 104a controls the amount of gas flowing from each gas source 102, and regulator valve 104b may be opened to increase the pressure of the gas, for example by providing an opening to the surrounding atmosphere.

Low pressure loop 118 includes flow meter 106 , active agent source 107 and vaporizer 108 . The mixture of gases from gas source 102 is provided by flow meter 106 , which controls the amount of each gas from gas source 102 . As illustrated in Figure 10, the active agent source 107 is a liquid. The active agent source 107 is vaporized by a vaporizer and added to the gas mixture.

The breathing circuit 120 includes an outlet controller 110 , two one-way valves 124 , 126 and an absorber 128 . The purge circuit 122 includes a valve 112a, a reservoir 112b, and a drain 112c. Subject 130 receives the gas mixture from vent controller 110 and the resulting gas is ventilated through purge circuit 122 . More specifically, vent controller 110 controls the amount of gas mixture delivered to subject 130 through one-way valve 124 . Exhaled air flows through one-way valve 126 to valve 112a and to reservoir 112b. The excess gas is discharged through the discharge port 112c of the remover 112 . Some of the gas may be recirculated and flow through absorber 128 and into breathing circuit 120 . Absorber 128 may be a carbon dioxide absorber tank for reducing carbon dioxide gas in exhaled breath. In this configuration, exhaled oxygen and/or active agent can be recycled and reused.

One or more sensors S may be added at various locations in the system 100A. Sensor S senses and/or monitors gas in system 100A. For example, if one of the gas sources 102 is oxygen, one of the sensors S may be an oxygen sensor programmed and placed in place to monitor oxygen in the system 100A so that the patient receives the proper amount of oxygen. Sensor S communicates with an alarm/monitoring system 114 (see FIG. 9). If undesired or dangerous gas levels are present in system 100, alarm/monitoring system 114 can alert the user of system 100A so that appropriate action can be taken, such as increasing the oxygen level provided to subject 130 or letting the subject Person 130 is disengaged from delivery system 100A.

With respect to Figure 11, a system 100B is shown in which an active agent source 107 is connected to two regulating valves 104b, 104a. If the active agent source 107 is a liquefied gas source, an optional vaporizer 108 is provided to vaporize the liquefied gas source. If the active agent source 107 is gaseous (eg, a high-pressure gas), then the vaporizer 108 may be omitted. Active agent from source 107 is mixed with other gas sources 102 in low pressure loop 118 in an amount controlled by flow meter 106 . Low pressure circuit 118 includes a gas reservoir 109 that contains any escaped gas mixture as it flows to breathing circuit 120 . It should be understood that the active agent source 107 and/or any gas source 102 may be provided to the vaporizer as a source of liquefied gas. The elements of system 100B illustrated in FIG. 11 are substantially the same as those described above with respect to FIG. 10 and will not be described otherwise.

A method according to an embodiment of the invention that may be implemented using the system 100 , 100A, 100B is illustrated in FIG. 12 . A mixture of one or more sources of breathable gas is provided (element 202). A source of breathable gas may be obtained from gas source 102 as described with respect to FIGS. 9-11. A predetermined amount of active agent is added to the gas mixture (element 204), such as shown with respect to active agent source 107 in Figures 9-11. The gas mixture is administered to subject 120 (block 306). The exhaled gases are ventilated and/or recirculated, eg, through the scavenger 112 (element 208). Although the method of FIG. 12 is described with respect to the systems 100 , 100A, 100B of FIGS. 9-11 , it should be understood that any suitable system or apparatus may be used to perform the steps in FIG. 12 .

2. Reduced pressure delivery system

An embodiment of a gas delivery system 300 is illustrated with respect to FIG. 13 . Gas delivery system 300 is placed on subject 302 . Gas delivery system 300 is particularly suitable for delivering an active agent in a gas mixture to tissue of subject 302, such as wounded tissue.

System 300 includes a reduced pressure chamber 304 having a shield 306 covering a treatment area of a subject 302 . The decompression chamber 304 is connected to a vacuum pump 310 through a pump outlet 310a. The decompression chamber 304 includes an air inlet 308 a and an outlet 308 b, which in turn are connected to an active agent source 307 . Controller 320 is connected to active agent source 307 and vacuum pump 310 . Decompression chambers and vacuum pump systems are discussed in US Patent Nos. 5,645,081 and 5,636,643, the contents of which are hereby incorporated by reference in their entirety.

Reduced pressure chamber 304 is configured to enclose an area of subject 302 to provide a fluid-tight or airtight enclosure for treatment of the area with reduced or negative pressure and source 307 of active agent. Pressure chamber 304 may be affixed to subject 302 with a covering (not shown), such as a flexible, adhesive, fluid-impermeable polymer sheet. The covering may have an adhesive backing for covering the skin around the edges of the treated area and for providing a generally air-tight or fluid-tight seal and for securing the chamber 304 in place.

Shield 306 is positioned over the treatment area of subject 302 . For example, if the treated area of subject 302 includes a wound, the shield 306 may be positioned over the wound to prevent its overgrowth. The size and configuration of the shield 306 can be adjusted to suit an individual treatment area, and it can be formed from a variety of porous materials. The material should be sufficiently porous to allow oxygen and any other gases, such as from the active agent source 307, to reach the treatment area. For example, the shield 306 may be in the form of an open cell polymer foam, such as polyurethane foam, that is sufficiently porous to allow gas to flow to and/or from the treatment area. Foams of varying thickness and firmness are available, but if the patient must lie on the device during treatment, a spongy material may be desirable for patient comfort. The foam may also be perforated to enhance airflow and reduce system 300 weight. The shield 306 may be cut to an appropriate shape and size to fit within the treatment area, or alternatively, the shield 306 may be large enough to overlap the surrounding skin.

Vacuum pump 310 provides a source of suction in decompression chamber 304 . Active agent source 307 provides a quantity of active agent to decompression chamber 304 . The controller 320 controls the amount of vacuum applied to the decompression chamber 304 through the vacuum pump 310 and the amount of active agent supplied to the chamber 304 through the active agent source 307 .

It should be understood that the controller 320 may apply the vacuum and/and active agent in a substantially constant manner, cyclically, or with various fluctuations or patterns, or any combination thereof. In some embodiments, the active agent is provided by active agent source 307 , optionally by the vacuum pumping action of vacuum pump 310 . That is, controller 320 may instead activate vacuum pump 310 when active agent source 307 is deactivated, and then activate active agent source 307 when vacuum pump 310 is deactivated. The pressure in the decompression chamber 304 is allowed to fluctuate. In other embodiments, a substantially constant pressure is maintained by vacuum pump 310, and active agent source 307 provides a substantially constant amount of active agent to chamber 304 in a reduced pressure environment. In some embodiments, a substantially constant pressure is maintained by vacuum pump 310 while the amount of active agent is varied in a periodic fashion. In other embodiments, the pressure in decompression chamber 304 is fluctuated by vacuum pump 310 and the amount of active agent provided by source 307 is also fluctuated. The vacuum pump 310 and resulting fluctuations in the pressure in the chamber 304, or fluctuations in the amount of active agent provided by the source 307 may be periodic or aperiodic.

A method according to an embodiment of the invention that may be performed with system 300 is illustrated in FIG. 14 . Chamber 304 is located on the treatment area (402 component) of subject 302 . The pressure in chamber 304 is reduced by vacuum pump 310 (404 item). A predetermined amount of active agent from active agent source 307 is applied to the chamber (406 component). Although the method of FIG. 14 is described with respect to system 300 in FIG. 12 , it should be understood that any suitable system or apparatus may be used to implement the steps in FIG. 14 . For example, the exhaust port 308b could be omitted, and the active agent could be supplied to the chamber 304 through a single inlet port 308a. Other gases may also be introduced into the chamber 304, eg, with a single inlet or an inlet and an outlet, as described with respect to the active agent source 307 and the inlet 308a and outlet 308b. In some embodiments, vacuum pump 310 is connected to an additional collection vessel between pump 310 and chamber 304 for collecting exudate from the treatment area, eg, as described in US Patent No. 5,636,643.

In some embodiments, negative pressure gas delivery system 500 , as illustrated in FIG. 22A , contains a source of active oxygen antagonist in container 502 connected to cover layer 504 via gas inlet 506 via conduit 508 . The cover layer forms a sealed envelope with respect to the tissue site 510, which may be a wound site. In some embodiments, the cover layer has a vent 512 in communication with a source of negative pressure 514 via a conduit 516 . In some embodiments, a waste tank 518, which may be a removable waste tank, communicates between the vent and the source of negative pressure. In some embodiments, circuit outlet 520 communicates with vessel 502 via conduit 522 . In some embodiments, as shown in FIG. 22B , vaporizer 524 is inserted into the communication between container 502 and cover 504 .

The conduit may be flexible and may be suitable as a hose of plastic like material. A source of negative pressure 514, which may suitably be a vacuum pump, is in some embodiments in fluid communication with exhaust port 512 via conduit 516 for facilitating fluid exhaust, as is known in the art. In some embodiments, waste tank 518 is placed under vacuum to collect expelled fluid through fluid communication. Preferably, a filter (not shown), which may be a hydrophobic membrane filter, is inserted between the waste tank and the source of negative pressure to prevent contamination of effluent fluid drawn into the waste tank. In some embodiments, cover layer 504 contains an elastic material so it can accommodate pressure changes across the tissue site area during intermittent operation of the negative pressure source. In some embodiments, the edges of the cover are covered with a pressure sensitive adhesive, which may be an acrylic adhesive, for sealing the cover to the tissue site.

The negative pressure gas delivery systems 300 and 500 as illustrated in Figures 12 and 22A-B can be used to treat various areas for therapy, and particularly for treating wounds. Wounds that may be treated using the system 300 include infected open wounds, decubitus ulcers, dehiscing incisions, partial thickness burns, and a variety of lesions to which flaps or grafts have been attached. Treatment of the wound may be performed by maintaining a substantially continuously reduced or periodically reduced pressure in the decompression chamber 304 with the gas delivery system secured at the treatment site as previously shown and described, and in a substantially continuous or periodic manner. The active agent is provided to chamber 304 until the wound achieves the desired improved condition. Selected states of improved condition may include formation of granulation tissue sufficient for attachment of the flap or graft, reduction of microbial infection at the wound, prevention or reversal of burn penetration, closure of the wound, separation of the flap or graft from the underlying injury Tissue integration, complete healing of a wound, or other stages of improvement or healing as appropriate for a given type of wound or wound syndrome. Especially when using a gas delivery system incorporating a shield on or in the wound, the gas delivery system may be changed periodically, for example at 48 hour intervals, during the treatment. The method may be practiced with negative or reduced pressure in the range of 0.01 to 0.99 atmospheres, or the method may be practiced with negative or reduced pressure in the range between 0.5 and 0.8 atmospheres. The period of time for using the method on a wound may be at least 12 hours, but may for example be extended to 1 or several days. There is no upper limit beyond which the method is used beyond which it is no longer effective; the method increases the speed of closure until the wound does close. Satisfactory treatment of many types of wounds can be accomplished by utilizing reduced pressure equivalent to about 2 to 7 Hg below atmospheric pressure.

Providing reduced pressure to a gas delivery system in an intermittent or periodic manner, for example as described above, can be used to treat a wound in the presence of an active agent. The intermittent or periodic provision of reduced pressure to the gas delivery system can be accomplished by manual or automatic control of the vacuum system. The cycle ratio, ie, the ratio of "on" time to "off" time in such intermittent decompression treatments, can be as low as 1:10 or as high as 10:1. A typical ratio is about 1:1, which is usually done with alternating 5 minute intervals of the on and off of reduced pressure.

Suitable vacuum systems include any suction pump capable of providing at least 0.1 pounds of suction to the wound, or up to 3 pounds of suction, or up to 14 pounds of suction. The pump may be any conventional suction pump suitable for medical purposes capable of providing the required suction force. The size of the conduit connecting the pump and the pressure relief device is governed by the pump's ability to provide the level of suction required for operation. A 1/4 inch diameter catheter may be suitable.

Embodiments of the invention also include methods of treating damaged tissue comprising the steps of applying negative pressure to a wound and applying an active agent for a selected time and at a selected amount sufficient to reduce bacterial density at the wound. Open wounds are almost always contaminated with harmful bacteria. In general, a bacterial density of ¹⁰⁵ bacterial organisms per gram of tissue is considered infected. It is generally believed that at this level of infection, the transplanted tissue will not adhere to the wound. These bacteria must be killed by the wound host's natural immune response or by some external means before the wound can close. Applying negative pressure and an active agent to a wound can reduce the bacterial density of the wound. It is believed that this effect may be due to the incompatibility of the bacteria with the negative pressure environment or a combination of increased blood flow in the wound area and exposure to the active agent as the blood brings it cells and enzymes to destroy the bacteria. Methods according to embodiments of the invention may be used to reduce the bacterial density of a wound by at least half. In some embodiments, it can be used to reduce bacterial density by at least 1,000-fold or at least 1,000,000-fold.

Embodiments of the invention also include a method of treating a burn comprising the step of applying a negative pressure and an active agent to an area of the burn at a predetermined negative pressure and for a time sufficient to inhibit the formation of a full thickness burn. A partial thickness burn is a burn with a superficial layer of dead tissue and an underlying stagnant layer, usually sufficiently infected that it will transform within 24-48 hours into a full thickness burn, ie a burn in which all epidermal structures have been destroyed. Application of negative pressure and an amount of active agent to the wound prevents the infection from becoming sufficiently severe to cause disruption of the underlying epidermal structure. The magnitude, pattern and duration of pressure application can vary with individual wounds.

Embodiments of the invention also include a method for enhancing the attachment of living tissue to a wound comprising the steps of first attaching living tissue to the wound to form a wound-tissue complex, and then applying a negative or reduced pressure of a selected magnitude and an amount of active agent to the wound-tissue complex on the area, said negative or reduced pressure and an amount of active agent sufficient to facilitate the migration of epithelial and subcutaneous tissue to the complex, allowing the negative pressure and exposure to the active agent to remain A period of choice sufficient to promote wound closure. Attachment of living tissue to a wound is a common method that can take many forms. For example, one common technique is to use a "flap," a technique in which skin tissue from the area adjacent to the wound is separated on three sides but remains attached on the fourth side, and then moved over the wound. Another frequently used technique is open skin grafting, in which the skin is completely separated from another skin surface and grafted over the wound. Application of negative pressure and active agent to the wound-graft complex reduces bacterial density in the complex and increases blood flow to the wound, thereby improving attachment of the graft tissue.

I. Other instruments

In certain embodiments of the invention, it is desirable to supplement the methods of the invention for treating a patient who will suffer or has suffered trauma with the ability to externally manipulate the patient's core body temperature. In this regard, the patient's core body temperature can be manipulated by invasive or non-invasive means in conjunction with the methods of the present invention. Invasive methods for manipulating core body temperature include, for example, utilizing a cardiopulmonary pump to heat or cool the patient's blood, thereby raising or lowering the patient's core body temperature. Non-invasive approaches to manipulating core body temperature include systems and instruments that transfer heat into or out of a patient's body.

J. Other Delivery Devices or Instruments

In some embodiments, it is contemplated that a method or composition will involve a specific delivery device or instrument. Any of the methods discussed herein can be accomplished with any device for delivery or administration, including but not limited to those discussed herein.

For topical administration of the active compounds of the invention, the active compounds of the invention may be formulated into solutions, gels, ointments, emulsions, suspensions, and the like, as is well known in the art. Systemic formulations may include those designed for administration by injection or infusion, such as subcutaneous, intravenous, intramuscular, intrathecal, or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral, or pulmonary administration. of those preparations.

For oral administration, the active compounds of this invention will be formulated as tablets, pills, lozenges, capsules, fluids, gels, syrups, slurries, mixes, for oral ingestion by the patient to be treated. suspensions, etc., or oral liquid preparations such as suspensions, elixirs, and solutions.

For buccal administration, the compositions may take the form of tablets, lozenges and the like prepared in conventional manner. Other intramucosal delivery can be by suppository or intranasal delivery.

For direct administration to the lungs by inhalation, the compounds of the invention are conveniently delivered to the lungs by a variety of different devices. For example,

Metered-dose inhaler (MDI): Metered-dose inhaler using a gas cartridge containing a suitable low-boiling propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. Devices ("MDIs") are used to deliver compounds of the invention directly to the lung. MDI devices are available from many suppliers, such as 3M Corporation (e.g. at 3m.com/us/healthcare/manufacturers/dds/pdf/idd_valve_canister_brochure.pdf- ), Nasacort from Aventis (e.g. at products.sanofi -aventis.us/Nasacort_HFA/nasacort_HFA.html-63k-), Boehringer Ingelheim, (e.g. at boehringer-ingelheim.com/corporate/home/download/r_and_d2003.pdf), from First Experiment Aerobid from Forest Laboratories (e.g. at frx.com/products/aerobid.aspx), Glaxo-Wellcome (e.g. at www.gsk.com/research/newmedicines/newmedicinespharma.html above) and Schering Plow, (available at www.schering-plough.com/schering_plough/pc/allergy_respiratory.jsp).

Dry Powder Inhaler (DPI): DPI devices typically employ a mechanism such as a puff of gas to form an aerosol of dry powder within a container, which can then be inhaled by the patient. DPI devices are also well known in the art and are commercially available from a number of vendors, including, for example, the Foradil aerolizer from Schering Corporation, (e.g., www.spfiles.com/piforadil.pdf), from Glaxo- Wellcome's Advair Diskus (eg www.us.gsk.com/products/assets/us_advair.pdf-). The most popular variant is the multiple dose DPI ("MDDPI") system, which enables the delivery of more than one therapeutic dose. MDDPI devices are available from several companies, such as the Plumicort Turbuhaler from AstraZeneca (e.g., www.twistclickinhale.com/), Glaxo Wellcome (e.g., www.us.gsk.com/products /assets/us_advair.pdf-) and Schering-Plough, eg www.schering-plough.com/schering_plough/pc/allergy_respiratory.jsp). It is further contemplated that such devices, or any other device discussed herein, may be adapted for single dose administration.

Electrohydrodynamic (EHD) aerosol delivery: EHD aerosol devices use electrical energy to aerosolize liquid drug solutions or suspensions (see, e.g., Noakes et al., U.S. Patent No. 4,765,539, Coffee, U.S. Patent No. 4,962,885 , Coffee, PCT Application, WO 94/12285, Coffee, PCT Application, WO 94/14543, Coffee, PCT Application, WO 95/26234, Coffee, PCT Application, WO 95/26235, Coffee, PCT Application, WO 95/32807 ). EHD Aerosol Device Delivers Drugs to the Lungs More Efficiently Than Existing Pulmonary Delivery Technologies

Nebulizers: Nebulizers generate aerosols from liquid drug formulations by using, for example, ultrasonic energy to form fine particles that can be easily inhaled. Examples of nebulizers include those provided by Sheffield/Systemic Pulmonary Delivery Ltd. (see Armer et al., U.S. Patent No. 5,954,047; van der Linden et al., U.S. Patent No. 5,950,619; van der Linden et al., U.S. Patent No. 5,970,974) , Intal nebulizer solution provided by Avans (eg, www.fda.gov/medwatch/SAFETY/2004/feb_PI/Intal_Nebulizer_PI.pdf).

To administer gases directly to the lungs by inhalation, a variety of delivery methods currently available on the market for delivering oxygen can be employed. For example, a resuscitator, such as a first aid bag (see, US Patent Nos. 5,988,162 and 4,790,327), may be employed. The first aid bag consists of a flexible squeeze bag attached to a mask which is used by the physician to introduce air/gas into the victim's lungs.

The portable, hand-held drug delivery device is capable of generating an aerosol suitable for inhalation by patients with respiratory diseases through a nebulizer. Furthermore, this delivery device provides a means by which the dosage of the inhalant can be remotely monitored and varied as needed by a physician or doctor. See US Patent No. 7,013,894. Delivery of the compounds of the invention can be accomplished by methods for delivering supplemental gas to humans, in combination with monitoring the person's ventilation, neither using a sealed face mask (such as that described in US Patent No. 6,938,619). Pneumatic oxygen conserving devices are used during respiratory therapy to efficiently dispense oxygen or other gases so that only the initial portion of the patient's breath contains oxygen or other therapeutic gases (see US Patent No. 6,484,721). Use a gas delivery device that activates when the patient starts inhaling. The exhaust flow is delivered to the patient after the initial inhalation timing period to prevent the delivery of gas pulses to the patient. In this way, gas is only delivered to the patient during the initial portion of inhalation, preventing gas delivery from merely filling the airways of the patient's lungs. By using oxygen efficiently, cylinders of oxygen used while the patient is ambulatory will last longer and can be smaller and easier to transport. Air is delivered to the patient pneumatically, without batteries or electronics.

All devices described herein may have an exhaust system to bind or neutralize the compounds of the invention.

Transdermal administration of the compounds of this invention can be accomplished by means of a medicated device or patch which is affixed to the patient's skin. The patch allows the medicinal compound contained within the patch to be absorbed through the layers of the skin and into the patient's bloodstream. Such patches are commercially available as Nicoderm CQ patches from GlaxoSmithkline (www.nicodermcq.com/NicodermCQ.aspx) and for example Ortho Evra from Ortho-McNeil Pharmaceuticals (www.ortho-mcneilpharmaceutical.com/healthinfo/ womenshealth/products/orthoevra.html). Transdermal drug delivery reduces the pain associated with drug injection and intravenous drug administration, as well as the risk of infection associated with these techniques. Transdermal drug delivery also avoids gastrointestinal metabolism of the administered drug, reduces hepatic elimination of the drug, and provides sustained release of the administered drug. Transdermal drug delivery also enhances patient compliance with medication regimens due to the relative ease of administration and sustained release of the drug.

Other variations of patches include ultrasonic patches, which are designed with materials such that ultrasound can be transmitted through the patch, enabling delivery of the drug stored in the patch, and can be used in conjunction with ultrasonic drug delivery methods (see U.S. Patent No. .6,908,448). A patch in a bottle (U.S. Patent No. 6,958,154) comprises a fluid composition, such as a spray in some embodiments, that can be applied to a surface as a fluid, but then dries to form a covering element on the surface of the host, Such as pills. The covering member thus formed has a non-adhesive outer surface covering and an underlying adhesive surface that facilitates adhesion of the patch to the substrate.

Another drug delivery system comprises one or more spherical semiconducting aggregates and facilitates the release of the drug stored in the reservoir. The first aggregate is used for perception and memory, while the second aggregate is used for control aspects, such as for pumping and dispensing drugs. The system can be in long-term communication with a remote control system, or operated independently with local power, for drug delivery based on patient demand, timed release under system control, or delivery in accordance with measured markers. See US Patent No. 6,464,687.

Pumps and Infusion Sets: An infusion pump or perfusor infuses fluids, drugs, or nutrients into a patient's circulatory system. Infusion pumps can administer fluids in a very reliable and inexpensive manner. For example, they may administer as little as 0.1 mL of injection per hour (too little for instillation), every minute of injection, repeated one-time dose injections as required by the patient, up to a maximum number per hour (e.g. in patient-controlled analgesia), or fluids whose volume varies with the time of day. Various types of infusion devices are described in the following patent applications pending with the United States Patent and Trademark Office. These include, but are not limited to, US Patent No. 7,029,455, US Patent No. 6,805,693, US Patent No. 6,800,096, US Patent No. 6,764,472, US Patent No. 6,742,992, US Patent No. 6,589,229, US Patent No. 6,626,329, US Patent No. .6,355,019, US Patent No. 6,328,712, US Patent No. 6,213,738, US Patent No. 6,213,723, US Patent No. 6,195,887, US Patent No. 6,123,524 and US Patent No. 7,022,107. Infusion pumps are also available from Baxter International (www.baxter.com/products/medication_management/infusion_pumps/), Alaris Medical Systems (www.alarismed.com/products/infusion.shtml), and from B Braun Medical (www. bbraunusa.com/index.cfm?uuid=001AA837D0B759A1E34666434FF604ED).

Oxygen/gas bolus delivery device: This device for delivering gas to patients with chronic obstructive pulmonary disease (COPD) is available from Tyco Healthcare (www.tycohealth-ece.com/files/d0004/ty_zt7ph2. pdf) obtained. It can also be used to deliver the compounds of the invention. The device described above is cost effective, lightweight, unobtrusive and portable.

Patches in bottles (U.S. Patent No. 6,958,154) include a fluid composition, such as a spray in some embodiments, that can be applied to a surface as a fluid, but then dries to form a covering element on the surface of the host, such as Pills. The covering member thus formed has a non-adhesive outer surface covering and an underlying adhesive surface that facilitates adhesion of the patch to the substrate.

Implantable drug delivery system: Another drug delivery system comprises one or more spherical semiconductor assemblies and facilitates the release of the drug stored in the reservoir. The first aggregate is used for perception and memory, while the second aggregate is used for control aspects, such as for pumping and dispensing drugs. The system can be in long-term communication with a remote control system, or operated independently with local power, for drug delivery based on patient demand, timed release under system control, or in accordance with measured marker delivery. See US Patent No. 6,464,687.

The contents of each cited patent and website discussed in this section are hereby incorporated by reference.

VIII. Combination Therapy

The compounds and methods of the invention are useful in many therapeutic and diagnostic applications. In order to increase the effectiveness of treatments with the components of the invention (eg, oxygen antagonists or other active compounds), it is desirable to combine these components with other agents effective in the treatment of those diseases and conditions (secondary therapy). For example, treatment for stroke (anti-stroke therapy) typically includes antiplatelet agents (aspirin, clopidogrel, dipyridamole, ticlopidine), anticoagulants (heparin, warfarin), or thrombolytic agents (tissue lysinogen activator).

Different combinations can be used, for example the active compound such as _H2S is "A" and the second treatment is "B".

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/AA/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/BA/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/AA/B/A/A A/A/B/A

Administration of the oxygen antagonists and/or other active compounds of the present invention to biological substances will follow the general regimen used to administer those particular second treatments, taking into account the toxicity, if any, of the oxygen antagonist (or other active compound) treatment . Cycles of treatment are expected to be repeated as needed. It is also contemplated that various standard therapies, as well as surgical interventions, may be used in combination with the described therapies.

IX. Embodiment

The following examples are included to illustrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice . However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the particular embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1:

Preservation of nematodes in carbon monoxide

The atmosphere contains 210,000 ppm of oxygen. Exposure to low levels of oxygen, or hypoxia, causes cell damage and death in humans. Oxygen concentrations between 100 ppm and 1000 ppm are also lethal in the nematode (C. elegans). By rigorously studying the response of nematodes to a series of oxygen tensions, it was found that oxygen concentrations below 10ppm and above 5000ppm are not lethal. In 10 ppm oxygen balanced with nitrogen, nematodes enter a reversible stasis state in which all aspects of the viability observable under a light microscope cease (Padilla et al., 2002). At 5000 ppm (balanced with nitrogen) and higher oxygen concentrations, nematodes progressed normally through their life cycle. In the search for drugs to protect nematodes from hypoxic damage, carbon monoxide was tested.

To obtain specific atmospheric conditions, the following devices are used: glass syringe barrels with a locking device such as LUER-LOK at the tip, the large opening of the syringe barrel is sealed with custom-machined steel and rubber fittings To create a hermetic seal, the syringe barrel is locked by a locking device to the inlet port of an environmental chamber having an inlet port and an outlet port, each port fitted with a locking device such as LUER-LOK Accessories. Determined gases were humidified and provided to the environmental chamber by first venting the gas from a compression tank (Byrne Specialty Gas, Seattle, WA) through a wash bottle (500 ml Kimex) filled with double distilled water. The scrubber bottle is connected to the environmental chamber via a gas flow meter. A gas flow meter was used to provide a regulated flow of 70 cc/min through the artificial environmental chamber throughout the 24 hour incubation period.

To test whether induced, reversible arrest can be achieved in C. elegans, 2-cell C. elegans embryos, L3 larvae, or adult nematodes were collected and exposed to an effective 100% CO environment at room temperature, 100 % _N2 environment, an environment containing 500 ppm oxygen balanced with carbon monoxide, or exposed to an environment containing 100, 500 or 1000 ppm oxygen balanced with nitrogen. Nematodes were visualized using differential interference contrast microscopy (also known as Nomarski optics). Images were collected and analyzed with NIH image and Adobe Photoshop 5.5. Embryos are about 50 μm long.

The results of these trials showed that 100% carbon monoxide was not lethal and induced reversible stagnation. Nematodes could not survive 500 ppm oxygen balanced with nitrogen, however, those treated with 500 ppm oxygen balanced with carbon monoxide entered paralysis and survived. See below:

Example 2:

Preservation of human skin in carbon monoxide

Carbon monoxide is very toxic to humans because it strongly competes with oxygen for binding to hemoglobin, the main molecule that distributes oxygen to tissues. The fact that hemoglobinless nematodes are resistant to carbon monoxide and are even protected against hypoxic damage by the drug raises the possibility that carbon monoxide will be present in human tissues in situations where blood is absent, such as in tissue grafts or Bloodless surgical field to prevent hypoxic damage. To test this hypothesis, human skin was used.

Obtain three human foreskins for this purpose. Foreskin tissue was maintained in keratinocyte growth medium (KGM) containing insulin, EGF (0.1 ng/ml), hydrocortisone (0.5 mg/ml) and bovine pituitary extract (approximately 50 micrograms/ml protein). The foreskins were rinsed in PBS and excess adipose tissue was removed. Divide each foreskin sample into 2 equal pieces. Each piece was placed in a separate container containing a PBS solution with 24 mg/ml dispase II (from Bacillus Polymyxa EC 3.4.24.4: Roche Diagnostics Corp., Indianapolis, IN). A container (containing foreskin pieces in PBS containing dispase II) was placed in a humid chamber in a fume hood. Another container (with the other half of the foreskin block in PBS containing dispase II) was placed in an environmental chamber filled with humidified 100% CO in the same fume hood. Both samples were maintained at room temperature for 24 hours. The methods used to establish the defined atmospheric conditions correspond to those used in Example 1.

After 24 hours of exposure to normoxia or 100% CO, keratinocytes were isolated from the foreskin as described by Boyce et al. (1983; 1985; each incorporated herein by reference in its entirety). Briefly, the epidermis from each foreskin sample was removed to a fresh vessel containing PBS. The epidermis was minced and homogenized prior to incubation in 3 ml of 0.05% trypsin, 1 mM EDTA for 5 min at room temperature to separate basal cells from the epidermis. After incubation, 6 ml of 400 μg/ml (microgram/ml) soybean trypsin inhibitor, 1 mg/ml of BSA were added, and the samples were centrifuged at 900 RPM. The supernatant of each sample was discarded and the sample pellet was resuspended in 10 ml of KGM. Each sample was divided into two 10 cm plates each containing 5 ml KGM and 100 μl of HEPES pH 7.3 (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid). Plates were incubated for 5 days at 37°C in an incubator filled with 95% room air, 5% carbon dioxide.

Cells were inspected visually using an inverted phase-contrast microscope. All three keratinocyte populations exposed to normoxia showed little or no growth. All three keratinocyte populations exposed to 100% CO showed significant growth. Quantification of the number of viable keratinocytes (judged by colony formation) was quantified in two of the three foreskins. see picture 1.

Table 1 - Quantification of colony formation

Foreskin atmosphere total number of colonies 1 100%CO 542 colonies (most of them are very large) 1 Normoxia 2 colonies (both small) 2 100%CO 780 colonies (most of them are very large) 2 Normoxia 0 colonies

Example 3:

Further preservation experiments with nematodes

The following examples contain information that overlaps and expands on the information disclosed in Example 1.

A. Materials and methods

Environmental chambers and devices . Hypoxia experiments were performed in a custom atmosphere chamber designed by W. Van Voorhies (Van Voorhies et al., 2000). The chamber was a 30 mL glass syringe (Fisher #14-825-10B) fitted with a custom made steel stopper lined with two viton o-rings to ensure a tight seal. The steel plug is drilled through and has a steel lure lock on the outer face to allow connection to a hose carrying compressed gas. A defined gas mixture is delivered into the chamber from a compression tank at a constant pressure and flow rate, the gas is first passed through a rotometer (Aalborg, flow tube # 032-41ST) or a mass flow controller (Sierra Instruments #810) to monitor the flow rate, The gas was then hydrated by passing through a 500ml scrubber bottle (Fisher #K28220-5001) containing 250ml of water. Utilizes 1/4" OD Nylon (Cole-Parmer #P-06489-06) or FEP (Cole-Parmer #A-06450-05) tubing and connections between tube and regulator and between tube and rotometer Brass John-Guest-type fittings (Byrne Gas) were used. All other connections were made with microfluidic quick-connect fittings (Cole-Parmer #A-06363-57, #A-06363-52) or standard lure fittings (Cole -Parmer #A-06359-37, #A-06359-17) Complete.

Viability of nematodes under low oxygen. Bristol strain N2 was continuously maintained at 20°C, taking care to ensure that the population did not starve. Adult C. elegans in the logarithmic phase were selected and placed on a glass plate in a drop of sterile water containing 100 μg/ml ampicillin, 15 μg/ml tetracycline and 200 μg/ml streptomycin. Adults were minced with a safety blade and 2-cell embryos were picked with a mouthpipet. 30-60 2-cell embryos were transferred into small glass boats (custom to fit atmosphere chamber, Avalon GlassWorks, Seattle WA) filled with 3 ml of 1% agarose in M9. The glass boats were then placed in a humid chamber for 2 hours to mature the embryos and then placed in an environmental chamber. The environmental chamber was continuously perfused with pure N ₂ (grade 4.5), 100 ppm O ₂ /N ₂ , 500 ppm O ₂ /N ₂ , 1000 ppm O ₂ /N ₂ or 5000 ppm O ₂ /N ₂ at room temperature at 70 cc/min 24 Hour. After exposure, the agarose block containing the embryos was cut out of the boat and placed embryos face up on medium size NGM plates inoculated with E. coli (OP50). Embryos were incubated for 24 hours post-exposure and hatched L1's were transferred to the surface of NGM plates and subsequently entered adulthood. Unexplained animals were removed from the population. All gases were provided by Byrne Gas (Seattle, WA). Guarantee that pure _N2 contains less than 10 ppm of impurities and demonstrate that all _O2 / _N2 mixtures are ±2% oxygen content (for example, demonstrate that 100 ppm of _O2 / _N2 contains between 98 ppm and 102 ppm of _O2 ). Parts per million to kPa conversions are based on parts per million = 101 kPa at 1 atmosphere.

Viability of nematodes in carbon monoxide-based atmospheres. 30-60 embryos were harvested from continuously maintained Bristol N2 and hif-2 (ia04) strains as described above. The environmental chamber was continuously perfused with pure CO (CP grade) or 500 ppm _O2 /CO at 70 cc/min for 24 hours at room temperature. To achieve 2500ppm O ₂ /CO or 2500ppm O ₂ /N ₂ , mix 5000ppm O ₂ /N ₂ in a 1:1 ratio with pure CO or with pure N ₂ using two mass flow controllers (Sierra Instruments 810 ) to accurately monitor the flow rate. Throughout the 24 hour exposure, 50 cc/min of each gas was delivered into a three-way valve (Cole-Parmer #A-30600-23) and the resulting mixture was passed through the wash bottle and into the environmental chamber. All gases were provided by ByrneGas (Seattle, WA). A 500ppm _O2 /CO mixture was certified for an oxygen content of ±2% and contained 7000ppm _N2 to ensure a consistent _O2 /CO ratio during use of the compression tank.

Cell biology analysis. To determine the extent of developmental progression in a nitrogen-based atmosphere (Table 2), 2-cell embryos were exposed to various degrees of hypoxia as described above and photographed immediately, or during a 12-hour recovery period in a humid chamber After taking pictures. To determine whether embryos were arrested in a carbon monoxide-based atmosphere, 2-cell embryos were matured in room air for 2 h and photographed immediately or placed in 100% carbon monoxide or 0.05 kPa _O2 /CO for 24 h and post-exposure. Take a picture now. In all cases, DIC microscopy was performed by placing embryos under a coverslip on a thin 1% agarose pad and viewing on a Zeiss axioscope. The photographs were then acquired with RS Image and Adobe Photoshop software.

B. Results

HIF-1 has previously been reported to be required in C. elegans in mild hypoxia (0.5 kPa O ₂ (Padilla et al., 2002) and 1 kPa O ₂ (Jiang et al., 2001)) and is known to be stagnant Possible in hypoxia (>0.001 kPa _O2 ) (Padilla et al., 2002). To precisely determine the extent to which each of these responses is active, the viability of wild-type C. elegans embryos was determined after 24 hours of exposure to various oxygen tensions between mild hypoxia and hypoxia. Embryos exposed to hypoxia entered stagnation as previously reported and thus survived exposure with high viability. Embryos in 0.5 kPa _O2 remained viable throughout the exposure and also survived with high viability. However, embryos exposed to oxygen tensions in the middle range between mild hypoxia and hypoxia (0.1 kPa O ₂ to 0.01 kPa O ₂ ) surprisingly did not survive ( FIG. 2 ).

Embryos did not hatch during exposure to this moderate range of hypoxia, suggesting that they did not successfully complete the HIF-1-mediated response. To determine whether they were arrested, it was examined whether embryos in this intermediate range ceased embryogenesis during exposure. Embryos in lethal oxygen tension did not cease embryogenesis, and increased oxygen levels correlated with increased developmental progression of embryos (Table 2). After reoxygenation, most of these embryos failed to hatch and many of those that did hatch ceased as abnormal L1s. These data show that this mid-range hypoxia is a unique stress in which oxygen levels are neither high enough to promote sustained viability nor low enough to induce stasis.

Based on these findings, it was hypothesized that if carbon monoxide, a competitive inhibitor of oxygen binding, could induce stasis in the presence of low levels of oxygen, it would confer protection against this lethal range of hypoxia. To test this possibility, the viability of C. elegans embryos in various concentrations of carbon monoxide was first measured. Although high levels of carbon monoxide can have toxic effects in some systems, it was found that C. elegans embryos are remarkably tolerant to a wide range of carbon monoxide tensions. Indeed, C. elegans embryos withstood continuous exposure to 101 kPa CO (100% CO) for 24 hours with high viability (81.5% survived to adulthood, Figure 3.) Notably, at 101 kPa In CO, embryos did not progress through embryogenesis during exposure, indicating that they entered stasis. To test whether carbon monoxide can protect embryos in the presence of lethal oxygen tension, the viability of embryos exposed to 0.05 kPa O ₂ equilibrated with carbon monoxide was determined. These embryos recovered to adulthood with 96.2% viability compared to embryos exposed to 0.05 kPa _O2 equilibrated with _N2 , most of which did not survive (Figure 3). Moreover, similar to embryos treated with 101 kPa CO, embryos in 0.05 kPa O ₂ equilibrated with CO stopped embryogenesis, indicating that they entered stasis. Thus, carbon monoxide may protect against hypoxic damage in the presence of lethal oxygen tension by inducing stasis.

To further investigate the range of oxygen tensions that can be protected by excess CO, embryos lacking HIF-1 function (hif-1(ia04) strain) were used to address whether protection from hypoxic damage is also possible in mild hypoxia . After examining various oxygen tensions between 0.1 kPa _O2 and 1 _kPaO2 equilibrated with nitrogen, it was found that the greatest demand for HIF-1 was at 0.25 kPa _O2 equilibrated with nitrogen. Under this atmosphere, wild-type embryos progressed normally to complete development and showed high viability, but hif-1(ia04) embryos failed to complete embryogenesis and showed 100% lethality (Table 3). Therefore, it was examined whether CO can protect hif-1(ia04) embryos in 0.25 kPa _O2 . In 0.25 kPa O ₂ equilibrated with carbon monoxide, wild-type and hif-1(ia04) embryos entered anaphysis and survived this exposure with high viability (78.7% and 84.0% survived to adulthood, respectively) ( table 3). Thus, induction of stagnation by CO is possible at oxygen tensions as high as 0.25 kPa _O2 , and CO protects against mild hypoxia even in the absence of HIF-1 function.

Table 2 - Quantification of developmental progression in hypoxia

atmosphere % of embryos in range Extent of embryogenesis (minutes after 2-cell stage) N

＞0.001kPaO ₂ /N ₂ 100%±0.0 20-40 minutes 35 0.01kPa O ₂ /N ₂ 92.9%±6.0 40-80 minutes 115 0.05kPa O ₂ /N ₂ 97.7%±2.0 100-140 minutes 108 0.1kPa O ₂ /N ₂ 91.4%±1.3 300-340 minutes 60

Wild-type 2-cell embryos were placed in various degrees of hypoxia for 24 hours and scored for the extent to which they developed to completion of embryogenesis. Exposure to an atmosphere containing increased amounts of oxygen resulted in an increase in the progression to completion of embryo formation. The percentage of embryos that ceased embryogenesis within a given range of 20-40 minutes was determined. Data are the results of 3 independent experiments.

Table 3 - Carbon monoxide protects hif-1 embryos against mild hypoxia

0.25kPa O ₂ /N ₂ n 0.25kPa O ₂ /CO N N2 94.2%±1.2 49 18.7%±21.9 109 hif-1(ia04) 0.0%±0.0 68 83.9%±13.8 108

Viability to adulthood was examined in wild-type and hif-1(ia04) embryos after 24 hours of exposure to 0.25 kPa O ₂ /N ₂ or 0.25 kPa O ₂ /CO. All data points are the result of at least 3 independent experiments and unexplained worms were removed from the population.

Viability of nematodes in response to hypothermia.

The viability of nematodes was also temperature sensitive, with 100% of the population dying after 24 hours of exposure to low temperature (4°C; Figure 15). However, if nematodes were induced to arrest by equilibrating into anoxic conditions (<10 ppm oxygen) for 1 hour before the temperature was lowered, a large proportion of nematodes survived 24 hours of exposure to 4° C. ( FIG. 15 ). In this experiment, nematodes were kept in arrest during hypothermia and for 1 hour after they had returned to room temperature. Anaerobic conditions (pure _N2 ), growth status and viability measurements are described below.

Example 4:

Decreased core body temperature and respiration in mice

A. Materials and methods

Implantation of the telemetry device. Female C57BL/6J mice (Jackson Laboratories-Bar Harbor, Maine) were implanted with a telemetry device (PDT-4000HR E-Mitter-MiniMitter Inc.-Bend, OR) according to the standard protocol provided by the manufacturer. Mice were allowed to recover for several weeks to allow body temperature and heart rate signals to stabilize. Core body temperature, heart rate and movement of the mice were continuously monitored by telemetry and recorded with VitalView software (provided by MiniMitter). Ambient temperature was monitored with HOBO (Onset Computer Corp. - Pocasset, MA), and data was analyzed with BoxCar software (supplied by Onset Computer Corp.).

Mice were exposed to a conditioned atmosphere. Each mouse was exposed to 1 L/min of (a) nitrogen containing 500 ppm H ₂ S balanced (Byrne Specialty Gas-Seattle, Washington) mixed with room air (using a 3-channel gas distributor from Aalborg-Orangeburg, New York (proportioner ) to achieve an atmosphere with a final concentration of 80 ppm H ₂ S and 17% O ₂ , or (b) nitrogen mixed with room air to achieve an atmosphere with a final concentration of 17% O ₂ . _H2S and _O2 measurements were performed with an Innova GasTech GT series portable gas monitor (Thermo Gas Tech-Newark, California).

Before and during exposure to conditioned and unconditioned atmospheres, the mice were placed in an air-filled chamber containing a glass cage (with drinking water, no food) equipped with The inlet and outlet tubes of FEP tubing from Parmer (Vernon Hills, Illinois) were used to introduce and exhaust the atmosphere. The cage was sealed with a lid using Dow Corning silicone vacuum grease (Sigma-St. Louis, Missouri.). Gas from each cage is exhausted through the exhaust tube into the chemical fume hood. To ensure the system is airtight, a GasTech GT handheld monitor is used to detect leaks.

Respirometry. In some experiments, oxygen consumption was measured using a PA-10a _O2 analyzer (Sable Systems) used according to the manufacturer's instructions. Similarly, carbon dioxide production by animals was monitored using a LI-7000 _CO2 / _H2O analyzer (Li-Cor Corporation) used according to the manufacturer's instructions. Place these devices in line with the environmental chamber so that they sample the gas input and output lines for inspection.

Regulation of ambient temperature. The mice were housed in an incubator (Sheldon Manufacturing Company-Cornelius, Oregon) with low temperature and day and night light in Shel Lab, and the temperature and photoperiod were adjusted for the mice (lights turned on at 8AM and lights off at 8PM). Mice were exposed to a conditioned atmosphere as described above. While the mice are exposed to the conditioned atmosphere, the temperature inside the incubator is lowered to the desired temperature, for example to 10°C or 15°C. Mice were maintained in a conditioned atmosphere and at reduced temperature for 6 hours. The atmosphere in the plenum chamber was replaced with room air, and the mice were returned to normal room temperature (22°C) and allowed to recover.

B. Results

baseline data. To determine the response of mice to sublethal doses of hydrogen sulfide, the inventors first determined core temperature, heart rate, and movement by recording data from four transceiver-implanted mice in an incubator over a one-week period. At baseline, the incubator was maintained at ambient temperature and filled with room air. The baseline data demonstrated that the mice had a circadian rhythm, with activity peaks in the evening just after the lights went out, and in the early morning just before the lights came on. Core temperatures varied from a high of 37°C during their activity to a low of 33.5°C during their inactivity. Heart rates varied from 750 bpm (beats per minute) during their active periods to 250 bpm during their inactive periods. Heart rate may correlate with core temperature (a higher temperature means a higher heart rate). Likewise, total locomotor activity was highest in the evening and shortly before dawn.

Mice were exposed to a conditioned atmosphere at room temperature. The first experiment in which mice were exposed to hydrogen sulfide consisted of first placing the mice in an air-filled chamber maintained at 27° C. for 1 hour in an incubator. After 1 hour, the chamber was filled with 80 ppm as generally described above, and the temperature of the oven was reduced to 18°C during the experiment. Although no immediate changes in heart rate and total motor activity were detected, a significant reduction in core temperature was observed. The experiment was allowed to run for 90 minutes, during which time the core temperature dropped to 28.6°C—5°C below the lowest temperature recorded in any of the four mice during the baseline study described above. During recovery after the chamber was filled with room air, the inventors noted that the animal was initially relatively immobile (easy to capture); however, within 60 minutes it had returned to a normal range of core temperature and activity. A second mouse was exposed to the same protocol; however this time with 80 ppm aeration for 3 hours. During this time period, the inventors noticed that the heart rate decreased significantly from 600 bpm to 250 bpm, the total motor activity showed little to no activity, and the core temperature dropped to 18.6°C.

Changes in respiration are accompanied by a decrease in core temperature. Exposure of mice to 80 ppm _H2S also resulted in a decrease in metabolic rate, as determined by measuring oxygen consumption and carbon dioxide production. For example, mice in which core temperature and carbon dioxide production had been measured simultaneously demonstrated that a rapid decrease in carbon dioxide production preceded a decrease in the animals' core temperature (Fig. 4A). An approximately 3-fold reduction in carbon dioxide production established a new baseline approximately 5 minutes after exposure to _H2S .

Table 4 shows _the results from experiments that simultaneously measured _O2 and _CO2 concentrations in mice exposed to room air that had been decontaminated (so the control had a value of 0), with or without _H2S (80ppm). The measurements were performed over a period of 15 minutes with mice in a 0.5 L sealed environmental chamber with an atmosphere flow rate of 500 cc/min. Oxygen consumption was obtained by subtracting the oxygen concentration when mice were present from the control when mice were not present. Likewise, carbon dioxide production was obtained by subtracting the carbon dioxide concentration in the presence of mice from the control in the absence of mice. RQ stands for respiratory quotient and is equal to the ratio of carbon dioxide produced to oxygen produced. The results demonstrate a 2-3 fold reduction in oxygen consumption and a 3-4 fold reduction in carbon dioxide production in the presence of _H2S . Changes in the respiratory quotient reflect differences in oxygen consumption and carbon dioxide production in mice in the presence or absence of _H2S .

Table 4 - H ₂ S exposure inhibited the respiration of mice

the presence of mice Presence of H ₂ S [O ₂ ]ppm [CO ₂ ]ppm RQ - - 207,000 0 + - 203,600 2800 Consume, produce 3,400 2800 0.82 - + 166,200 0 + + 164,900 750 Consume, produce 1300 750 0.58

The different parameters of arrest (reduced oxygen consumption, reduced carbon dioxide production, or reduced mobility) can be assessed by a wide variety of assays and techniques. For example, probably the easiest way to measure the induced arrest in mice administered _H2S is by observing their respiration. Indeed, this encompasses all three parameters as it represents reduced oxygen consumption, carbon dioxide production and mobility. A normal mouse in room air under standard conditions will breathe about 200 times per minute. If mice were administered 80 ppm _H2S and the core temperature was lowered to 15°C, respiration was reduced by at least an order of magnitude between about 1-10 breaths per minute. Indeed, mice under these conditions were observed not breathing for periods longer than 1 hour, suggesting that deep levels of arrest are achievable. Thus, this represents at least about a 1-20-fold reduction in cellular respiration (ie, oxygen consumption and carbon dioxide production).

Mice were exposed to a conditioned atmosphere at reduced ambient temperature. To begin to determine the limits of hydrogen sulfide's ability to reduce activity in mice, the inventors performed several experiments using mice without telemetry devices and then obtaining data by exposing mice with telemetry devices. The first experiment involved mice without telemetry receiving a conditioned atmosphere of 80 ppm _H2S at a reduced chamber temperature of 10 °C, essentially as described above in Materials and methods, except that after exposure to Mice were placed in an air-filled chamber at 27° C. for 1 hour prior to gassing and lowering the ambient temperature. Mice without the telemetry device performed well on this treatment and returned to activity within approximately 90 minutes after removal from the air-filled chamber. Mice with the telemetry device that received the same conditions also did well and showed a reduction in core temperature to about 12.5°C. The inventors could not accurately determine this temperature because the electronic equipment does not work at 15.3°C. Therefore, the drop in temperature to 12.5°C is an estimate based on the slope of the decline prior to inactivity and the amount of time the animals were indoors after the electronics were inoperative.

Due to instrument limitations, the inventors next tested 4 mice each with a telemetry device, essentially as described above, in a plenum with a conditioned atmosphere containing about 80 ppm hydrogen sulfide, or with room air. 6 hours. The temperature of the incubator was reduced to a constant 15° C. at the start of the experiment (time 0 of exposure to the conditioned atmosphere, or time 0 for mice exposed to room air). At the end of the 6 hour period, mice were returned to an atmosphere of room air and an ambient temperature of 22°C as described generally above. Core body temperature decreased significantly in all 4 mice dependent on the utilization of 80 ppm hydrogen sulfide (Fig. 4B). There was also a marked decrease in heart rate and total motor activity associated with a decrease in body temperature. The mice were maintained for 4 weeks, and the behavior of the animals did not change significantly.

Example 5

Rat study on reducing radiation damage

A. Fundamentals of Science

While aspects of radiation injury models can and have been evaluated in cell culture, testing the ability of an experimental drug to affect injury and healing processes requires inclusion of all response systems affected. At present, the only way to achieve it is in intact animals. The present inventors propose the use of mice as the most suitable model for this study. C57BL/6 mice have been chosen for the study because this strain of mice is prone to radiation lung injury, the level of radiation tolerated by this strain has been established, and the inventors have recently shown that _H2S reduces this small Core temperature of the mouse strain.

Two consistent experiments were designed following this protocol. Each trial will investigate the effect of _H2S -induced hypothermia on the development of radiation-induced lung injury. Ten mice per group will be exposed to four experimental conditions ( _H2S /17.5Gy chest irradiation, _H2S /no chest irradiation, no _H2S /17.5Gy chest irradiation, or no _H2S /no chest irradiation irradiation), followed by 13 weeks. Groups of 12 animals will be similarly exposed for 26 weeks (increased n is needed to offset the increased mortality that occurs later in the disease course.)

For these experiments, analysis of variance (ANOVA) will be used as the statistical model for data analysis. A fully crossover and _randomized two-way ANOVA with 4 groups (irradiated mice that received or did not receive _H2S or non-irradiated mice) and two time intervals (13 or 26 weeks) will be used Bronchoalveolar lavage inflammatory cell counts and total protein concentrations and temporal changes in lung hydroxyproline levels were analyzed. Assuming a power of 80%, a significance of 5%, and a two-tailed test, 5 surviving mice per combination of injury group, intervention group, and time point would allow a detectable difference between group means greater than or equal to the potential 1.7 times the within-group standard deviation. The within standard deviation is expected to be equal to about 25%. Thus, changes in inflammatory cell counts or lung collagen content of 35-50% of control values should be discernible in these assays.

_H2S exposure and chest irradiation will be performed at the SLU AHR in the Linear Accelerator Facility. Bronchoalveolar lavage and lung harvesting at necropsy will be performed in the AHR mouse necropsy room. Bronchoalveolar lavage cell counts and protein concentrations and measurements of lung hydroxyproline levels will be performed in another laboratory (D3-255). Wild genotype C57BL/6 mice will receive 17.5Gy chest irradiation. Mice will be anesthetized intraperitoneally with Avertin, placed in individual cloth mouse restraints, and irradiated by a linear accelerator with 8.5 Gy at a dose rate of 3 Gy/min through a calibrated only Both lateral regions of the chest were targeted (total chest dose of 17.5 Gy).

B. Program

anaesthetization. Wild genotype C57BL/6 mice will be anesthetized with intratracheal administration of isoflurane. Depth of anesthesia will be monitored by respiration rate in response to tactile stimuli. An intraperitoneal injection (0.4-0.7 ml/mouse ip) of Avertin will be used to anesthetize the animals for the chest irradiation procedure. Depth of anesthesia will be monitored by breathing rate and response to tactile stimuli.

Exposure to hydrogen sulfide. Mice were placed in a sealed Perspex plenum chamber (IR1606) similar to that previously used for mice. The chamber will have two ports (input and output). A gas containing _H2S (80 ppm) equilibrated with room air was passed into the chamber at a rate of 1 liter per minute. Exhaust air from the room using a room ventilation system with a hose extending from the output vent to the room's exhaust vent.

Administration of Hazardous Agents. Mice were irradiated with a linear accelerator at a total dose of 17.5 Gy while they were in an air-filled chamber. This irradiation dose will induce subacute lung injury in mice that progresses to fibrosis. The mice would not be radioactive, which would cause injury to workers or other animals. Due to this exposure, no special monitoring, containment or handling is required.

Scheduled euthanasia. Approximately 13 and 26 weeks after chest irradiation, animals were euthanized by deep anesthesia (with Avertin 0.4-0.7 ml ip) followed by exsanguination by puncture of the inferior vena cava. Bronchoalveolar lavage was performed for determination of inflammatory cell numbers, differential counts, and eluate protein concentrations. Lung and esophageal tissues were removed for histological evaluation and analysis of collagen content.

dying animal. Chest irradiation was associated with limited mortality in mice, with 15% of mice dying 10 weeks after irradiation and 50% of mice dying at 22 weeks after irradiation. The investigators will monitor the animals daily for adverse effects (initially 2-3 times a day until they appear stable, then once a day until the disease begins to progress, at which point the inventors will revert to multiple daily observations). If the animal lost weight, was out of grooming, exhibited severe respiratory distress, and/or clumsy or significantly reduced activity, it was euthanized with avertin overdose. When practical, these occasional euthanized animals will be subjected to bronchoalveolar lavage and tissue harvested for histology.

Chest irradiation should produce lung damage, which is painless in itself but may manifest itself as increased respiratory rate, mild loss of appetite, mild weight loss and/or loss of grooming (week 10). Animals will be monitored daily for this adverse effect by the investigator and animal facility staff. Provide soft food and fluid support if the animal does not appear to be feeding. If the animal was perceived to be in pain, butorphanol (0.2 mg/kg i.p.) or buprenorphine (1.0 mg/kg bid s.q.) was administered as needed for analgesia. Animals were euthanized immediately if they showed distress and palliative measures did not result in improvement. Lung and esophageal tissue was collected at scheduled necropsy for histopathological evaluation and analysis of collagen content.

Post-irradiation management. In order to minimize the risk of retransmitting any pathogens to the rest of the facility, and to protect these animals in the event that they are somewhat immune-compromised, all handling of these animals will be performed first of the day (in the laboratory before any other animals in ) and will be performed in a biosafety room. To minimize the risk of exogenous infection, mice will use autoclaved cages and bedding. Also, they will be fed a standard rodent chow that has been irradiated to kill pathogens.

Wild genotype C57BL/6 mice will receive 17.5Gy chest irradiation. Mice will be anesthetized intraperitoneally with avertin, placed in individual cloth mouse restraints and moved into a sealed plexiglass plenum chamber (IR1606) similar to that previously used for mice. The chamber will have two ports (input and output). A gas containing _H2S (80 ppm) equilibrated with room air was passed into the chamber at a rate of 1 liter per minute. Exhaust air from the room using a room ventilation system with a hose extending from the output vent to the room's exhaust vent. Once inside the plenum, mice were irradiated by a linear accelerator with 8.5 Gy at a dose rate of 3 Gy/min through a calibrated target to only two lateral regions of the thorax (total thoracic radiation dose of 17.5 Gy). After completion of chest irradiation, animals were returned to their monitored micro-isolation cages until recovery from anesthesia.

A scheduled autopsy. A cohort of animals will be necropsied at week 13 after irradiation to assess the inflammatory phase of the injury. A second group of animals will be euthanized at week 26 to assess the fibrotic stage of injury. Animals were anesthetized with avertin and exsanguinated. Lungs were lavaged with 1000 μl PBS and the eluate was kept on ice for total and differential cell counts. The right lung was then harvested for hydroxyproline content analysis, and the left lung was perfused with 10% NBF through the trachea at a pressure of 25-30 cm. The esophagus, trachea, left lung and heart were immersed in 10% NBF and sent to the FHCRC Histology Shared Resource Laboratory (FHCRC) for processing and pathological evaluation.

Chest irradiation should produce lung lesions, which are not painful per se but may manifest themselves as increased respiratory rate, mild loss of appetite, mild weight loss and/or loss of grooming (week 10). Animals will be monitored daily for this adverse effect by the investigator and animal facility staff. Provide soft food and fluid support if the animal does not appear to be feeding. If the animal was perceived to be in pain, butorphanol (0.2 mg/kg ip) or buprenorphine (1.0 mg/kg bid sq) was administered as needed for analgesia. If the animal showed distress and palliative measures did not result in improvement, it was immediately euthanized by _CO2 asphyxiation.

The main problems may be esophagitis (leading to decreased food and water intake) and respiratory insufficiency (reduced oxygen intake). The inventors will check these animals 2-3 times a day until they are confident that they are stable and doing well, at which point the inventors can reduce the frequency of checks to once a day until the disease begins to progress, at which point revert to daily checks repeatedly. Supportive care will be provided in a variety of ways. If the animal is not eating or drinking well (manifested by weight loss and grooming behavior problems), the inventors will provide soft food and try fluid supplements (Lactated Ringer's solution, 1-2ml/mouse, Use a small-bore needle (>20G) for subcutaneous injection, 1-2 times a day). If the animal was perceived to be in pain, butorphanol (0.2 mg/kg ip) or buprenorphine (1.0 mg/kg bid sq) was administered as needed for analgesia. If the animal showed distress and palliative measures did not result in improvement, it was immediately euthanized by _CO2 asphyxiation. If the animal experiences significant pain or distress during chest irradiation, the animal will be euthanized by _CO2 asphyxiation.

A third experiment was essentially as described above, with telemetry mice subjected to a conditioned atmosphere of 80 ppm _H2S at a reduced room temperature of 10.5°C. During the experiment, the mouse was observed visually and its activity was recorded by a webcam, and telemetry measurements were recorded as described above. Mice were exposed to a conditioned atmosphere of 80 ppm _H2S and the temperature in the chamber was reduced to a constant 10.5°C. At the end of the approximately 6 hour period, heat was applied to the chamber by setting the chamber temperature to 25°C. Mice were warmed in a conditioned _H2S atmosphere until the mice's core temperature was between 17°C and 18°C, after which time point the conditioned atmosphere was replaced with room air. The core body temperature of the mice in the conditioned atmosphere was significantly lowered to 10.5°C, which was accompanied by a significant decrease in total locomotor activity. Respiration rate decreased to an undetectable rate by visual inspection for approximately 1 hour and 15 minutes. After warming the chamber, weak respiration was observed when the core body temperature of the mice reached 14°C. During the warming phase, the conditioned atmosphere was replaced with room air when core body temperature rose to between 17°C and 18°C and mice showed breathing and movement. Normal movement and respiration are fully evident when core body temperature returns to 25°C. The mice showed no significant changes in behavior compared with untreated animals.

Embodiment 6 :

Cellular and Mammalian Research

A. Canine Research

Canine studies will be conducted with dogs that have been surgically implanted with telemetry devices to monitor their core body temperature. Animals will be studied for 10 hours in the presence or absence of sublethal doses of hydrogen sulfide. During this period, their vital signs will be continuously monitored via telemetry. The ambient temperature will also be lowered to 15°C for 30 minutes to determine if this has any effect on the core body temperature of the animals.

The procedure will be performed with 2 groups of 2 dogs (total of 4). Due to the expense of telemetry equipment, the inventors will continue to perform these experiments. If the results from the first group show that the hypothesis is wrong, the study will be repeated with two dogs from the second group. If the results from the second group do not support the hypothesis, the project will be discarded.

Toxicology studies have shown that when _H2S levels are above the OSHA limit for humans (10ppm), it was previously shown that rats and mice exposed to 80ppm _H2S for 6 hours per day, 5 days per week for 90 days showed No adverse effects were observed. This includes gross and histopathological examination of intestines, lungs, heart, liver, kidneys or other organs at the conclusion of treatment. To the knowledge of the present inventors, no information is available regarding the exposure of dogs to hydrogen sulphide.

A key issue in working with _H2S is not to exceed the dose (80 ppm) described by others who have published studies exposing rodents to hydrogen sulphide and found no deleterious effects. There is considerable experience available in gas science, and the inventors were able to deliver prescribed doses of gas to mice. Numerous precautions are taken to ensure that neither animals nor researchers are harmed. These precautions include continuous monitoring of the gas mixture, alarms set to OSHA limits and a sensitivity of 1 ppm, and multiple devices capable of mixing and delivering gases according to instructions without leaking into or out of the system.

The timeline of the program is given in Table 5.

Table 5 - Timeline of Trials

sky Activity detail -1 Before surgery CBC/Chem will be administered; dogs will be fasted in the afternoon but will be allowed free access to water. 0 Operation A fentanyl transdermal patch was applied the afternoon before surgery for preemptive analgesia. Head catheterization prior to surgery; premedication with acepromazine, buprenorphine, glycopyrrolate; induction with ketamine: diazepam or propofol to enable intubation; maintenance of anesthesia with isoflurane and oxygen . Lay dog on its back and trim abdomen/surgical prep and drape. Monitoring of pulse, respiratory rate, end-tidal carbon dioxide, percent anesthetic inhaled, and _SpO2 is performed and recorded every 15 minutes or more frequently. Fluid support will be given during and after the procedure. Once the dog is stable and properly prepared for the procedure, an abdominal midline laparotomy will be performed, starting caudally to the umbilicus and extending 5-10 cm caudally. Place a sterile syringe into the peritoneal cavity. The placement is checked to ensure the emitter can move freely; momentum will be replaced and closure of the peritoneal cavity will be done in 3 layers. The dog will be monitored until it is removed from the tube, is able to withstand temperature regulation and lies on its chest. Dogs were monitored daily for incision site, abdomen (by palpation and ultrasound, if indicated), appetite, temperature (first 3-5 days post-surgery), weight, and activity.

7 determine the baseline The date is flexible. Only this step is performed with approval. Four animals will be placed on the receiver facility (this does not involve removing the animals from their cages and will be done in the AHR) and a baseline of vital signs will be determined for all four animals. 8 Exposure to H ₂ S Animals will be transferred to the room where the assay will be performed, where they will be placed in cages with food and water in an enclosed atmosphere. After establishing a baseline, two of the four animals will receive _H2S at a concentration of 80 ppm. After 10 hours of exposure, the atmosphere will return to room air temperature and the animals will be returned to their cages. Exposure to _H2S will be repeated weekly to initially determine whether any data set is reproducible.

B. Human platelets

To test the idea that the use of inhibitors of oxidative phosphorylation could be used to benefit humans, the inventors induced a state of stasis in human tissues to protect them from lethal exposure to oxygen. In a pilot test, the inventors placed human skin in a 100% CO environment. The inventors observed that after 24 hours the survival of skin cells in CO was 100 times better than those in room air. These results are very exciting: they demonstrate that inhibitors of oxidative phosphorylation may be effective in human tissues.

Another set of experiments demonstrated the protective effect of induced stagnation on platelets. Divide a unit of platelets in half. The first half was stored under standard storage conditions, which included keeping the platelets at room temperature (22-25° C.) with constant shaking. The other half was placed in an anoxic environment (<10 ppm oxygen) from which oxygen was removed by standard methods. These two groups of platelets were compared on day 0, day 5 and day 8. In a panel of 5 different in vitro assays, platelets stored under anaerobic conditions performed as well or better than those stored under standard conditions, including aggregation capacity, cell morphology, annexin-V Staining (phosphatidylserine flipped to the outer membrane as an early apoptosis marker), etc. This suggests that control of metabolic activity, particularly oxidative phosphorylation, can be accomplished by removal of oxygen and have protective effects on cellular function during prolonged stasis.

H2S binds cytochrome c oxidase like CO and terminates oxidative phosphorylation when required. It is so effective in blocking oxidative phosphorylation that if a person takes one breath in an atmosphere containing 0.1% hydrogen sulfide, they should not take another. Otherwise, they immediately fall to the ground - an event often referred to as a "knockdown" in an industrial context. This also appears to be reversible, as these individuals can sometimes resuscitate and go on living without neurological problems if removed quickly to fresh air (and the fall is uninjured). This is a substance that is not only common in our world, in fact, even produced in our own cells, but is a potent reversible inhibitor of oxidative phosphorylation without oxygen delivery.

C. Rat Studies

A hibernation-like state was induced with _H2S . By definition, warm-blooded animals maintain a core body temperature 10-30°C above the ambient temperature. For these animals to do this, they must generate heat from energy produced through oxidative phosphorylation. The terminal enzyme complex in oxidative phosphorylation is cytochrome c oxidase. Since hydrogen sulfide inhibits this complex (Petersen, 1977; Khan et al., 1990), the inventors predicted that exposing warm-blooded animals to hydrogen sulfide would prevent such animals from maintaining their core body temperature above ambient temperature.

To test this hypothesis, the inventors continuously monitored the core body temperature and activity level of homeothermic animals (mice). A telemetry device implanted intraperitoneally in a mouse can do both of these things and has the advantage of not introducing bias to the readings due to handling of the mouse (Briese, 1998). Additionally, they can remotely monitor mice during exposure to hydrogen sulfide gas. It has previously been shown that hydrogen sulfide doses of 80 parts per million (ppm) are not harmful to mice for exposures lasting up to 10 weeks (CIIT 1983; Hays, 1972). Therefore, for these experiments the inventors used a hydrogen sulfide dose of 80 ppm to test our hypothesis. Generating an atmosphere containing 80 ppm hydrogen sulfide is no small matter. Over time, hydrogen sulfide will be oxidized to sulfate in the presence of oxygen. To this end, in order for the inventors to continuously expose mice to an atmosphere containing 80 ppm hydrogen sulfide, the inventors continuously mixed room air with a tank of 500 ppm hydrogen sulfide equilibrated with nitrogen.

Characterization of core temperature control

Exposure of mice to 80 ppm _H2S lowered their core temperature to approximately 2 degrees Celsius above ambient temperature (Figure 5). This effect was highly reproducible, as the mean core body temperature of 7 mice exposed to 80 ppm hydrogen sulfide for 6 hours followed a similar pattern (Figure 5). In an ambient temperature of 13°C, the minimum mean core body temperature of these 7 mice was 15°C. All of these mice recovered successfully after rewarming when the atmosphere was switched to that containing only room air. As a control, the inventors replaced hydrogen sulfide with nitrogen and found no substantial decrease in core body temperature.

Although these mice appeared normal (despite a temporary decrease in core body temperature and respiration rate), the inventors performed a battery of behavioral tests to rule out the possibility that exposure to hydrogen sulfide gas, a dramatic decrease in core body temperature, and a decrease in respiration rate , or a combination of these effects causes neurological damage. All experiments were performed on mice before and after exposure to hydrogen sulfide. These behavioral tests were selected from the SHIRPA method developed by the Mouse Models for Human Disease consortium (Rogers et al., 1997). There were no detectable behavioral differences in the mice following gas exposure. From this, the inventors concluded that entering a hibernation-like state is not harmful.

Preliminary optimization of _H2S dosage. The experiment above describes the effect of 80 ppm hydrogen sulfide on core body temperature in mice. To determine the concentration of hydrogen sulfide sufficient to lose thermoregulation, the inventors exposed mice to a range of hydrogen sulfide concentrations (20 ppm, 40 ppm, 60 ppm and 80 ppm) (Figure 6). While 20 ppm and 40 ppm hydrogen sulfide were sufficient to cause a drop in core body temperature in mice, this was minor compared to the drops observed with 60 ppm and 80 ppm hydrogen sulfide. From this experiment, the inventors concluded that the loss of thermogenesis is directly dependent on the concentration of hydrogen sulfide provided to the mice. This preliminary study of the dose range and pharmacokinetics of hydrogen sulfide highlights the need for a more comprehensive analysis.

Preliminary determination of the low core temperature boundary. The inventors were also interested in establishing a more complete understanding of the tolerance of both the range of core body temperature and the length of time mice are allowed to remain in this state. The above experiments showed that the inventors could repeatedly lower the core body temperature of mice to 13-15°C when necessary. Furthermore, the mice appeared to tolerate the treatment for several hours. Using the same protocol, the inventors succeeded in achieving a core body temperature of 10.7° C. in mice while reducing the ambient temperature ( FIG. 7 ). Further attempts to lower core body temperature even lower, and for longer periods of time, will be undertaken in the future. Although preliminary, these results demonstrate that there is a significant range of biologically tolerated core body temperatures in mice and that this range can be explored by loss of thermoregulation due to exposure to hydrogen sulfide.

Regulation of endogenous _H2S levels. It is well known that mammalian cells endogenously produce hydrogen sulfide (Wang 2002). Since this chemical is dynamically produced in cells, it is extremely important to understand the basal levels under different conditions, as this can significantly affect the pharmacokinetics of externally administered hydrogen sulfide. To address this important aspect of our research, the inventors have set out to examine endogenous hydrogen sulfide levels in mice. The present inventors used an extractive alkylation technique combined with gas chromatography and mass-specific detection to quantify hydrogen sulfide (Hyspler et al., 2002). Using this method, the inventors observed hydrogen sulfide levels in undisturbed mice. Figure 8A shows the presence of significant amounts of hydrogen sulfide in this mouse. Additionally, hydrogen sulfide levels appeared to depend on the mice's ambient temperature. Specifically, when the mice were in a cold environment, they decreased endogenous sulfide levels and when the mice were in a warmer ambient temperature, they increased endogenous sulfide levels. From this, the inventors concluded that mice regulate their sulfide levels in response to ambient temperature.

Changes in endogenous levels affect the potency of _H2S . Because ambient temperature alters endogenous levels of sulfide in mice, the inventors hypothesized that ambient temperature might affect changes in core body temperature upon exposure to extrinsic hydrogen sulfide. Acclimatization of mice to low temperature ~12°C produced a long-lasting plateau that the inventors observed after core temperature had started to decline (Fig. 8B). Thus, this suggests that this cold adaptation makes the mice more resistant to core body cooling induced by the action of hydrogen sulfide gas. However, acclimatizing the mice to warm thermoneutral temperatures prior to exposure to the gas abolished this plateau. In fact, normothermic mice cooled much more rapidly when exposed to hydrogen sulfide than cold-adapted mice (Fig. 8B). These data suggest that endogenous levels of hydrogen sulfide in mice have a direct effect on the potency of exogenous hydrogen sulfide.

_H2S protects mice from hypoxia. Normal indoor air contains about 21% oxygen. In a preliminary experiment investigating the protective effects of stasis against hypoxia in a mouse model, mice exposed to 80 ppm hydrogen sulfide survived 11 minutes of 5.2% oxygen, and 3 weeks later, the mice were still doing well. Previously published work showed that 90% of these animals (C57B1) exposed in this way without hydrogen sulfide did not survive (Zhang et al., 2004). The test consisted of pre-equilibrating the mice in 80 ppm _H2S for 3 hours and then reducing the oxygen tension in the chamber as described in the experiment above. The same flow rate was used as described above (ie 500 cc/mL in a 0.5 L chamber). It is well established by those familiar with the field that if a group of mice is exposed to 4% oxygen, 100% will die within 15 minutes. However, mice in which _H2S was administered during the reduction in oxygen tension to 4% remained viable under these hypoxic conditions, even for extended periods of time (up to 1 hour). The mice appeared unaffected by these conditions after recovery and were alive and responding normally when tested 24 hours later. This assay differs from the one above in that mice remain in _H2S at the end of hypoxic exposure until oxygen tension returns to normal levels (21% _O2 ).

Example 7

Additional Animal Studies

A. Protection from Adverse Conditions

This test was performed to test the ability of a mouse to survive in a 'hibernation-like' state under conditions where it would normally die. A disadvantage is hypoxia, for which it has been reported that mice (C57BL6/J males) can survive up to 20 minutes in 5% oxygen (Zhang et al., 2004).

As shown in Table 6, experiments consisted of exposing mice to 80 ppm (unless otherwise stated) _H2S for the indicated times, after which the oxygen tension in the chamber was reduced, while still under _H2S . The time of exposure to hypoxia (as described below) was recorded and the viability of the mice was determined.

Short exposure of mice to _H2S (at least 80ppm) was less successful in protecting mice from hypoxia, but at least one mouse survived 50 minutes after only 8 minutes in _H2S under hypoxic exposure. Furthermore, it was observed that a mouse exposed to 90 ppm _H2S for only 10 minutes survived longer at 5% oxygen, although it eventually died.

Exposure of mice to 80 ppm _H2S for longer periods of time had a strong effect on protecting mice from hypoxia for up to 1 hour.

Table 6

Ambient temperature Time in _H2S before exposure to hypoxia Oxygen% time in low oxygen result 20℃ 5 hours 5.20% 11 minutes Survive 20℃ 5.5 hours 5.00% 25 minutes Survive 20℃ 5 hours 5.00% 60 minutes Survive 20℃ 5 hours 4% 28 minutes Survive 24°C H ₂ S free 5% 14 minutes die 24°C Simultaneously 5.10% 10 minutes die 24°C 8 minutes 5% 20 minutes die 24°C 8 minutes 4.00% 8 minutes die 24°C 8 minutes 4.50% 23 minutes die 30℃ 8 minutes 4.50% 6 minutes die 24°C 10 minutes (90ppm) 5% 56 minutes die 24°C 8 minutes 5.00% 50 minutes Survive

B. Enhance hypoxia tolerance

1. Background

The use of carbon dioxide ( _CO2 ) and hydrogen sulfide ( _H2S ) to enhance the survival of the complex metazoan Drosophila melanogaster under hypoxia was investigated. These experiments showed that these substances, especially _H2S , increase hypoxia tolerance in adult Drosophila.

C. elegans embryos survive hypoxia (<10 ppm _O2 ) by entering stagnation, and development can proceed in 0.5% _O2 . However, there is a 10-fold range of lethal oxygen concentrations (0.01-0.1% _O2 ). Furthermore, blocking oxygen utilization with carbon monoxide prevents hypoxic damage to embryos. Therefore, it is better not to have (or use) any oxygen if there is not enough oxygen available for efficient biological activity.

In more complex metazoans, the cellular oxygen concentration is not necessarily the same as the oxygen level of the environment. In C. elegans, oxygen is delivered to tissues by diffusion. However, in higher organisms there are proteins such as hemoglobin that bind oxygen in order to transport it to tissues. Therefore, when the oxygen level of the environment drops, there may be residual oxygen in the cells.

Most organisms are not immune to exposure to environmental hypoxia. One possibility is that residual oxygen at the cellular level is toxic, corresponding to the range of lethal oxygen observed in C. elegans embryos. In this case, survival under hypoxia should increase if residual oxygen is removed or becomes unavailable. _CO2 promotes the release of _O2 from hemoglobin and _H2S is a potent inhibitor of oxidative phosphorylation.

2. Materials and Methods

Basic experimental protocol. Adult flies were introduced into 35 mL tubes made of glass (Balsh tubes) with airtight rubber stoppers. This is usually done by anesthetizing flies with CO ₂ , moving groups of flies into vials with food to recover for at least 2 hours, and then transferring them into Balsh tubes. To exchange the gas environment in the Balsh tube, insert two 18-gauge needles into the rubber stoppers, and let gas blow at 100 mL/min into one injection needle. To prevent drying, the gas was humidified by bubbling it through 10 mL of water before passing through the Balsh tube. The water in the bubbler was equilibrated with gas for at least 20 minutes before the test began.

For "stopped-flow" experiments, gas exchange was performed for 60 minutes before sealing the tubes. For the "low-flow" test, the gas flow was continued throughout the test. CO ₂ was from a room source (100%), and an anoxic environment was established by blowing the room air with 100% nitrogen (N ₂ ). When switching the atmosphere from _CO2 to _N2 , take care to prevent introducing room air into the system.

After the hypoxic treatment, oxygen was reintroduced into the Balsh tubes by flooding with room air for 20 min. The rubber stopper was then removed and a food bottle was placed upside down on top of the Balsh tube with parafilm. Flies were scored as alive if they regained mobility. Viability was assessed at least 18 hours after the end of the hypoxic treatment. After 2 weeks, flies were considered fertile if the food vial contained larvae and/or pupae.

3. Results

Treat with _CO2 before exposure to hypoxia. If adult flies were first pretreated with _CO2 , they showed higher hypoxic survival. Adult flies pretreated with _CO for 30 or 90 min showed 54% or 28% survival, respectively, after 19 h of hypoxic exposure in a stop-flow test. No survivors were observed in controls exposed to hypoxia without _CO2 preconditioning or exposed to _CO2 followed by no exposure to hypoxia. Furthermore, no flies pre-treated with CO ₂ survived the hypoxic exposure if flies were also exposed to CO ₂ immediately after the 20 min hypoxic exposure.

Short-term exposure to _CO2 is sufficient to enhance survival in hypoxia. In the stop-flow test of 22 hours hypoxic exposure, the proportion of surviving flies was highest if _CO2 was administered 0.5-5 minutes before switching to nitrogen atmosphere (Figure 16). Therefore, for subsequent experiments, the standard protocol is to treat with _CO for 10 min prior to exposure to hypoxia. In slow-flow assays using this protocol, 6% of adult flies survived 20 hours of hypoxic exposure, and this survival required _CO2 preconditioning.

Trials have shown that it is important to prevent re-introduction of _O2 between _CO2 treatment and establishment of _N2 environment. When the water in the bubbler used to humidify the gas was not equilibrated with _N2 before blowing out the _CO2 , no flies survived the 13 h hypoxic exposure in these trials, regardless of whether _N2 was given at 10, 50 or 100 mL/ Minutes introduced. Under these _conditions , the _CO2 atmosphere was purged out with a _N2 / _O2 mixture produced by _{dissolving O2} in water.

A series of slow-flow experiments were performed to determine the duration of hypoxic exposure for _CO2 exposure compared to no pretreatment, and each condition was tested in duplicate (Figure 17). In these data, the trend was that _CO2 pretreatment resulted in greater survival. An important note is that these trials deviate from the standard protocol, except that flies are anesthetized with _CO2 and transferred to Balsh tubes, and are only allowed to recover for 10-20 min before starting _CO2 treatment (except for trial 2, p. 18 hour time point).

Several other trials were performed which did not provide information on whether pretreatment with _CO2 would be beneficial. For example, in one trial no survivors were observed after 17, 22 and 24 hours of hypoxia in a slow-flow trial with a 10 min _CO2 preconditioning phase. In other experiments, however, many flies survived 17 hours. This could explain that in some cases other factors influenced the results, such as the age of the adult, changes in circadian rhythm or room temperature. In another trial comparing stop-flow and slow-flow protocols, no flies survived 17 or 19.5 hours of hypoxic exposure; however, mold contamination may have contributed to the death of flies in this case.

Treatment with _H2S prior to hypoxic exposure . Including _H2S in the pretreatment regimen more significantly enhanced the ability of adult flies to survive hypoxia. In a series of experiments similar to those shown in Figure 17, the addition of 50 ppm _H2S to the _CO2 pretreatment ( _H2S / _CO2 ) increased the proportion of flies that survived the treatment (Figure 18). The flies appeared healthy and produced offspring after exposure. However, in similar experiments, no flies survived 18, 20, 25 or 30 hours of hypoxia after 10 minutes in _H2S / _CO2 . The reason for this difference is unclear. Consistent with the beneficial effects of _H2S treatment, after 15 h of hypoxic exposure, 50% of flies pretreated with _H2S survived, whereas control flies not exposed to _H2S did not recover. In this test, flies were treated with CO ₂ for 10 min, followed by H ₂ S/CO ₂ for 10 min, followed by N ₂ /H ₂ S for 10 min, and finally N ₂ for the continuous slow-flow test. duration.

_H2S -dependent increase in hypoxic survival does not require _CO2 treatment. 25% of the flies treated with 50 ppm _H2S in room air 18.5 hours before inducing hypoxia survived. The proportion of surviving flies was unaffected if the exposure to H ₂ S/CO ₂ was increased for 10 min before establishing the hypoxic environment. In a control experiment in which flies were treated with CO alone before hypoxic exposure, only 11% of flies recovered.

The timing of _H2S administration appears to be important in increasing hypoxic survival. No flies recovered if _H2S was present throughout the hypoxic exposure (20 h), regardless of the presence or absence of _H2S during _CO2 pretreatment. However, if 50ppm _H2S was present in the _CO2 pretreatment and subsequently removed when the hypoxic environment was established, 35% of the flies survived. In a parallel experiment, 6% of flies exposed to hypoxia survived pretreatment with _CO2 (without _H2S ) for 10 minutes.

Initial experiments with larvae and embryos. Increased hypoxic survival was also observed in embryos and larvae after treatment with CO and HS-containing CO. After 24 h exposure to hypoxia, 7 pupae formed from a group of 0-19 h old embryos. However, 20 pupae were observed in paired pools pretreated with _CO for 10 min. Similarly, larvae exposed to 24.5 h of hypoxia were reactivated upon reoxygenation only if they were pretreated with _CO2 or _H2S / _CO2 . Embryos aged 0-24 h survived and developed to the adult stage after 18.5 h of hypoxic exposure, with or without pretreatment with _CO2 or _H2S / _CO2 .

Cold treatment during hypoxia. Lowering the ambient temperature increases the length of time adult flies can survive hypoxic exposure. At room temperature, no flies survived the 15.5 hours of hypoxic exposure in the stop-flow protocol, but 20% of those kept at 4°C while remaining hypoxic recovered. Similarly, flies that were switched to hypoxia at 4°C and subsequently moved to room temperature for 16.5 hours did not survive. However, flies kept at 4 °C throughout the exposure recovered and were fertile at 16.5 h and even after 40 h. Pretreatment with _CO2 prior to establishment of the hypoxic environment did not make a significant difference in these trials.

Example 8:

drop in core body temperature

In both rats and mice, studies have shown that with _H2S and _CO2 , metabolic output can be reduced, manifested by a decrease in core body temperature. Figures 20A-B show that at time 0 when _H2S or _CO2 was first applied, the core body temperature of the animals began to drop. After 6 hours, when the _H2S or _CO2 was removed, body temperature began to return to normal. Apparently larger mammals require more _H2S to affect metabolism.

Prophetic Example 9:

gas matrix

To determine the concentration of each constituent gas in a custom mixed atmosphere that provides the greatest ability to control metabolic flexibility in mammals, the following experiments can be performed. Gases include oxygen (O ₂ ), nitrogen (N ₂ ), carbon dioxide (CO ₂ ), hydrogen sulfide (H ₂ S), and helium (He). While _H2S may reduce oxygen demand in mitochondria, _CO2 may further reduce oxygen demand. Additionally, it has been found that a reduction in core body temperature is necessary to reduce metabolism. Therefore, helium, with its high heat capacity, offers a simple and non-invasive cooling method. Furthermore, with 100% _O2 , normoxic 20.95% oxygen can be maintained in any gas mixture where the other components make up less than 79.05% of the total. And finally, nitrogen was used to equilibrate the mixture to 100%.

These experiments describe a progressive approach to single and combined detection of gases first in mice, then in rats, and subsequently in dogs. The goal of the gas matrix is to develop a basis upon which to study with multiple variables in a logical order. The experimental design is depicted in Figure 21.

One of the features of this gas matrix is that it indicates that a test will only be performed if the preceding tests (connected by arrows) are completed. Mixing trials will not be performed in any animal model without first optimizing the constituent gases. Furthermore, a gas or gas mixture will not be used in rats without first optimizing the dose in mice, and will not be used in dogs without first optimizing in rats. Thus, it shows the progression of experiments with single gases to multiple gas mixtures (read from top right to bottom left) and from mice to rats to dogs (read from top left to bottom right). Mice will always be used first to determine the concentrations of the constituent gases that provide the best control over metabolic flexibility. Once the most effective dose of gas has been determined in mice, the same experiment will be performed in rats. In parallel, the next gas or gas mixture will be assayed with mice. Once concentrations are established in rats, the gas will be tested in dogs. The table below provides a slightly different way of viewing the gas matrix and determining the sequence of trials:

order mouse rat dog 1 H ₂ S+CO ₂ CO ₂ 2 He/ _O2 H ₂ S+CO ₂ CO ₂ 3 _H2S + _CO2 +He He/ _O2 H ₂ S+CO ₂ 4 _H2S + _CO2 +He He/ _O2 5 _H2S + _CO2 +He

Procedure 1. Carbon Dioxide (CO ₂ )

Mice: 15% _CO2 was found to provide control over metabolic flexibility in mice. However, we were not able to test higher concentrations due to limitations in our mixing capabilities. This notion is no longer true, and we can test _CO2 concentrations up to 80%. So we'll start with 15% _CO2 and increase to 40% in 5% increments, then 80% in 10% increments. Animals will be exposed for 6 hours. Mice will be exposed with a 375 ml glass chamber into which the animals will be provided with water and into which a premixed gas atmosphere will flow at a rate of 500 ml per minute. These glass chambers will be contained within an incubator so that the ambient temperature can be controlled. This table provides the framework for our high-concentration _CO2 experiments, but additional experiments may be needed to better understand the effects. Mice can be used in multiple experiments, but we will not use an animal more frequently than once a week. In addition, animals can be used as their own controls in subsequent experiments.

%CO ₂ 15 20 25 30 35 40 50 60 70 79 %O ₂ twenty one twenty one twenty one twenty one twenty one twenty one twenty one twenty one twenty one twenty one % N ₂ 64 59 54 49 44 39 29 19 9 0

Metabolism ( _O2 consumption and core body temperature) and activity will be monitored. At 15% _CO2 , tidal volume increased but respiratory rate remained constant. The mice showed no "panting". Increasing _CO2 concentrations should enhance the anesthetic effect.

These experiments will be performed in an incubator so that we can then lower the ambient temperature to 10°C to examine the relationship between core body temperature and metabolic output.

Rats: Experiments in rats will begin with a nitrogen atmosphere balanced with 3% _CO2 and 21% _O2 . This is considered normocapnic because it is the exhaled _CO2 concentration. Starting at 3%, we will increase to 15% in 2% increments. This increase can be done in a single baseline trial where we increase the _CO2 concentration until we observe a change in metabolism. Metabolism will be monitored by measuring _O2 consumption and core body temperature. A single rat in one trial can be used to determine this minimum _CO2 dose. In subsequent trials, where the effect of 6 hours of _CO2 exposure will be examined, a single rat will be used for only one _CO2 dose (ie, the level will not change during the course of the trial). Rats may be used for multiple experiments; they will be used no more than once per week. Animals will be exposed in 2,800 ml glass containers into which water will be provided and premixed gas will flow at a rate of 3 liters per minute. This table shows the structure of the initial _CO2 experiment with rats, but additional experiments are needed to fully study the effects.

%CO ₂ 3 5 7 9 11 13 15 %O ₂ twenty one twenty one twenty one twenty one twenty one twenty one twenty one % N ₂ 76 74 72 70 68 66 64

From 15% to 80%, the test will progress as performed with mice (15%-40% in 5% increments, 40%-80% in 10% increments). Metabolism and behavior will be monitored. Once the effective dose of _CO2 for rats is determined, the ambient temperature will be lowered to investigate whether metabolism is further reduced by core temperature reduction as done with _H2S . These tests will be performed in an incubator that will provide cooling at the start and duration of the test and heating at the end of the test.

After completing the _CO2 study in rats, it will be known whether rats need more _CO2 than mice (true for _H2S ), or the same, or less _CO2 for reducing them metabolism. Understanding this key allometric trend will provide better assumptions about effective _CO2 dosage in dogs.

The termination point for these procedures in mice and rats would be a 99% reduction in _O2 consumption or a 99% reduction in _CO2 production.

Dogs: The experimental design for dogs will be the same as for rats and mice (using a flow rate of 10 L/min). Trials will start with 3% _CO2 and increase until a physiological response is observed. Dogs were exposed to the gas mixture using an anesthesia mask to which the dogs had been pre-conditioned prior to exposure. _O2 consumption and core body temperature will be monitored. The same one or two dogs can be used for these trials.

Procedure 2. Hydrogen sulfide and carbon dioxide (H ₂ S+CO ₂ )

By mixing _H2S and _CO2 , we will be looking for a synergistic effect of these two gases. Namely, can _H2S and _CO2 be used together to reduce metabolism as deeply as higher concentrations of either gas. In the initial experiments, using mice, 20 ppm of _H2S will be used and titrated into _CO2 to 15% (or other concentration determined in procedure 1). One animal can be used to vary the concentration of _CO2 while keeping _H2S constant. Second, we will use 40ppm _H2S and add _CO2 to reach 15%. Third, we'll use 80ppm _H2S and let the _CO2 go to 15%. One animal can be used for each of these experiments. _O2 consumption, core body temperature, and behavior will be monitored. The experiments listed in the table provide the basis for studying the effect of the _H2S + _CO2 mixture and additional experiments will be required to understand the effect.

test O ₂ % H ₂ S ppm _CO2 % 1 twenty one 20 5-10-15 2 twenty one 40 5-10-15 3 twenty one 80 5-10-15

Once an effective mixture is determined, the ambient temperature will be lowered to investigate whether the optimized gas mixture can act as a synergist with low core body temperature to further reduce metabolic rate. In these temperature-dependent tests, the concentration of the gas in the mixture will not change. Again, one animal can be used for multiple experiments, no more than one experimental procedure per animal per week.

Experiments with rats and dogs will be performed in the same way. _CO concentrations for rats and dogs will be optimized in Procedure 1, but it is assumed that they will be between 5% and 15%. For dogs, high _H2S concentrations greater than 400 ppm have not been determined.

Procedure 3 Helium (He)

Helium is an effective heat dissipator; it conducts heat six times greater than nitrogen. It has been used in many mammals including rats, dogs and humans to promote cooling. It is non-toxic, inexpensive, and easy to handle. Helium is expected to be used like other gases in an 80%/20% mixture with oxygen (He- _O2 ) to enhance thermal conductivity through respiration. Five preliminary experiments were presented to analyze the effects of He- _O2 on metabolism and behavior. A widely used standard 80%-20% blend will be used. A mixture containing 60% He will also be tested to better reflect the minimum He- _O2 mixture we will use in Scheme 5 where we mix _H2S , _CO2 and He.

test animal temperature He-O ₂ -N ₂ 1 mouse 23°C 80-20-0 2 mouse 10℃ 80-20-0 3 mouse 23°C 60-20-20 4 mouse 10℃ 60-20-20 5 rat 23°C 80-20-0 6 rat 10℃ 80-20-0 7 rat 23°C 60-20-20 8 rat 10℃ 60-20-20 9 dog 23°C 80-20-0 10 dog 23°C 60-20-20

Oxygen consumption, carbon dioxide production, core body temperature and behavior will be monitored to see if the same effect that other gases have can be induced with He- _O2 .

Procedure 4: Oxygen (O ₂ )

Reducing oxygen concentration reduces core body temperature has been shown by Gellhorn and Janus in 1936 with guinea pigs. It is expected that these experiments will be replicated first in mice, then in rats, and finally in dogs to investigate whether reduced _O2 concentrations reduce metabolism.

Mice: Experiments in which mice will be exposed to reduced O _{concentrations} as low as 6% are presented. Trials will progress in 5% increments. _O2 consumption, _CO2 production, core body temperature, and behavior will be measured. If convulsive behavior indicative of extreme hypoxia is observed, restore _O2 to 21%. The table below provides a summary of this _O2 test. The exposure time was 6 hours in a control chamber (with water) into which the premix gas flowed. Due to the evidence of hypoxic preconditioning, four separate animals were used for these four experiments.

test %O ₂ % N ₂ 1 twenty one 79 2 16 84 3 11 89 4 6 94

Once the relationship between _O2 partial pressure and metabolism has been established, it may be important to repeat the experiment in a cold environment. These tests will be carried out exactly as the previous tests except that the temperature of the oven will be lowered to 10°C.

Rats: Rats are expected to perform the same experiments previously performed with mice. If convulsive behavior indicative of extreme hypoxia is observed, return the _O2 concentration to 21%.

Dogs: If a positive correlation between reduced oxygen tension and reduced metabolic rate is observed in mice and/or rats, it would be desirable to perform the same series of experiments with dogs.

Procedure 5: Hydrogen sulfide, carbon dioxide, helium and oxygen (H ₂ S+CO ₂ +He+O ₂ )

These experiments were the goal of this matrix of gases; to determine the mixture of _O2 , _CO2 , _H2S and He that provides the most robust and reversible control of the metabolism, combined with an optimized ambient temperature. Therefore, using the concentrations of the individual gases determined in the previous procedure as a basis, mixtures of these four gases were assayed to find the mixture that provided the best control over metabolic flexibility. _O2 , _CO2 and _H2S will change relative to each other while helium will be used to equilibrate the mixture. In the mouse experiment shown below, _CO2 was varied while _O2 and _H2S were held constant; helium will be varied to maintain a constant flow. We will use the same metabolic assays including oxygen consumption and core body temperature. One animal can be used for multiple experiments. Mice will be exposed for 6 hours. These experiments were then repeated at lower ambient temperatures to see how much lower core body temperature with the gas mixture affected metabolism.

mouse:

O ₂ H ₂ S CO ₂ He test Concentration% Concentration ppm Concentration% balance 1 twenty one 20 5-10-15 2 twenty one 40 5-10-15 3 twenty one 80 5-10-15 4 16 20 5-10-15 5 16 40 5-10-15 6 16 80 5-10-15 7 11 20 5-10-15 8 11 40 5-10-15 9 11 80 5-10-15 10 9 20 5-10-15 11 9 40 5-10-15 12 9 80 5-10-15 13 6 20 5-10-15 14 6 40 5-10-15 15 6 80 5-10-15

Rats: After knowing what the optimal gas mixture is for mice, the same experiments as those performed with mice will be performed with rats, except that the _H2S concentrations are 100, 200 and 300 ppm. Rats will be treated for 6 hours.

Dogs: Experiments using dogs will be consistent with those using mice and rats. The concentration will start at 300ppm and go to the concentration to be measured. An animal can be used for multiple experiments but no more than once per week. Mice and rats will be treated for 6 hours and dogs will be treated for 2 hours.

Prophetic Example 10:

Choice of hydrogen sulfide dose in humans

Arrest can be induced by administering hydrogen sulfide to animals or humans by any of a variety of dosage forms and routes of administration, including, but not limited to, inhalation gas form or intravenous administration of hydrogen sulfide solution. Methods are described for determining dosage forms and routes of administration of hydrogen sulfide sufficient to induce arrest in whole organisms in need of arrest. Expose test organisms (e.g., rats, dogs, pigs, monkeys) to increasing concentrations of hydrogen sulfide administered intermittently or continuously as a single dose and monitor physiology while removing blood samples (0.5 mL) at multiple time points State, including but not limited to core body temperature, oxygen consumption, carbon dioxide production, heart rate, blood pressure, respiration rate, blood pH, motion and wakefulness. The concentration of hydrogen sulfide present in plasma derived from the blood of the test animals is measured by methods known in the art including, but not limited to, X derivatization, Y extraction, and quantification using gas chromatography and mass spectrometry.

The correlation of steady-state plasma levels of hydrogen sulfide produced by specific dosing regimens in test animals with the achievement of arrest to varying degrees in test animals determines the effective dose of hydrogen sulfide sufficient to induce arrest in test animals. Effective dose for inducing arrest in humans in need of arrest by determining the dose, route of administration of hydrogen sulfide to obtain the same stable plasma concentration of hydrogen sulfide in humans as obtained in experimental animals under conditions in which arrest is induced and dosing regimen determination. The effective concentration of hydrogen sulfide to achieve stagnation in humans depends on the dosage form and route of administration. For inhalation, in some embodiments, effective concentrations range from 50 ppm to 500 ppm for continuous delivery. For intravenous administration, in some embodiments, effective concentrations range from 0.5 to 50 milligrams per kilogram of body weight delivered continuously.

The range in each case is characterized by the increasing degree of stagnation obtained with increasing doses of hydrogen sulfide. A dose of hydrogen sulfide sufficient to cause a sustained, 12-24 hour reduction in core body temperature of 3-5°C to 32-34°C in an unconscious person suffering cardiac arrest and cardiac resuscitation and recovery outside the hospital is expected relative to unexposed Similar humans to hydrogen sulfide have a significant survival advantage, as described in Bernard et al. 2002.

Example 11:

Animal Preconditioning Studies

The studies shown in Example 7 demonstrate that pre- and continuous treatment of male C57Bl/6 mice with _H2S enhances their ability to survive hypoxic conditions of 5% oxygen or 4% oxygen.

To determine the effect of pretreatment with H ₂ S alone on viability under hypoxic conditions (no continuous exposure to H ₂ S during hypoxia), mice were exposed to 5% O ₂ (5%), 4 % O ₂ (4%), 4% O ₂ after 1 hour of 5% O ₂ (4% + 1 hour 5%) or 3% O ₂ after 1 hour of 5% O ₂ (3% + 1 hour 5%) before exposure to room air for 30 minutes (without pretreatment), or after exposure to room air for 10 minutes to 150 ppm hydrogen sulfide in room air (pretreatment) for 20 minutes, and determine their survival time. The test was terminated at 60 minutes and the animals that were still alive were returned to their cages. As shown in Figure 23, all mice in a group of animals pre-exposed to 150 ppm _H2S in room air for 20 minutes survived the subsequent exposure to 5% _O2 , whereas all controls exposed to room air only Animals all died within 15 minutes of exposure to 5% _O2 . Thus, pre-exposure of mice to _H2S establishes a physiological state in the mice that enables prolonged survival of otherwise lethal hypoxia. The protective effect observed in _H2S -pretreated mice far exceeds the known protective effect of whole-body hypoxic preconditioning that has been reported in the literature in which 5% _O2 Viability was only extended 2-fold (Zhang et al., 2004). Although not shown in Figure 23, some _H2S -pretreated mice were able to survive longer than 4 hours in 5% _O2 and were able to recover without significant motor or behavioral deficits.

To determine whether _H2S pretreatment enhanced survivability to even lower oxygen tensions, mice were exposed to lower _O2 concentrations. As shown in Figure 24, _H2S pretreatment greatly enhanced survival in the presence of 5% _O2 . In comparison, _H2S pretreatment only slightly increased survival in the presence of 4% _O2 . However, if _H2S -pretreated mice were exposed to gradually decreasing _O2 levels such that they were first pretreated and then exposed to 5% _O2 for 1 h, subsequently exposed to 4% _O2 or 3% _O2 , their survival time was enhanced to the same level as that observed when they were exposed to 5% _O2 after _H2S pretreatment (Figure 28). Thus, pre-exposure to _H2S established a physiological state in which mice could survive a stepwise reduction in oxygen tension (21% normoxia to 3% _O2 ) over 80%. Furthermore, in some experiments, stepwise reduction of oxygen tension after _H2S pretreatment showed that mice could survive oxygen tension as low as 2.5% for 1 hour.

These data, and those described in Example 7, demonstrate that exposure to _H2S has pharmacological effects, with greatly improved survival in otherwise lethal hypoxia. In this regard, the pharmacological effects of _H2S depend on the dose level and duration of exposure to _H2S , parameters that can be varied by those skilled in the art to obtain optimal survival to lethal hypoxia. Those skilled in the art will appreciate that the route of administration (eg, inhalation versus parenteral administration) may also be altered to achieve the desired lethal hypoxic tolerance effect in mammals. Additionally, the pharmacological effects described can be observed when _H2S exposure is limited to preconditioning or extended to the hypoxic period. Likewise, the timing of exposure to _H2S relative to the onset of lethal hypoxia can be varied to maximize enhanced viability. These data are consistent with the hypothesis that the reduction in oxygen demand resulting from pretreatment with active compounds such as oxygen antagonists enables survival of a reduction in oxygen supply that would otherwise be lethal to the animal.

To characterize the metabolic changes present in an environment of enhanced survivability to lethal hypoxia produced by H ₂ S treatment, during exposure to H ₂ S and then termination of H ₂ S treatment and subsequent exposure to 5% During the O ₂ process, measure the CO ₂ production of the mice. The change in _CO2 production is shown in Figure 25. Measurement of _CO2 upon switching to 5% _O2 or 4% _O2 in mice exposed to room air for 30 minutes (no pretreatment) or room air 10 minutes followed by 150 ppm _H2S for 20 minutes (pretreatment) resulting changes. In addition, the change in _CO2 production upon a gradual transition to 5% _O2 for 1 h and then to 4% _O2 was measured. The results of these experiments are provided in FIG. 29 .

_CO2 production was reduced approximately 2- to 3-fold within the first 5-10 min of _H2S pretreatment, indicating that arrest was induced in mice during 20 min pretreatment with 150 ppm _H2S in room air. However, _O2 consumption and core body temperature of the animals were not significantly altered during _H2S pretreatment (data not shown), suggesting that during _H2S exposure, a survival response to lethal hypoxia was established in mice. A physiological state of heightened vitality rather than stagnation. This state can be characterized by a reduction in metabolism within the biomaterial below the magnitude defined as stagnation. In order to achieve stagnation with an active compound, the biomass must necessarily transition through a progressive hypometabolic state in which oxygen consumption and _CO2 production in the biomass are reduced by less than 2-fold. This continuous state in which metabolism or cellular respiration is reduced to less than twofold by the active compound is described as a "pre-arrested" state. Continuous monitoring of CO production after cessation of H ₂ S pretreatment and induction of lethal hypoxia, shown in Figure ₂₅ , demonstrated an approximately 50-fold reduction in CO production, _suggesting that the stagnation was during exposure to lethal hypoxia. acquired. The concomitant decrease in _O2 consumption and strong attenuation of activity in mice during exposure to lethal hypoxia further supports the observation that arrest is subsequently acquired during exposure to lethal hypoxia.

Changes in _CO2 production associated with switching to hypoxic conditions of 5% _O2 or 4% _O2 after _H2S pretreatment or without pretreatment were measured. As shown in Figure 28, mice exposed to 5% _O2 in the absence of _H2S pretreatment or mice exposed to 4% _O2 in the presence of _H2S pretreatment exhibited CO ₂ produces a large reduction. In contrast, H ₂ S-pretreated mice exposed to either 5% O ₂ subsequently, or 4% O ₂ after 5 _% O ₂ , had an increase in did not show any significant change in _CO2 production. These results demonstrate an association between reduced metabolic activity and mortality associated with exposure to 5% _O2 without _H2S pretreatment, or to 4% _O2 with _H2S pretreatment. Furthermore, these data demonstrate that exposure to 5% O ₂ after H ₂ S pretreatment, or a gradual reduction from 5% O ₂ to 4% O _{2 ,} did not result in additional reductions in metabolic activity. To summarize these results, the decrease in _CO2 evolution that occurs upon transition from normoxia to lethal hypoxia was retarded in mice pretreated with _H2S . Switching from normoxia to lethal hypoxia resulted in a 40% reduction in _CO2 evolution, but pretreatment with _H2S , which itself resulted in a 50-60% reduction in _CO2 emission to a new, lower baseline, prevented the Any further reduction in _CO2 evolution upon transition to lethal hypoxia. These data demonstrate that pretreatment with _H2S alone prevents the additional reduction in metabolic activity normally associated with the transition to lethal hypoxia, thereby improving survival under hypoxic conditions. Furthermore, these data support a model in which pre-exposure of biological material to active compounds is sufficient to enhance viability and/or reduce damage from injury or disease damage.

Example 12:

Hydrogen selenide reduces core body temperature in mice at reduced concentrations

It has been previously reported in the literature that _H2Se greater than 1 ppm is lethal to animals. Experiments were performed according to the materials and methods described in Example 4, except that an even lower concentration of _H2Se than _H2S was used. The _H2Se used had an initial concentration of 20 ppm in nitrogen from a source tank, which was then diluted with room air to approximately 10 or 100 parts per billion (ppb). Animals are then exposed to the mixture.

Two mice were exposed to 100 ppb _H2Se for less than 10 minutes. Figure 26 shows a reduction in core body temperature and a 3-fold reduction in metabolic activity as evidenced by respiration in one mouse.

The concentration of _H2Se was reduced even further to 10 ppb. Mice exposed to 10 ppb _H2Se also experienced a reduction in core body temperature and respiration (Figure 27).

Furthermore, the effect of _H2Se appears to be fully reversible based on experiments used to assess reversibility using _H2S (Blackstone et al., 2005, which is hereby incorporated by reference).

Example 13:

Hydrogen sulfide protection against lethal bleeding

The studies shown in Examples 7 and 11 demonstrate that treatment of mice with hydrogen sulfide ( _H2S ) enhanced their ability to survive hypoxic conditions of 5% oxygen or 4% oxygen. To determine whether _H2S treatment could also be used to reduce mortality and/or tissue damage associated with a more clinically relevant model of ischemic hypoxic acute injury, _H2S treatment was administered during a controlled lethal hemorrhage In rats, the lethal hemorrhage reduces oxygen supply to tissues and leads to death (Blackstone et al., 2005). In this study, rats treated with _H2S survived lethal blood loss and fully recovered.

Rats were treated with _H2S during controlled lethal bleeding (60% blood loss). Following surgical implantation of the catheter and recovery, blood was removed from conscious animals within 40 min. A small amount (300 ppm) of _H2S mixed with room air was administered to the treated animals 20 minutes after the start of exsanguination (ie after 30% blood loss). Animals were returned to _H2S -free room air at the end of the exsanguination. Three hours after the end of exsanguination, the surviving animals were administered intravenously with the outflowing blood volume of Ringer's lactate.

Most (6/7) of the _H2S -treated rats survived the hemorrhage and the 3-hour shock period and fully recovered (Table 7). None of these surviving rats showed behavioral or functional deficits after recovery. One _H2S -treated rat died 174 minutes after the end of bleeding. All untreated animals died within 82 minutes after bleeding ended; mean survival time of untreated animals was 35+/-26 minutes. The p-value was 0.0047 using a two-tailed Fishers exact T-test.

During the first 20 minutes of bleeding (before 30% blood loss), rats increased respiratory rate and tidal volume to compensate for the reduced oxygen carrying capacity due to blood loss. This increase in ventilation resulted in a decrease in respiratory carbon dioxide production (V _CO2 ) (Table 7). After 60% blood loss, both _H2S -treated and untreated animals showed a decrease in _VCO2 . Arterial lactate increased while pCO ₂ , bicarbonate ([HCO ₃ ⁻ ]), pH, and base excess decreased (Table 7). Hemorrhage thus leads to metabolic acidosis with respiratory compensation. However, these changes were smaller in magnitude in _H2S -treated rats, indicating a reduction in metabolic acidosis. Furthermore, in _H2S -treated animals, _VCO2 did not continue to decrease after hemorrhage. In untreated animals, V _CO2 decreased steadily until the animals stopped breathing. _H2S administration was shown to prevent the progression of the shock response to death.

Table 7. Survival and Physiology of Rat Bleeding Model Using _H2S

H ₂ S treated unprocessed Survive Full recovery to the time of non-survivor death 85.7% (6/7) 174 (1/7) 0%(0/7)35+/-26 CO ₂ generation (VCO ₂ ) in ml/kg/min: Bleeding before bleeding Middle bleeding ends 25+/-420+/-216+/-2 26+/-621+/-311+/-3

15 minutes after bleeding 17+/-3 7+/-5 Blood CO2 content in mmHg (pCO2) Bleeding ends before bleeding 45+/-635+/-6 44+/-321+/-3 Blood bicarbonate content in mmol/L ([HCO3-]) Bleeding ends before bleeding 32+/-321+/-3 30+/-112+/-3 blood pH Bleeding ends before bleeding 7.46+/-0.037.41+/-0.02 7.45+/-0.027.35+/-0.06 Blood base excess in mmol/L Bleeding ends before bleeding 8+/-2-5+/-4 6+/-1-14+/-3 Blood lactate in mmol/L Bleeding ends before bleeding 1.4+/-0.56.6+/-1 1.2+/-0.211+/-3

Example 14:

Benefits of short-term exposure to hydrogen sulfide during bleeding

Male Sprague-Dawley rats weighing 275-350 g were purchased from Charles River Laboratories and acclimated one week prior to each experiment. On the day of the test, catheters were surgically implanted in the right femoral artery and vein. The catheter emerges behind the scapula. Rats were administered buprenorphine post-surgery and allowed to recover.

The anticoagulant drug heparin (80-100 units) is administered intravenously in a one-time dose to reduce the blood's ability to clot and enhance bleeding. After heparin administration, unrestrained rats were placed individually in 2.75 liter crystallization dishes with glass lids. Pass the conduit, temperature probe and gas sampling line through the hole drilled in the center of the cover. The temperature was kept approximately constant (27+/-2°C).

The bleeding model was defined as the removal of 60% of the total body blood during a 40 minute bleeding session. Blood was removed with a peristaltic pump. To determine the amount of blood constituting 60% of the total body, rats were weighed and blood volume calculated using the following equation: (0.06X body weight)+0.77 (Lee et al., (1985)).

Treatment groups received exposure to room air containing hydrogen sulfide (test animals), or room air containing nitrogen (control animals), administered at a rate of 3 liters per minute through a thermal mass flow controller (Sierra Instruments).

Hydrogen sulfide ( _H2S ) (20,000 ppm, balanced with nitrogen) (Byrne Specialty Gas) was diluted into the room air to reach a concentration of 2000 ppm for treatment. Blood was removed via the femoral catheter at the calculated rate. Blood was weighed as it was removed. After 20 minutes (or at 50% of the 40 minute exsanguination), the test animals were exposed to room air containing 2000 ppm hydrogen sulfide. The exposure was terminated when the animal showed apnea and dystonia. The average length of exposure to hydrogen sulfide ( _H2S ) is generally between 1 and 2 minutes. The maximum _H2S concentration in the chamber is estimated to be between 1000 and 1500 ppm. Animals were exposed to room air when apnea and dystonia were observed. The test animals resumed a regular breathing pattern within 20 to 30 seconds of exposure. Control animals were bled at the same rate as the test animals but did not receive treatment with hydrogen sulfide. Control animals showed no apnea or dystonia during the test.

Metabolic rate was determined by measuring _CO2 production (Licor Li7000). Temperature and _CO2 data were collected (ADI PowerLab). Arterial blood values were measured (I-Stat blood chemistry analyzer). After bleeding, the animals were caged for 3 hours and observed. At the end of 3 hours, Lactated Ringer's solution was given to surviving rats ad libitum. For animals that did not survive, the time of death was declared when the animal ceased breathing and _CO2 production ceased. After resuscitation, rats were transferred to clean cages with food and water and housed at 30°C for approximately 16 hours. Catheters were surgically removed and animals were allowed to recover at 30°C for several hours before being transferred back to the colony. Behavioral and functional tests were selected from the panel described in the SHIRPA protocol (Rogers et al., 1997).

In these experiments, 7 out of 8 (88%) animals treated with hydrogen sulfide ( _H2S ) during the hemorrhage survived the hemorrhage. Two control animals that did not receive treatment died during the 3 hour observation period.

Embodiment 15 :

Additional results from Example 2

As discussed in Example 2 above, human foreskins were used to assess the preservation of cells and tissues in carbon monoxide. A total of 8 human foreskins were finally evaluated (3 reported in Example 2). The number of viable keratinocytes was assessed with trypan blue (Table 8 and Figure 30). This shows that carbon monoxide exposure increases the number of viable cells.

              Table 8. Tests of exposure to indoor air (RA) by trypan blue (tb) staining

Viability of isolated keratinocytes from foreskin or CO for 24 hours.

RA CO Live (tb-) dead(tb+) alive score Live (tb-) dead (tb+) alive score SUM 010070109 31003412142 0.000.500.170.000.330.000.18 104105492431106 14314263161 0.910.500.770.830.540.800.500.500.63

Untreated CO #recoverycell51167 Ratio of alive 0.180.63 t-test 0.000883884

Example 16:

Low-level chronic _H2S exposure increases viability

Using the methods and apparatus described in Example 1, C. elegans nematodes were exposed to low levels of _H2S (<100 ppm). Nematodes adapted to this treatment displayed increased lifespan and resistance to heat stress, however, there was no discernable reduction in metabolic activity as would be the case with induced arrest.

In C. elegans, failure to perform aerobic metabolism (by reducing the ambient oxygen concentration or adding CO) leads to the induction of stagnation or stasis (see Example 1). However, stasis cannot be induced by exposing them to <100 ppm _H2S in room air. At doses greater than 100 ppm, _H2S can cause considerable lethality in exposed nematode populations. Interestingly, even in cases where most of the worms were killed by _H2S , those that survived behaved normally and were apparently not harmed by the substance. Worms grown in about 50 ppm _H2S completed embryogenesis, developed to sexual maturity and produced offspring at the same rate as siblings raised in the absence of _H2S . In contrast, in oxygen concentrations (below 3.5% _O2 ) where the metabolic rate is reduced, all these processes are slowed down. Furthermore, worms reared in _H2S produced the same number of offspring as controls in room air, indicating that there are no deleterious effects from these conditions. These data indicate that _H2S did not reduce the metabolic activity of C. elegans under these conditions.

Nematodes grown in _H2S were more resistant to heat stress than age-matched controls in room air alone. In this assay, worms reared in 50 ppm _H2S were exposed to high temperature in _H2S , and worms reared in room air were exposed to room air. Thus, _H2S -induced resistance to stress was not associated with reduced metabolic activity. However, this resistance to heat-stress requires adaptation of the nematodes to the _H2S environment. Worms reared in room air and exposed to heat-stress in _H2S died more rapidly than if they were exposed in room air. Adaptation to _H2S was sustained to the extent that worms reared in H2S and exposed to heat stress in room air survived better than controls _reared in room air. Additionally, worms adapted to non-toxic low concentrations of _H2S (eg, 50 ppm) can counteract higher concentrations of _H2S that are lethal to non-acclimated worms.

These data are consistent with data that Drosophila briefly exposed to _H2S subsequently survived hypoxia better than untreated controls (see, Example 7 and Example 11). Protection from heat stress (in worms) and hypoxic stress (in Drosophila) suggests that _H2S can enhance survival under a variety of adverse or stressful conditions that may be encountered clinically.

Worms adapted to 50 ppm _H2S had increased lifespan compared to isogenic untreated controls (Figure 32). This is consistent with the idea that they are in a state that is generally more tolerant to the various stresses associated with aging. In fact, aged worms grown in _H2S appeared more robust and healthy than those of a similar age that were not treated with _H2S . Furthermore, this is also true when comparing worms from each population at mid-life (i.e., the room-air control and _H2S -treated worms were not age-matched in time, but 50 of each population % is dead which matches). Thus, adaptation to _H2S slows aging-associated chronic cellular damage in C. elegans.

Example 17:

Implementation of Gas Matrix Test

Metabolic flexibility was assessed in mice, rats and dogs using altered gaseous environments. Three parameters were used to define this reduction in metabolism, including carbon dioxide production as measured by respirometry, changes in oxygen consumption, and changes in core temperature as measured by telemetry. In experiments with mice and rats, the animals were placed in a sealed chamber with one gas input and one gas outlet. For dogs, a mask is placed over the animal's snout, to which two hoses (inlet and outlet) are attached. Gas flow rates for each animal: mice - 500 cc per minute, rats - 2 liters per minute, and dogs - 40 liters per minute. Each atmosphere was constructed from compressed gases by dilution into room air, unless otherwise noted. For the rat and mouse tests, the ambient temperature during exposure to the test gas was 7-10°C. For dogs, the ambient temperature was room temperature (22°C).

Table 9: Construction for detection of metabolic flexibility in mice, rats and dogs

A description of the gaseous environment.

mouse rat dog Hydrogen Sulfide Hydrogen Selenide Phosphine Carbon Dioxide H ₂ S+CO ₂ CO ₂ +Low O ₂ CO ₂ +Low O ₂ +He+77% 0.01% with 0.0001% without 0.016% with 15% with 0.01%+15% with 15%+8% with 15%+8%+77% 0.03% 0.003% N/D 15% N/D 15%+8% 15%+8%+77% Without 0.85% N/DN/D without 9% N/DN/D with 9%+15%

Table 9 shows the amount of each gas given as a percentage of the room air atmosphere unless otherwise stated. Evidence of inhibition of metabolic rate greater than 5-fold as judged by carbon dioxide production or oxygen consumption during 6 hours of treatment is described by "yes"; "absent" (decrease in these values or less than 5-fold reduction is described as such); "N/D" indicates that the test was not performed. In the dog test (carbon dioxide + low oxygen + helium) the temperature dropped about 1.5°C during the 30 minute exposure. Such a drop in temperature was considered significant because the dogs were 12 kg and such a drop in temperature was not observed during extensive baseline recordings of the animals in room air.

Animals exposed to various constructed atmospheres displayed metabolic flexibility as evidenced by changes in core body temperature (CBT) close to ambient temperature (Figures 33-40). Figure 33 demonstrates that rats exposed to an atmosphere containing 15% _CO2 , 8% _O2 , and 77% He have accelerated metabolic depression compared to rats exposed to 300 ppm _H2S under similar conditions. Figure 34 demonstrates a significant reduction in CBT in mice exposed to 1.2 ppm _H2Se . Rats exposed to room air at an ambient temperature of 10°C did not show a significant reduction in CBT (Figure 35). Rats exposed to 80% He, 20% _O2 at 7°C also did not show a significant reduction in CBT (Figure 36). Rats exposed to an atmosphere of 15% _CO2 , 20% _O2 , 65% He at an ambient temperature of 7°C showed a significant reduction in CBT (Figure 37). Likewise, Figure 38 shows a significant reduction in CBT in rats exposed to an atmosphere of 15% _CO2 , 8% _O2 , 77% He at 7°C. A significant reduction in CBT was also demonstrated in dogs exposed to an atmosphere of 9% _CO2 , 20% _O2 , 71% He (Figure 39). The magnitude of this reduction was lower, likely due to the animal's larger size and limitations on thermal diffusion. Similar reductions were observed in dogs exposed to different concentrations of _CO2 .

Example 18:

Compound Screening

A compound screen was performed to identify test compounds capable of causing a reversible decrease in subcutaneous temperature in mice. The identified test compounds were then tested for their ability to provide protection from lethal hypoxia (measured at 4% _O2 as compared to a typical environment of nitrogen atmosphere balanced by 21% _O2 ). The whole screening procedure consists of 3 steps:

1) a first step (1°) screening to determine the minimum effective dose of the test compound that will cause a measurable decrease in the subcutaneous temperature of the test mice;

2) A second step (2°) screen to determine the reversibility of temperature reduction, as defined by test mice with normal behavior at 24 hours post treatment and return to normal subcutaneous temperature within 24 hours or less; and

3) A third step (3°) screening to assess the ability of test mice to survive lethal hypoxia (4% O ₂ ) compared to untreated control subjects under the same hypoxic conditions.

The mice used in these studies were 5-6 week old, jugular vein cannulated (JVC) male C57BL/6 mice (Taconic) with a subcutaneous RFID temperature sensor (IPTT-300, Bio Medic Data Systems , Inc. (BMDS)) and allowed to recover for at least 24 hours. Mice were dosed by infusion of test compounds through an indwelling catheter using a 1 or 5 ml Luer-Lok syringe (Becton Dickinson) and infusion pump (Harvard Apparatus). The DAS-6008 data acquisition module from BMDS records the subcutaneous temperature of the mice via a transponder, and this data is entered into a computer spreadsheet and plotted against time.

The first step (1°) screening:

For the first step of the screen, infusions of test compounds were prepared at concentrations considered to be the maximally optimal concentrations. The pH is adjusted to 6-8 with NaOH or HCl, the osmolarity is adjusted to 250-350 mOsm with NaCl, and the total dose of test compound administered (mg) divided by the test subject's body weight (kg) does not exceed 400% of its published mg/kg LD50 in mice.

Mice were placed in tall glass bottom containers with opaque walls and infused via the jugular vein. Test compounds were infused using a step-wise regimen with increasing infusion rates over the course of 2 hours (Table 10).

Table 10. Step-by-Step Infusion Protocol for Test Compounds

time (minutes) Infusion rate (μl/min) Infused microliters Total infused microliters 0-2020-4040-6060-8080-100100-120 0.81.63.26.312.725.4 15.87531.7563.5127254508 15.87547.625111.125238.125492.1251000.125

During the infusion, read the subcutaneous temperature of the mouse every 3-5 min and record any changes in the behavior of the mouse. The results of the first step screen show whether the test compound has the ability to lower the subcutaneous temperature to 33°C or lower, and indicate the effective dose required to lower the subcutaneous temperature, which is obtained by first observing The infusion rate of the test compound is measured as the steady temperature decreases.

The second step (2°) screening:

Test compounds that cause a decrease in the subcutaneous temperature of mice to 33°C or lower are tested in a second screen. In the second screen, mice are infused with test compound for 60 minutes at a rate of 50% of the effective infusion rate determined in the first screen. During the infusion, monitor the subcutaneous temperature of the mice by taking measurements every 3-5 min. If the subcutaneous temperature does not decrease within the first 60 minutes, double the infusion rate and continue the infusion for an additional 60 minutes. When the subcutaneous temperature of the mice dropped to 33°C or lower, the infusion was stopped immediately, and the recovery of the mice was evaluated by measuring the subcutaneous temperature and observing the behavior of the mice. The temperature and behavior of the mice were observed and recorded 24 hours after treatment. The results of the second screen determine whether the test compound causes a reversible decrease in subcutaneous temperature without lethality.

Third Step/Lethal Hypoxic (3°) Screen: In the third step screen, mice are infused with the test compound at the rate determined in the second step screen. The subcutaneous temperature of the mice was measured every 3-5 minutes until it decreased to 33°C, as in the second screening step. The infusion was stopped and mice were immediately transferred to a hypoxic (4% O ₂ ) chamber along with control mice infused with vehicle (saline solution, 148 mM, osmolarity=300) or without deal with. The airtight glass chamber was perfused with a continuous flow of air and nitrogen to achieve the desired hypoxic atmosphere of 4% _O2 . If the mouse survived the hypoxic atmosphere for 60 minutes, it was transferred back to room air and its recovery was monitored for 24 hours by recording subcutaneous temperature and by behavioral observation.

Control mice usually died within 6-15 minutes.

Mice infused with sodium sulfide (effective dose 0.79 mmol/kg), sodium thiomethoxide (effective dose 4.61 mmol/kg), or sodium thiocyanate (effective dose 4.67 mmol/kg) survived exposure to lethal Sexual hypoxia for 60 minutes. Mice infused with cysteamine (effective dose 7.58 mmol/kg) survived lethal hypoxia for 45 minutes; mice infused with cysteamine-S-phosphate sodium salt survived lethal hypoxia for 31 minutes; and mice infused with thiopyran-4-ol survived lethal hypoxia for 15 minutes. These survival rates were compared to those of control mice, which typically died within 6-15 minutes in a hypoxic environment.

In contrast, some other test compounds identified in the first screen as having the ability to lower body temperature did not protect animals against lethal hypoxia. Thioacetic acid, selenourea, and S-(2-((3-aminopropyl)amino)ethyl)phosphorothioate all lowered body temperature but did not improve survival in hypoxia. 2-Mercapto-ethanol, mercaptoethylene, and 2-mercaptoethyl ether all lowered body temperature but were toxic at doses effective at lowering temperature. Thiourea, dimethyl sulfide, sodium selenide, sodium methanesulfinate, and N-acetyl-L-cysteine did not reduce subcutaneous temperature at the highest dose given in this study. Dimethyl sulfoxide was excluded as the effective dose (10% DMSO) was considered too high for medicinal purposes.

These studies established that the developed screening program can be successfully used to identify compounds that can protect animals subjected to lethal hypoxia. In addition, the results of this study suggest that the identified compounds, and others to be identified using this procedure, can be used to protect patients from hypoxia-induced injury as well as ischemic injury.

Example 19:

daily exposure to active compounds

The ability to physiologically adapt to chronic treatment with hydrogen sulfide ( _H2S ) and the time required for adaptation have been examined in mice. Adaptation was defined as animals not showing a decrease in core body temperature (greater than 4° C.) during chronic exposure to 80 ppm hydrogen sulfide in room air. Long-term treatment was defined as exposure to 80 ppm hydrogen sulfide in room air for 4 hours per day, 4 days per week, over a six-week period.

Mice used in these studies were 5-6 week old male C57BL/6 mice or male C129 mice. A telemetry device was implanted in the body cavity of the mice prior to the experiment to record core temperature.

Mice (8 per treatment) were exposed to hydrogen sulfide at a flow rate of 10 liters per minute in a Plexiglas box with a separate chamber for each mouse. Carbon dioxide production and oxygen consumption of animals were measured in 500cc glass bowls with a flow rate of 0-5 liters per minute.

Over the course of one week, the mice adapted to the hydrogen sulfide exposure on average. Adaptation was defined as when animals did not show a decrease in core temperature (greater than 4° C.) when exposed to 80 ppm hydrogen sulfide in room air for 4 hours. Mice that did not show a decrease in core temperature were considered to have physiological adaptations to hydrogen sulfide. H2S-treated mice that developed adaptations showed increased oxygen consumption (vO2) compared to carbon dioxide production (vCO2) when compared to untreated control mice (Figure 42). _H2S -adapted mice showed lower respiratory quotient (RQ ratio), defined as the ratio of vCO2/vO2 or the ratio of carbon dioxide produced to oxygen consumed (Figure 43).

Example 20:

Application to thalassemia

Based on current results in other model systems presented here, it is expected that erythrocytes from animals with a hematological disorder (thalassemia) will have an increased ability to resist oxidative damage when they are treated with sulfide, resulting in prolonged erythrocyte survival . The following experiments will be carried out to confirm that treatment with active compounds can protect animals with thalassemia from oxidative damage.

In the first series of trials, animals with thalassemia will be treated by chronic exposure to the active compound. Following an initial trial to establish a baseline, treatment will be initiated according to the protocol summarized below. If erythropoiesis or erythrocyte survival is improved, effects may be observed as early as 1-2 weeks, as the half-life of thalassemia erythrocytes is estimated to be 4-7 days. We will look at the smear and get a reticulocyte count at two weeks and start a more extensive study after another two weeks. If an improvement in red blood cells (as identified by elevated reticulocyte counts and blood smears) is detected in mice, the study will continue until steady state is observed in the metrics used in the monthly studies. The study has a projected final endpoint of one year. At one year, the erythrocyte survival study will be completed and the animals will be sacrificed. If no improvement is observed, exposure will continue until another year, at which time survival studies will be completed and animals will be sacrificed.

plan 1:

1) Animals will undergo initial testing.

2) Within 1 week of the initial study, the animals are similarly housed and:

A) Exposure to 80ppm _H2S for 8 hours/day;

B) not exposed; or

C) Give water with 0.25% dimethyl sulfide (DMSO) and allow to drink ad libitum (estimates from previous studies suggest that mice will consume 5-10 cc/day/mouse with 2.5-25 micrograms/day DMSO content , using an average body weight of 18 g per mouse, consumption was estimated at 700-1,400 ug/kg/day).

In a second series of experiments, the in utero effects of treatment with the active compounds will be determined according to the protocol summarized below.

Scenario 2:

1) Three days after conception, pregnant dams (Plugged dams) will be treated in one of the following groups:

A) Exposure to 80ppm _H2S for 8 hours/day;

B) not exposed; or

C) Water given with 0.25% dimethyl sulfide (DMSO) and allowed to drink ad libitum (estimates from previous studies suggest that mice will consume 5-10 cc/day/mouse with 2.5-25 micrograms/day DMSO content. Using an average body weight of 18 g per mouse, consumption was estimated at 700-1,400 ug/kg/day).

2) Pregnant dams will be allowed to give birth spontaneously and pups will be genotyped shortly after birth and sacrificed for detailed analysis.

Test animals will be monitored throughout the course of these experiments, as indicated below.

monitor:

1) Initial research:

1. Reticulocyte count;

2. Blood smear;

3. Computed tomography (CT) of the spleen and bones;

4. _O2 consumption and _CO2 production;

5. Weight;

6. Sulfide metabolites and

7. Hematocrit (60 μl of total blood).

2) At two weeks: reticulocyte count and blood smear (5 μl blood).

3) Monthly research:

1. Reticulocyte count;

2. Computed tomography of the spleen and bones;

3. _O2 consumption and _CO2 production;

4. Weight; and

5. Sulfide metabolites (less than 30 μl of blood drawn).

4) One month before sacrifice: erythrocyte survival study.

5) Sacrifice and detailed in vitro analysis.

Example 21:

Uses for Sickle Cell Anemia

To test this hypothesis, a mouse model of sickle cell anemia (SCD) will be used in which a strain has been engineered so that it no longer expresses mouse Hba and Hbb, but expresses human HBA and HBB (Patsy , et al., 1997). It mimics the genetic, hematological and histopathological features found in persons with sickle cell anemia, including irreversible sickling of red blood cells, anemia and multiorgan pathology. A significant percentage of sickle cell mice do not survive to adulthood.

Using this mouse model, various agents and sulfide-containing compounds will be tested for their efficacy against SCD. Exposure will be acute and chronic, and animals will be exposed at birth or in utero. Survival to birth or survival to adulthood for pups exposed in utero will be an endpoint for measuring efficacy. Phenotypic effects will be assessed by reticulocyte count, hematocrit, and red blood cell (RBC) half-life measurements (which are typically 20-fold less for SCD compared to wild-type controls).

Example 21:

Cyanide Exposure Test

This example shows that when mice were exposed to 80 ppm cyanide in room air, they gradually decreased their core body temperature to about 34°C.

One goal of these metabolic flexibility studies is to identify compounds that can reduce oxygen consumption and protect animals from hypoxic injury. Previously, hydrogen sulfide (H ₂ S), a potent inhibitor of oxygen consumption, has been shown to reduce metabolism and protect mice and rats from hypoxic injury. Hydrogen cyanide (HCN) is similar to _H2S in many respects, and we wanted to use our assay of metabolic output to see if it could be used to regulate metabolism. Like _H2S , HCN is widely used in industrial chemical synthesis and it is found in many biological systems, including humans. It is not known whether HCN is simply a by-product of carbon-nitrogen metabolism or whether it has specific biological activities. Like _H2S , HCN is thought to act by reacting with transition metal-containing proteins such as oxidases and dehydrogenases. HCN does not react strongly with components of hemoglobin.

In humans, the NIOSH IDLH (immediately hazardous to life and health) value is 50 ppm. OSHA PEL (Permissible Exposure Limit) TWA (8 hour time weighted average) is 10ppm. The LC50 for rats is 143 ppm for 60 minutes.

To measure the effect of HCN on metabolism, mice were exposed to increasing concentrations of HCN (starting at 1 ppm). Measure or assess oxygen (O ₂ ) consumption, carbon dioxide (CO ₂ ) production, core body temperature (BCT) and behavior. The concentration of HCN was increased in 10 ppm increments until an effect on metabolism was observed or the animal appeared to show signs of distress. The effect on metabolism was defined as a 10% change in less than 10 minutes for any of the measurements described above.

It was found that when mice were exposed to 80 ppm of cyanide in room air at room temperature, they gradually lowered their core temperature to approximately 34°C. This differs from the decrease seen with 80 ppm hydrogen sulfide, where the core temperature decreased to approximately 28°C. Furthermore, recovery of core temperature was very slow in mice exposed to cyanide (approximately 14 hours) compared to exposure to hydrogen sulfide (approximately 2 hours).

tested the hypothesis that in cyanide, slow recovery (judged by core temperature) could be rescued by a brief exposure to 80 ppm hydrogen sulfide. This is based on the idea that a conserved enzyme, thiocyanate generating enzyme (and others like it), might use hydrogen sulfide and cyanide to produce the relatively less toxic substance thiocyanate. It has been shown that thiocyanate generating enzymes can use cyanide and thiosulfate to produce thiocyanate. (Chen 1933) Furthermore, intravenous administration of thiosulfate is the standard of care for the treatment of cyanide poisoning in the United States. found that the time to recover core body temperature after exposure to cyanide was reduced by brief treatment with hydrogen sulfide. These results suggest that hydrogen sulfide exposure may be used to treat conditions in which patients suffer from cyanide poisoning.

       ***************

All of the compositions and methods disclosed and claimed herein can be made and performed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that changes may be made in the compositions and methods described, as well as in the steps of the methods described herein. Changes may be made in the sequence or order of steps without departing from the concept, spirit and scope of the invention. More particularly, it will be apparent that certain substances, both chemically and physiologically related, may be substituted for the substances described herein, while the same or similar results will be obtained. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Claims (149)

CLAIMS 1. A method for enhancing the viability of biological material, the method comprising providing said biological material with an effective amount of at least one active compound. 2. The method of claim 1, wherein the active compound is an oxygen antagonist. 3. The method of claim 1, wherein the substance is exposed to an active compound. 4. The method of claim 1, wherein the active compound is a chalcogenide. 5. The method of claim 1, wherein the active compound has the chemical structure of Formula I or Formula IV. 6. The method of claim 5, wherein the substance is exposed to a precursor of the chemical structure of Formula I or Formula IV. 7. The method of claim 1, wherein said substance is provided with a combination of active compounds. 8. The method of claim 1, wherein said active compound is provided to said substance before, during or after an injury, disease onset or progression, or bleeding in said substance. 9. The method of claim 8, wherein said injury comprises bleeding. 10. The method of claim 9, wherein said damage is from an external physical source. 11. The method of claim 8, wherein the active compound is provided prior to injury or prior to onset or progression of a disease. 12. The method of claim 8, wherein said injury or disease is associated with a decrease in metabolism or temperature of said substance. 13. The method of claim 12, wherein said injury is a surgical procedure. 14. The method of claim 11, wherein the active compound is not provided during or after the onset or progression of the injury or disease. 14.2. The method of claim, wherein the active compound is provided during the progression of the disease. 14.3. The method of claim 14.2, wherein the disease is thalassemia, sickle cell disease, or cystic fibrosis. 15. The method of claim 11, wherein said active compound is provided to said substance in an amount and for a duration of said providing sufficient to reduce _CO2 evolution by at least 25%. 16. The method of claim 11, wherein said active compound is provided to said substance in an amount and for a duration of time that does not cause the substance to enter stagnation. 17. The method of claim 11, wherein said active compound is provided to said substance, the amount of the compound and said duration of provision protect the substance from damage or death, wherein said damage or death is caused by damage or disease onset or progression. 18. The method of claim 11, wherein said active compound is provided to said substance in an amount and for a duration sufficient to increase the rate at which the substance enters a stasis following onset or progression of injury or disease. 19. The method of claim 11, wherein said active compound is provided to said substance in an amount and for a duration sufficient to prevent further reduction in _CO2 evolution following onset or progression of injury or disease. 20. The method of claim 11, wherein said active compound is provided to said substance in an amount and for a duration sufficient to cause the substance to enter stasis. 21. The method of claim 1, wherein said active compound is provided to said substance under hypoxic or anoxic conditions or prior to exposure to hypoxic or anoxic conditions. 22. The method of claim 21, wherein said hypoxic or anoxic conditions damage said substance in the absence of said active compound. 22.4. The method of claim 22, wherein said substance is not exposed to said active compound during exposure to hypoxic or anoxic conditions. 23. The method of claim 21, wherein said substance is provided with said active compound in the form of a gas, semi-solid liquid, liquid or solid. 24. The method of claim 23, wherein said substance is provided with at least one of said active compounds in gaseous form. 25. The method of claim 23, wherein said substance is provided with said active compound in at least one semi-solid liquid form. 26. The method of claim 23, wherein said substance is provided with at least one of said active compounds in liquid form. 27. The method of claim 26, wherein said active compound is effervescent in said liquid. 28. The method of claim 26, wherein said active compound is dissolved in said liquid. 29. The method of claim 1, wherein at least two active compounds are provided sequentially. 30. The method of claim 1, wherein at least two oxygen antagonists are provided together. 31. The method of claim 1, wherein said substance is exposed to one of said oxygen antagonists for a period of time ranging from about 30 seconds to 30 days. 32. The method of claim 7, wherein said combination comprises an oxygen antagonist having the formula of at least one selected from the group consisting of: a) a compound of formula I; b) a compound of formula II; c) a compound of formula III; d) a compound of formula IV; or a salt or precursor thereof. 33. The method of claim 32, wherein the combination comprises more than one active compound from the same group. 34. The method of claim 32, wherein at least one active compound is from group a). 35. The method of claim 32, wherein at least one active compound is a chalcogenide or a chalcogenide salt. 36. The method of claim 35, wherein at least one active compound comprises sulfur. 37. The method of claim 35, wherein at least one active compound comprises selenium. 38. The method of claim 35, wherein at least one active compound is a chalcogenide salt. 39. The method of claim 38, wherein the chalcogenide salt is selected from the group consisting of _Na2S , NaHS, _K2S , KHS, _Rb2S , _Cs2S , ( _NH4 ) _2S , ( _NH4 )HS, The group consisting of BeS, MgS, CaS, SrS and BaS. 40. The method of claim 33, wherein said combination comprises more than one active compound from group a). 41. The method of claim 40, wherein the combination of oxygen antagonists comprises _CO2 . 42. The method of claim 32, wherein at least one active compound is from group b). 43. The method of claim 42, wherein the active compound has the formula II(a), Formula II(b) or Formula II(c). 44. The method of claim 32, wherein at least one active compound is from group c). 45. The method of claim 44, wherein the active compound has formula III(a), formula III(b), formula III(c), formula III(d), formula III(e), formula III(f), A chemical formula of formula III(g) or formula III(h). 46. The method of claim 1, wherein said active compound is provided to said biological material by inhalation, injection, catheterization, maceration, lavage, perfusion, topical application, absorption, adsorption, or oral administration. 47. The method of claim 1, wherein intravenous, intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular, intraprostatic, intrapleural, intratracheal, intranasal, intrathecal, intravitreous, vaginal Internal, intrarectal, superficial, intratumoral, intramuscular, intraperitoneal, intraocular, subcutaneous, subconjunctival, intracapsular, transmucosal, intrapericardial, intraumbilical, intraocular, oral, topical, topical, by inhalation, via Administration to the biological substance by injection, by infusion, by continuous infusion, by local infusion, via catheter, or via lavage provides the active compound to the biological substance. 48. The method of claim 1, comprising exposing said biological material to a controlled temperature and/or pressure environment. 49. The method of claim 48, wherein the biomass is exposed to a controlled temperature environment. 50. The method of claim 49, wherein the biological matter reaches a non-physiological core temperature. 51. The method of claim 50, wherein the biomass is exposed to a controlled temperature environment below about 20°C. 52. The method of claim 49, wherein the biological material is exposed to a controlled temperature environment prior to and/or simultaneously with providing the one or more active compounds. 53. The method of claim 1, wherein the one or more active compounds comprise a cationic structure capable of targeting the one or more active compounds to mitochondria. 54. The method of claim 1, wherein said biological material is to be preserved. 55. The method of claim 54, wherein said biological material comprises platelets. 56. The method of claim 54, wherein said biological material is to be transplanted. 57. The method of claim 1, wherein said biological substance is at risk of ischemia-reperfusion injury. 58. The method of claim 57, wherein cardioplegia is induced within said biological substance. 59. The method of claim 1, wherein said biological substance is an organism at risk for hemorrhagic shock. 60. The method of claim 1, further comprising identifying the biological material in need of treatment. 61. The method of claim 1, further comprising monitoring said biological material for toxicity from said active compound. 62. The method of claim 1, wherein said active compound is provided to said biological substance in the form of a pharmaceutical composition. 63. A method for enhancing the viability of a biological material comprising providing said material with an effective amount of i) an active compound or combination of active compounds and ii) hypoxic conditions. 64. A method for enhancing the viability of a biological substance, the method comprising providing said substance with an effective amount of a composition having one or more compounds of formula II, or a salt or precursor thereof. 65. A method for enhancing the viability of a biological substance, the method comprising providing said substance with an effective amount of a composition having one or more compounds of formula III, or a salt or precursor thereof. 66. A method for enhancing the viability of a biological substance, the method comprising providing said substance with an effective amount of a composition having one or more compounds of formula IV, or a salt or precursor thereof. 67. A method for enhancing the viability of a biological substance, the method comprising administering to said substance an effective amount of a compound of formula I, formula II, formula III and/or formula IV, or a salt or prodrug thereof. 68. A method for preventing or reducing damage to biological material under adverse conditions, the method comprising providing said biological material with an effective amount of an active compound, wherein damage is prevented or reduced. 69. A method for producing a deposited stock of an organism, the method comprising exposing said organism to an effective amount of one or more compounds selected from the group consisting of: a) a compound of formula I; b) a compound of formula II; c) a compound of formula III; d) a compound of formula IV; or a salt or prodrug thereof. 70. A method for reversibly inhibiting metabolism in an organism, the method comprising providing said biological substance with an effective amount of an active compound other than rotenone. 71. The method of claim 69, further comprising exposing said organism to hypoxic conditions. 72. The method of claim 69, wherein the organism is a fly, fish, frog, or an embryo thereof. 73. A method for inducing sleep in an organism, the method comprising providing said organism with an effective amount of an active compound, wherein the effective amount is less than an amount that induces sleep in the organism. 74. A method for anesthetizing a biological substance, the method comprising providing said substance with an effective amount of an active compound, wherein the effective amount is less than an amount that would induce stasis in the organism. 75. A method for protecting a biological substance from injury, onset or progression of disease, or death, comprising providing an effective amount of an active compound to said substance before said injury, onset or progression of disease, or death, wherein The effective amount is less than the amount that induces stagnation in the biological material. 76. The method of claim 75, wherein the biological substance is bleeding. 77. A method of preventing an organism from bleeding to death, the method comprising administering to said bleeding organism an effective amount of an active compound to prevent death. 78. The method of claim 77, wherein the organism goes into hemorrhagic shock. 79. A method for inducing stagnation in a biological substance, the method comprising: a) identification of the biological material in which stagnation is desired; and b) providing the biological substance with an effective amount of at least one active compound to induce stagnation of the biological substance in vivo. 80. The method of claim 79, wherein said biological material is provided with an effective amount of a combination of different active compounds. 81. A pharmaceutical composition comprising at least one active compound-containing mixture in a pharmaceutically acceptable formulation. 82. The pharmaceutical composition of claim 81, wherein said active compound is an oxygen antagonist. 83. The pharmaceutical composition of claim 82, wherein said oxygen antagonist is a direct oxygen antagonist. 84. The pharmaceutical composition of claim 82, wherein the oxygen antagonist is _H2S . 85. The pharmaceutical composition of claim 84 comprising an effective amount of _H2S that, when administered to a patient, provides a _Cmax or a steady state plasma concentration of 10 [mu]M to 10 mM. 86. The pharmaceutical composition of claim 81, wherein the active compound has the structure of Formula I, Formula II, Formula III, Formula IV, or a salt or precursor thereof. 87. The pharmaceutical composition of claim 81, wherein said active compound is a chalcogenide. 88. The pharmaceutical composition of claim 72, wherein the chalcogenide is _H2S or a salt or precursor thereof. 89. The pharmaceutical composition of claim 66, wherein said composition is a liquid. 90. An article of manufacture comprising packaging material and an active compound contained in the packaging material, wherein said packaging material comprises a label indicating that said active compound can be used to induce stagnation in biological material in vivo. 91. The article of manufacture of claim 90 comprising a pharmaceutically acceptable diluent. 92. The article of manufacture of claim 91, wherein said active compound is provided in a first sealed container and said pharmaceutically acceptable diluent is provided in a second sealed container. 93. The article of manufacture of claim 92, further comprising instructions for mixing said active compound and said diluent. 94. The article of manufacture of claim 91, wherein said active compound is reconstituted for inducing stasis in biological material in vivo. 95. The article of manufacture of claim 90, further comprising a buffer. 96. The article of manufacture of claim 90, wherein the active compound has the chemical structure of Formula I, Formula II, Formula III, Formula IV, or a salt or prodrug thereof. 97. The article of manufacture of claim 90, wherein said active compound is an oxygen antagonist. 98. The article of manufacture of claim 90, wherein the active compound is a chalcogenide. 99. The article of manufacture of claim 98, wherein the chalcogenide is _H2S or a salt or precursor thereof. 100. The article of manufacture of claim 90, wherein said label indicates that said oxygen antagonist may be used to induce arrest in a patient in need of such treatment. 101. An article of manufacture comprising, packaged together: an active compound, instructions for using the active compound comprising: (a) identifying an in vivo tissue in need of arrest treatment; and (b) administering to said in vivo tissue Biological substances administer an effective amount of the active compound. 102. An article of manufacture comprising a medical gas containing an active compound and a label containing details or usage and administration for inducing stagnation in a biological substance. 103. A kit for delivering an active compound to a tissue site in need of stagnant treatment, comprising: A covering suitable to form an airtight wrap around the tissue site; a container containing an oxygen antagonist; and an inlet in said wound cover; wherein the container containing the active compound communicates with the inlet. 104. The kit of claim 103, further comprising an outlet in said covering, wherein the outlet is in communication with a source of negative pressure. 105. The kit of claim 104, wherein said outlet is positioned in fluid communication with a source of negative pressure. 106. The kit of claim 105, further comprising a flexible conduit communicating said outlet with a source of negative pressure. 107. The kit of claim 106, further comprising a canister in fluid communication between said outlet and a source of negative pressure. 108. The kit of claim 107, wherein the canister is a removable canister. 109. The kit of claim 106, wherein said container containing an active compound is in gaseous communication with said inlet. 110. The kit of claim 104, wherein said container contains the gaseous active compound. 111. The kit of claim 106, wherein said container contains a liquefied gas active compound. 112. The kit of claim 111, further comprising a vaporizer in communication between said container containing the active compound and the inlet. 113. The kit of claim 106 comprising a return outlet in communication with a container containing said active compound. 114. The kit of claim 103, wherein said active compound is carbon monoxide, carbon dioxide or hydrogen sulfide. 115. The kit of claim 103, wherein said tissue comprises a wound site. 116. The kit of claim 103, wherein said covering comprises an elastic material. 117. The kit of claim 116, further comprising a pressure sensitive adhesive covering edges of said covering. 118. The kit of claim 103, wherein said source of negative pressure is a vacuum pump. 119. A method for delivering an oxygen antagonist to a biological substance, the method comprising using the kit of claim 103. 120. A method for screening candidate active compounds, the method comprising: a) exposing zebrafish embryos to a substance; b) measuring the heart rate of said embryo; c) Comparing the heart rate of the embryo in the presence of the substance with the heart rate in the absence of the substance, a reduction in the heart rate identifies the substance as a candidate active compound. 121. The method of claim 120, wherein the embryo's heart rate is measured by counting heartbeats. 122. The method of claim 120, wherein the zebrafish embryo is observed under a dissecting microscope while heart rate is being measured. 123. The method of claim 120, further comprising d) exposing mice to the candidate active compound and determining one or more of the following parameters: i) core body temperature; ii) oxygen consumption; iii) athleticism; or iv) Carbon dioxide generation. 124. The method of claim 120, wherein said compound has the structure of Formula I or Formula IV. 125. A method for screening candidate active compounds, the method comprising: a) exposing the nematode to the substance; b) Determination of one or more of the following cellular respiration factors: i) core body temperature; ii) oxygen consumption; iii) athleticism; or iv) carbon dioxide production; c) Comparing the cellular respiration factors of nematodes in the presence of said substance to the absence of said substance, wherein a reduction of said characteristic identifies the substance as a candidate active compound. 126. The method of claim 125, wherein locomotor power is determined. 127. The method of claim 125, further comprising identifying said substance. 128. The method of claim 124, wherein said compound has the structure of Formula I or Formula IV. 129. A method of protecting a mammal from cellular damage caused by surgery, the method comprising providing said mammal with hydrogen sulfide in an amount sufficient to induce said mammal into pre-arrest prior to surgery. 130. The method of claim 129, wherein the surgery is selected from elective surgery, planned surgery, or emergency surgery. 131. The method of claim 129, wherein the hydrogen sulfide is administered intravenously. 132. The method of claim 129, wherein the hydrogen sulfide is administered by inhalation. 133. The method of claim 129, wherein said surgical procedure is cardiopulmonary surgery. 134. A method of protecting a mammal from cellular damage caused by a disease or adverse medical condition, the method comprising providing said mammal with sulfur in an amount sufficient to induce the mammal to enter pre-arrest prior to the onset or progression of said disease hydrogen. 135. The method of claim 134, wherein the disease or adverse medical condition is selected from the group consisting of: hemorrhagic shock, myocardial infarction, acute coronary syndrome, cardiac arrest, neonatal hypoxia/ischemia, ischemic revascularization Perfusion injury, unstable angina, post angioplasty, aneurysm, trauma, and blood loss. 136. A method for inducing apnea in a subject, the method comprising administering to said subject an effective amount of hydrogen sulfide. 137. The method of claim 136, wherein the subject is bleeding or at risk of bleeding. 138. The method of claim 137, further comprising obtaining a blood sample from said subject. 139. The method of claim 138, further comprising evaluating said blood sample for hydrogen sulfide exposure. 140. The method of claim 136, the method further comprising identifying a subject in need of treatment. 140.3. The method of claim 136, wherein said patient is provided with more than 3,000 ppm hydrogen sulfide. 140.4. The method of claim 136, wherein hydrogen sulfide is provided to the patient for about 5 minutes or less. 140.5. The method of claim 140.4, wherein hydrogen sulfide in excess of 3,000 ppm is provided to the patient for about 5 minutes or less. 140.6. The method of claim 140.5, wherein hydrogen sulfide is provided to the patient for about 3 minutes or less. 141. A method for treating hemorrhagic shock in a patient, the method comprising providing an effective amount of hydrogen sulfide. 142. The method of claim 141, wherein hydrogen sulfide is provided to the patient using a nebulizer. 143. The method of claim 141, wherein hydrogen sulfide is provided to the patient in excess of 3,000 ppm. 144. The method of claim 141, wherein hydrogen sulfide is provided to the patient for about 5 minutes or less. 145. The method of claim 144, wherein hydrogen sulfide in excess of 3,000 ppm is provided to the patient for about 5 minutes or less. 146. The method of claim 145, wherein hydrogen sulfide is provided to the patient for about 3 minutes or less. 147. The method of claim 141, wherein hydrogen sulfide is provided to said patient continuously. 148. The method of claim 141, wherein a single dose of hydrogen sulfide is provided to said patient. 149. The method of claim 141, wherein said patient is provided with multiple doses of hydrogen sulfide.

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