RS96904A - Carbon monoxide as a biomarker and therapeutic agent - Google Patents

Abstract

The present invention relates to the use of carbon monoxide (CO) as a biomarker and therapeutic agent of heart, lung, liver, spleen, brain, skin and kidney diseases and other conditions and disease states including, for example, asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung diseases, idiopathic pulmonary diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including lung, larynx and throat cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular thrombotic diseases such as pulmonary embolism. CO may be used to provide anti-inflammatory relief in patients suffering from oxidative stress and other conditions especially including sepsis and septic shock. In addition, carbon monoxide may be used as a biomarker or therapeutic agent for reducing respiratory distress in lung transplant patients and to reduce or inhibit oxidative stress and inflammation in transplant patients.

Description

CARBON MONOXIDE AS BIOMARKERS THERAPEUTIC AGENT

Field of invention

The present invention relates to the use of carbon monoxide (CO) as a biomarker and therapeutic agent for diseases of the heart, lungs, liver, spleen, brain, skin and kidneys, as well as for other conditions or diseases including, for example, asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung disease, idiopathic lung diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including cancer lungs, larynx and pharynx, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular disease with thrombus formation, such as pulmonary embolism. CO can be used to provide anti-inflammatory effects in patients suffering from oxidative stress and other conditions, particularly those including sepsis and septic shock. Additionally, CO can be used in organ storage prior to transplantation. Additionally, CO may be used as a biomarker or therapeutic agent to reduce respiratory distress in lung transplant patients, to reduce or inhibit oxidative stress, inflammation, or graft rejection in transplant patients.

State of the art

Heme oxygenase (HO) catalyzes the first step of heme degradation, which determines the rate of the reaction, which yields equimolar amounts of biliverdin Ka, carbon monoxide (CO) and iron (Choi et al, Am. J. Respir. Cell Mol. Biol. 15: 9-19; and Maines, Annu. Rev. Pharmacol. Toxicol. 37: 517-554). There are three isoforms of HO; HO-1 is highly inducible, while HO-2 and HO-3 are constitutively expressed (Choi et al., supra; Maines, supra; and McCoubrev et al., E. J. Bioch. 247: 725-732). Although heme is the major substrate for HO-1, various agents other than heme, including heavy metals, cytokines, hormones, endotoxin, and heat shock proteins, are also strong inducers of HO-1 expression (Choi et al., supra; Maines, supra; and Tenhunen et al., J. Lab. Clin. Med. 75 : 410-421). This diversity of HO-1 inducers provides further support for the hypothesis that, in addition to its role in heme degradation, HO-1 may also have a vital function in maintaining cellular homeostasis. Furthermore, HO-1 is strongly induced by various agents that induce oxidative stress, including hydrogen peroxide, glutathione-depleting agents, UV radiation, endotoxin, and hyperoxia (Choi et al., supra; Maines, supra; and Keyse et al., Proc. Natl. Acad. Sci. USA 86:99-103). One interpretation of this finding is that HO-1 may serve as a key biological molecule in adaptation and/or defense against oxidative stress (Choi et al., supra; Lee et al., Proc. Natl. Acad. Sci. USA 93: 10393-10398; Otterbein et al., Am. J. Respir. Cell. Mol. Biol. 13: 595-601; Poss et al., Proc. Natl. Acad. Sci. USA 94 : 10925 - 10930; Vile et al., Proc. Natl. Acad. Sci. USA 91 : 2607 - 2610; Abraham et al., Proc. Natl. Acad. Sci. USA 92 : 6798 - 6802; i Vile & Tvrrell, J. Biol. Chem. 268 : 14678 - 14681). Our laboratory, as well as others, has shown that induction of endogenous HO-1 provides in vivo and in vitro protection against oxidative stress associated with hyperoxia and lipopolysaccharide-induced tissue injury (Lee et al., supra; Otterbein et al., supra; and Tavlor et al., Am. J. Phvsiol. 18: L582-L591). We have also shown that exogenous administration of HO-1 via gene transfer can provide protection against oxidative stress injury and induce tolerance to hyperoxic stress (Ottebein et al, Am. J. resp. Crit. Care Med. 157 : A565 (abstract)).

Carbon monoxide (CO) is a gaseous molecule with known toxicity and lethality to living organisms (Haldane, Biochem. J. 21: 1068-1075; and Chance et al., 1970, Ann. NY Acad Sci. 174: 193-204). However, in contrast to this well-known paradigm of CO toxicity, there has been renewed interest in recent years for the role of CO as a regulatory molecule in cellular and biological processes, based on several key observations. Mammalian cells possess the ability to generate endogenous CO primarily from heme through the catalytic action of the enzyme heme oxygenase (HO) (Choi et al., supra; and Maines, supra). Total cellular production of CO is primarily achieved through the degradation of heme by HO (Marilena, Biochem. Mol. Med. 61: 136-142; and Vermaet al., 1993, Science 259: 381-384).

Moreover, CO, similar to the gaseous molecule nitric oxide, has important roles as a mediator of neuronal transmission (Verma et al, supra; and Xhuo et al., Science 260 : 1946 - 1950) and in the regulation of vasomotor tone (Morita & Kourembanas, 1995, J. Clin. Invest. 96 : 2676 - 2682; Morita et al., 1995 Proc. Acad. USA 92 : 1479, J. Clin. Other publications relating to the biological effects of CO include Pinsky et al, US Pat. 6,316,403; Sato et al., J. Immunol. 166: 4185 - 4194 (2001); Fujita et al., Nat. Med. 7 (5): 598 - 604 (2001); Nachar et al., High Altitude Medicine & Biology 2: 377-385 (2001); Vassalli et al., Crit. Care Med. 29: 359 - 366 (2001); Otterbein et al., A. J. Phvsiol. Lung Cell Mol. Phvsiol. 276: L688 - L694 (1999); cardel et al, Brit. J. Pharmacol. 124: 1065 - 1068 (1998); Otterbein et al., Nat. Med. 6 (4): 422 - 428 (2000); and Otterbein et al., Am. J. Physiol. Lung Cell Mol. Phvsiol. 279: L1029-1037 (2000)).

Septic shock and sepsis syndrome, resulting from overstimulation of immune cells, especially monocytes and macrophages, remain one of the leading causes of death in hospitalized patients (Parillo et al., Ann. Intern. Med. 113 : 991-992 (1992)). The pathophysiological alterations observed in sepsis are often not caused by the infectious organisms themselves, but rather by the uncontrolled production of proinflammatory cytokines and chemokines including TNF-ct, IL-1 and MIP-1, which cause leukocyte recruitment, capillary permeability and ultimately participate in the lethal outcome in sepsis (Beutler et al., 232 : 977 - 980 (1986); Netea et al., Immunology

94: 340-344 (1998); and Wolpe et al, J. exp. Med. 167: 570 - 581 (1988)). Lipopolysaccharide (LPS), which is a constituent of the cell wall of gram-negative bacteria, is a leading agent of sepsis and when administered experimentally to macrophages or in mice, mimics the inflammatory response that occurs in sepsis. Following LPS administration, there is a rapid, albeit transient, increase in the levels of these pro-inflammatory mediators, whose expression is subsequently reduced by the action of a number of anti-inflammatory cytokines, including interleukin-10 (IL-10) and interleukin

- 4 (IL-4), which inhibit the synthesis of proinflammatory cytokines and chemokines (J. Exp. Med. 177: 1205 - 1208 (1993)). LPS initially binds to CD14 and toll-like receptor (TLR) 2 (or 4) on the cell surface (Yang et al., Nature 395:284-288 (1998); and Chow et al., J. Biol. Chem. 274:10689-10692 (1999)) and has subsequently been shown to activate pathways involving mitogen-activated protein (MAP) kinase, including p38, 042/p44 ERK and JNK (MAP) kinases (Liu et al., J. Immunol. 153 : 2642 - 2652 (1994); Hambleton et al., Proc. Natl. Acad. Sci. USA 93 : 2274 - 2778 (1996); Han et al., J. Biol. Chem. 268 : 25009 - 25014 (1993); Han et al., Science 265:808-811 (1994); Sanghera et al., J. Immunol. 156: 4457 - 4465 (1996); and Raingeaud et al., J. Biol. Chem. 270: 7420 - 7426 (1995)). The relationship between the activation of these signaling

molecules, reduction of cytokine expression and physiological function is an area of active research.

The United States Government provided support for the research that led to the subject invention, under one or more NIH grant numbers HL60234, AI42365, and HL55330. Accordingly, the Government reserves certain rights with respect to the invention in question.

The essence of the invention

The present invention provides novel pharmaceutical compositions for use in patients suffering from the effects of oxidative stress, wherein the compositions contain effective concentrations of carbon monoxide in a gaseous mixture containing oxygen and optionally nitrogen in a gaseous state (as well as other optional gaseous components in a smaller amount). An additional solution according to the subject invention relates to the procedure of delaying the occurrence, inhibition or mitigation of the consequences of oxidative stress, which implies the delivery of therapeutic gas containing carbon monoxide in an amount and over time that is effective in slowing down the occurrence, inhibition or mitigation of the consequences of oxidative stress in the patient. Unexpectedly, it has been found that providing a therapeutic gas containing low concentrations (ie concentrations in the range of about 1 ppb (part per billion) to about 3000 ppm (preferably above 0.1 ppm within this range), preferably from about 1 ppm to about 2800 ppm, more preferably from about 25 ppm to about 750 ppm, even more preferably from about 50 ppm to about 500 ppm, e.g. about 250 ppm) of carbon monoxide is an extremely effective procedure for delaying the onset, inhibition or manifestation of the opposite effects of the consequences of oxidative stress in the patient. This is an unexpected result. It should be noted here that in the process according to the present invention, sometimes the amount of carbon monoxide in the gaseous therapeutic preparation above 0.3% may be used, depending on the condition or disease being treated.

In another solution, the present invention provides for the use of gaseous carbon monoxide in the process of obtaining a medication used in the treatment of a patient suffering from or at risk of developing emphysema, bronchitis, cystic fibrosis, pneumonia, interstitial lung disease, wound healing, arthritis, Parkinson's disease and/or Alzheimer's disease.

In another solution, the subject invention provides the use of gaseous carbon monoxide in obtaining a medication that is used in the treatment of a patient suffering from or at risk of developing localized inflammation of the kidneys, spleen and/or skin.

The present invention also provides methods for using carbon monoxide as a biomarker in assessing whether a patient is suffering from oxidative stress or a number of conditions or diseases that result from or lead to oxidative stress, such as, for example, asthma, emphysema, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung disease, idiopathic lung disease, other lung diseases, including primary pulmonary hypertension, secondary pulmonary hypertension, cancers, including lung, throat and pharyngeal cancer, arthritis, wound healing, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vascular disease with thrombus formation, such as pulmonary embolism, among others. The method according to the subject invention includes the detection of carbon monoxide in the patient's breath, in order to determine whether there are detectable levels of carbon monoxide in the exhaled air. If there are detectable levels of carbon monoxide in the exhaled air, the patient can be diagnosed with oxidative stress or the existence of a risk of oxidative stress. Manifestations of oxidative stress can be in the form of one or more of the above conditions or diseases. It is possible to take appropriate therapeutic steps after making such a diagnosis, in order to alleviate or treat the condition responsible for the occurrence of oxidative stress in the patient. In one solution, the method according to the present invention includes measuring carbon monoxide in the air exhaled by the patient, wherein the amount of carbon monoxide in the exhaled air of at least 1 ppm indicates that the patient has a risk of developing sepsis or septic shock.

Another solution of the present invention relates to the finding that in certain patients the application of carbon monoxide in effective amounts can be used to induce HO-1 and thus prevent or limit the consequences of oxidative stress in the patient, especially that caused by hyperoxia or sepsis. It is indicated that HO-1 is involved in maintaining homeostasis in the patient's cells.

Another solution according to the present invention relates to the use of carbon monoxide to delay the onset or inhibit or alleviate the effects of oxidative stress occurring in transplant patients, especially organ transplant patients, especially, but not limited to, lung transplant patients.

Another solution according to the present invention relates to the process of inhibiting the production of pro-inflammatory cytokines, such as TNF-α, IL-1p, IL-6 and MIP-1(3), as well as promoting the production (expression) of the anti-inflammatory cytokines IL-10 and IL-4 in the patient, whereby the process implies the application of an effective amount of CO to the patient.

Another solution according to the present invention relates to a procedure for preserving organs or tissues for transplantation, which includes adding a protectively effective amount or concentration of carbon monoxide to the medium in which the organs or tissues are preserved. In this solution according to the present invention, the use of carbon monoxide in effective amounts reduces, inhibits or enhances the formation of reactive oxygen compounds in the organ or tissue being stored, thus extending the period during which organs for transplantation can be effectively stored without suffering the consequences of oxidative stress. In one embodiment, the method includes providing a substrate containing carbon monoxide and storing the organ in the substrate in which carbon monoxide is present in an amount sufficient to improve organ storage stability.

Another solution according to the present invention relates to a procedure for preventing the occurrence or reducing the probability of damage caused by oxidative stress associated with hyperoxia in patients, which involves the application of an effective amount of carbon monoxide in a hyperoxic patient.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning common to those skilled in the art to whom this invention is intended. Although suitable methods and materials for practicing or testing the subject invention are described in the text that follows, methods and materials that are similar or equivalent to those described herein and that are well known in the art may be used. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the subject specification, including definitions, shall prevail. Additionally, the materials, procedures and examples are purely illustrative and in no way limit the scope of protection of the subject invention.

Detailed description of the invention

The following definitions are used in the description of the subject invention.

The term "carbon monoxide" (or "CO"), as used herein, means the molecular form of CO in the gaseous state, compressed into a liquid or dissolved in an aqueous solution. The terms "carbon monoxide preparation" or "pharmaceutical preparation containing carbon monoxide" are used in the present specification to describe a gaseous or liquid preparation containing CO, which may be administered to a patient and/or an organ. An experienced expert knows what the form of the pharmaceutical preparation is, e.g. gaseous, liquid or gaseous and liquid, preferred in a given solution.

The terms "effective amount" and "therapeutically effective" as used herein refer to the administration of that amount or concentration of CO used over a period of time, including acute or chronic administration, as well as periodic or continuous administration, which is effective in the context of producing a desired effect or physiological outcome. For gases, effective amounts of CO are generally in the range of about 0.0000001% to about 0.3% (mass percent), e.g. from 0.0001% to about 0.25% (mass percent), preferably at least from about 0.001%, for example, at least 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%, 0.20%, 0.22% or 0.24% (mass percent) CO. Ranges from, e.g. 0.001% to about 0.24%, about 0.005% to about 0.22%, about 0.005% to about 0.05%, about 0.010% to about 0.20%, about 0.02% to about 0.15%, about 0.025% to about 0.10%, or about 0.03% to about 0.08%o, or about 0.4% to about 0.06%. For liquid CO solutions, effective amounts generally range from about 0.0001 to about 0.0044 g CO/lOOg liquid, for example, at least 0.0001, 0.0002, 0.0004, 0.0006, 0.0008, 0.0010, 0.0013, 0.0014, 0.0015, 0.0016, 0.0018, 0.0020, 0.0021, 0.0022, 0.0024, 0.0026, 0.0028, 0.0030, 0.0032, 0.0035, 0.0037, 0.0040 or 0.0042 g CO/100 g liquid solution. Preferred amounts include, for example, from about 0.0010 to about 0.0030 g CO/100 g liquid, from about 0.0015 to about 0.0026 g CO/100 g liquid, or from about 0.0018 to about 0.0024 g CO/100 g liquid. An effective amount of carbon monoxide in the context of reducing the production or effect of inflammatory cytokines can be, for example; an amount sufficient to inhibit the production and/or action of, among others, TNF-a, IL-1, IL-6 and MIP-1. Alternatively, it may be an amount sufficient to induce or increase the production of anti-inflammatory cytokines, such as, inter alia, IL-10. In the context of patients undergoing transplantation, an effective amount of carbon monoxide is that amount administered to a patient undergoing transplantation that reduces the likelihood of rejection via an adverse immune reaction. In the context of preservation of stored organs to be used in transplantation, an effective amount of carbon monoxide may be that amount that is added, for example, bubbled through the medium in which the organs for transplantation are stored, in order to improve the preservation of the organ and reduce the likelihood that the organ will be exposed to a certain degree of oxidative damage. The skilled artisan will recognize that doses outside of the stated ranges may also be used, depending on the given solution.

The term "patient" is used in the present specification to describe an animal, human, or non-human organism for which treatment is provided according to the methods of the present invention. Applications in veterinary medicine are clearly covered by the subject invention. Said term includes, without limitation, mammals, for example, humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats.

The term "treat/treat" is used herein to describe delaying the onset, inhibition, or amelioration of the effects of a condition, such as, for example, emphysema, bronchitis, arthritis, cystic fibrosis, pneumonia, interstitial lung disease, Parkinson's disease, Alzheimer's disease, or inflammation of the kidneys, spleen, or skin. In the case of wound healing, this term means promoting wound healing, e.g. promoting the healing of skin wounds. Individuals who are considered to be at risk for developing any of the conditions described herein may particularly benefit from the present invention because prophylactic treatment can begin before any signs of the pathological condition appear. One of skill in the art knows that determining whether a patient is at risk of the conditions described herein can be accomplished using any procedure known in the art, e.g. by establishing a diagnosis by a doctor.

The term "biomarker" is used herein to describe carbon monoxide that is present in a patient's breath in small, detectable amounts, and which is evidence that the patient is at risk of, or is in the early stages of, or suffering from the effects of oxidative stress and is at risk of or suffering from one or more conditions or diseases that occur as a result of or may lead to the occurrence of oxidative stress. The amount of carbon monoxide in a patient's breath that can serve as a biomarker can be as low as 0.001 ppm, but is generally at least around 0.1 ppm.

The gases of those preparations are rejected

The CO preparation can be a gaseous CO preparation. Compressed gas or gas under pressure that can be used in the processes according to the present invention can be obtained through any commercial source, as well as in any type of vessel used to store compressed gas. For example, compressed or pressurized gas can be obtained from any source to produce compressed gases, such as oxygen, for medical applications. The pressurized gas, including CO used in the processes of the present invention, can be obtained in a form in which all the gases (eg, CO, He, NO, CO2, O2, N2) that are desired in the final form of the preparation are contained in the same vessel. Optionally, the processes of the present invention can be carried out using multiple vessels containing individual gases. For example, a vessel may be provided containing CO, with or without other gases, the contents of which may optionally be mixed with room air or with the contents of other vessels, for example, vessels containing oxygen, nitrogen, carbon dioxide, compressed air, or any other suitable gas or mixture of gases.

Gaseous preparations administered to a patient according to the present invention typically contain from 0% to 79% nitrogen (percentage by mass), from about 21% to about 100%> oxygen (percentage by mass) and from about 0.0000001%) to about 0.3%> (percentage by mass) CO (corresponding to from about 1 ppb or 0.001 ppm to about 3000 ppm). Preferably, the amount of nitrogen in the gaseous preparation is about 79% (mass percent), the amount of oxygen is about 21%> (mass percent), and the amount of CO is from about 0.0001%) to about 0.25% (mass' percent), preferably at least about 0.001%, for example, at least about 0.005%, 0.010%, 0.02%, 0.025%, 0.03%, 0.04%, 0.05%, 0.06%, 0.08%, 0.10%, 0.15%), 0.20%), 0.22%) or 0.24% (mass percentages). A preferred amount ranges from about 0.005% to about 0.24%, from about 0.01% to about 0.22%, and from about 0.08% to about 0.20%.

(mass percentages). It has been noted that gaseous CO preparations having a CO concentration greater than 0.3%) (such as a conc. of 1%o or greater) can be used for a short period (eg, one or two days), depending on the solution.

A gaseous CO preparation can be used to create an atmosphere containing CO gas. An atmosphere containing appropriate levels of CO gas can be formed, for example, by providing a vessel containing pressurized gas containing CO and releasing the pressurized gas from the vessel into a chamber or space to create an atmosphere containing CO gas within the chamber or space. Alternatively, the gases may be released into an apparatus that terminates in a breathing mask or breathing tube, thereby creating an atmosphere containing CO gas in the mask or breathing tube, ensuring that the patient is the only person in the room exposed to significant levels of CO.

Atmospheric CO levels can be measured or monitored using any method known in the art. Said procedures include electrochemical detection, gas chromatography, radioisotope determination, infrared absorption, colorimetry, and electrochemical procedures based on selective membranes (see, for example, Sunderman et al, Clin. Chem. 28: 2026-2032, 1982; Ingi et al. Neuron 16: 835-842, 1996). Sub-ppm levels of CO can be detected, for example, by gas chromatography and measurement of radioisotope radioactivity. Further, it is known in the art that CO levels in the sub-ppm range can be measured in biological tissue by a gas sensor operating in the mid-infrared region of the spectrum (see, e.g., Morimoto et al, Am, J. Phvsiol. Heart Circ. Phvsiol. 280 : H482 - H488, 2001). CO sensors and gas detection devices are widely available from a number of commercial sources.

Refusal of liquid preparations with carbon monoxide

The CO preparation can also be a liquid CO preparation. A liquid CO preparation can be obtained by any process known in the art to render gases soluble in liquids. For example, the liquid can be placed in a so-called "CO2 incubator" and exposed to a continuous flow of CO, preferably one balanced with carbon dioxide, until the desired concentration of CO in the liquid is reached. Another example is passing CO gas in the form of bubbles directly through the liquid until the desired CO concentration is reached. The amount of CO that can dissolve in a given aqueous solution increases with decreasing temperature. Another example is that a suitable liquid can be passed through a tube that allows gas diffusion, where the tube is exposed to an atmosphere containing CO (for example, using a device such as a membrane extracorporeal oxygenator). CO diffuses into the liquid to create a liquid CO preparation.

In one embodiment, the liquid can be any liquid known to those skilled in the art and suitable for administration to a patient (see, for example, Oxford Textbook of Surgery, Morris & Malt, editors, Oxford University Press (1994)). In general, the liquid can be an aqueous solution. Examples of suitable solutions include phosphate buffered saline (PBS), Celsior™, Perfadex™, Collins solution, citrate solution, and University of Wisconsin solution (UWS).

(Oxford Textbook of Surgery, Morris & Malt, editors, Oxford University Press (1994)).

Any suitable liquid can be saturated to the appropriate CO concentration by means of a gas diffusion device. Alternatively, previously made solutions that have been quality controlled to have the appropriate CO levels can be used. Precise dose control can be achieved by measurements with a gas-permeable, liquid-impermeable membrane connected to a CO analyzer. Solutions can be saturated to desired effective concentrations that are maintained at these levels.

Treatment of patients with carbon monoxide preparations

The patient may be treated with the CO preparation using any procedure known in the art for administering gases and/or liquids to patients. CO preparations may be used in a patient who has been diagnosed with, or is at risk of, developing emphysema, bronchitis, arthritis, cystic fibrosis, pneumonia, interstitial lung disease, Parkinson's disease, Alzheimer's disease, or inflammation of the kidneys, spleen, or skin; or to promote wound healing, e.g. to promote the healing of a wound on the skin that is not related to surgical intervention. The present invention includes the systemic application of liquid or gaseous CO preparations to patients (for example, by inhalation and/or ingestion), as well as the topical application of the preparation to the patient's lungs (by inhalation or endotracheal application), to the joints (e.g. by infusion or transdermal application), to the skin (e.g. by injection or application of the preparation to the surface of the skin), as well as to other organs (e.g. by ingestion, insufflation and/or introduction into the abdominal cavity).

Systemic application of carbon monoxide

Gaseous CO preparations can be administered systemically to the patient. Gaseous CO preparations are typically administered by inhalation through the mouth or nasal passages to the lungs, where the CO can exert its effects directly or be rapidly absorbed into the patient's circulation. The concentration of active compound (CO) used in a therapeutic gaseous preparation depends on the rate of absorption, distribution, inactivation, and excretion (generally, via respiration) of CO, as well as other factors known to those skilled in the art. It should further be noted that for each subject, over time, specific dosage regimens should be adjusted according to the individual needs and professional judgment of the person administering or supervising the administration of the preparation, and it should also be noted that the concentration ranges listed here are purely illustrative in nature and do not in any way limit the scope of protection or application of the preparation according to the subject invention. The scope of protection of the subject invention includes acute, subacute and chronic application of CO, which depends, for example, on the severity or duration of the patient's pathological condition. CO can be administered to a patient for a period of time (including indefinitely prolonged administration) sufficient to treat the condition and to produce the desired pharmacological or biological effect.

The following are examples of some of the procedures and devices that can be used to administer gaseous CO preparations to patients.

Respirators

CO for medical applications (concentrations may vary) can be ordered mixed with air or other oxygen-containing gas in a standard compressed gas tank (eg a mixture of 21% O2 and 79% N2). The gas is non-reactive, and the concentrations required for the procedures according to the present invention are well below the flammability limit (10% in air). In a hospital environment, the gas is usually administered at the patient's bedside, where it is mixed with oxygen or room air in a mixing vessel until the desired concentration is reached in ppm (parts per million). The patient inhales the gas mixture through a respirator, where the flow rate is adjusted in relation to the patient's comfort and needs. This is determined from a lung function chart (ie respiratory frequency, inspiratory and expiratory volume, etc.). Safety mechanisms can be designed into the administration system to prevent the patient from unnecessarily receiving more CO than desired. The level of CO received by the patient can be monitored by observing: (1) carboxyhemoglobin (COHb), which is determined in venous blood, and (2) exhaled CO, which is collected at the side port of the ventilator. CO exposure can be adjusted based on the patient's health status and other indicators. If necessary, CO can be expelled from the patient's body using inhalation with 100% O2. CO is not metabolized; therefore, the amount that is inhaled will eventually be completely exhaled, except for a very small percentage that is converted to CO2. CO can be mixed in any ratio with O2 to provide therapeutic CO administration without resulting hypoxic conditions.

Face mask and inhalation tent

A gas mixture containing CO can be prepared as described above to allow passive inhalation by a patient using a face mask or inhalation tent. The inhaled concentration can be changed and can be forced out of the patient's body by simply switching to 100% O2. Monitoring of CO levels can be performed on or near the mask or tent with a safety mechanism that prevents too high a concentration of CO from being inhaled.

Portable inhaler

Compressed CO can be packaged in a portable inhalation device and inhaled in a metered dose, for example, to provide intermittent treatment to a recipient in a non-hospital setting. Different concentrations of CO can be stored in the containers. The device can be simple, in the form of a small tank (eg capacity under 5 kg) with suitably diluted CO, with an open/close valve and with a tube from which the patient breathes CO according to the standard mode or as needed.

Intravenous artificial lung

An artificial lung (catheter device for blood gas exchange) designed to deliver O2 and remove CO2 can be used to deliver CO. Once implanted, the catheter is located in one of the larger veins and is capable of delivering CO in given concentrations systemically or locally. Delivery can be a local delivery of high concentrations of CO during a short period at the site of the intervention, e.g. near the spleen or kidney (this high concentration is rapidly diluted in the circulation) or over a long period at a lower concentration (see, for example, Hattler et al., Artif. Organs 18 (11) : 806 - 812 (1994); and Golob et al, ASAIO J„ 47 (5): 432 - 437 (2001)).

Normal pressure chamber

In certain cases, it is desirable to expose the entire patient to CO. The patient should be inside a hermetically sealed chamber into which CO has been injected (at a concentration that does not endanger the patient, or at a concentration that carries an acceptable risk, without the risk of bystanders being exposed to the gas). After the exposure is complete, the chamber can be purged with air (eg, 21% O2, 79% N2), and samples can be analyzed using a CO analyzer to ensure that no CO remains in the chamber before the patient is removed from the exposure system.

Liquid preparations

The subject invention further includes the creation of liquid CO preparations for systemic administration to the patient, e.g. orally and/or by injection, e.g. intravenous, intra-arterial, intraperitoneal and/or subcutaneous.

Topical treatment of organs with carbon monoxide

Alternatively, or in addition, CO preparations can be applied directly to the organs, e.g. on the skin, spleen, lungs and/or kidneys. Gaseous preparations can be applied directly to the internal and/or external surface of the patient's body to treat the patient's organs. The gaseous preparation can be applied to the patient's internal organs using any procedure known in the art for insufflating gases into the patient's body. For example, gases, such as carbon dioxide, are often insufflated into the patient's abdominal cavity to facilitate laparoscopic procedures (see, for example, Oxford Textbook of Surgery, Morris & Malt, editors, Oxford University Press (1994)). The skilled artisan knows that similar procedures can be used to deliver the CO preparation directly to the patient's internal organs. The skin and overlying joints can be treated topically with a gaseous preparation, for example, by exposing the affected skin to the gaseous preparation in a normal pressure chamber (described above) and/or by injecting the CO preparation directly into the skin.

Aqueous CO preparations can also be applied topically to the patient's organs. Aqueous formulations may be administered using any method known in the art for administering fluids to a patient. As in the case of gaseous preparations, aqueous preparations can be applied directly internally and/or externally to the patient's body. For example, the aqueous form of the formulation can be administered orally, for example, by the patient swallowing an encapsulated or non-encapsulated dose of the aqueous CO formulation. In another example, fluids, eg, saline containing dissolved CO, can be injected into the abdominal cavity of a patient during laparoscopic procedures. Alternatively, or in addition, in situ organ exposure can be reported, e.g. kidney and spleen, using any procedure known in the art, eg, in situ irrigation of the organ with a liquid preparation of carbon monoxide (see Oxford Textbook of Surgery, Morris & Malt, editors, Oxford University Press (1994)). The skin can be treated topically with a liquid preparation, e.g. by injecting a liquid preparation into the skin. Another example is that the skin covering the joints can be treated topically by applying a liquid preparation directly to the surface of the skin, e.g. pouring or spraying the liquid on the skin and/or immersing the skin in the liquid preparation.

Disorders and conditions

Carbon monoxide gas can be used in the preparation of medications used in the treatment of conditions or diseases such as asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis, cystic fibrosis, pneumonia, interstitial lung disease, idiopathic lung disease, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, cancers including lung, throat and pharyngeal cancer, arthritis, Parkinson's disease, Alzheimer's disease, peripheral vascular disease and pulmonary vasculature diseases with thrombus formation, such as pulmonary embolism; as well as in the treatment of a patient suffering from or at risk of developing localized organ inflammation, e.g. kidney, spleen and/or skin. The subject invention can also be used to promote wound healing, e.g. healing of skin wounds. Of particular interest is the treatment of wounds that were not caused by surgical intervention.

The subject invention may also be used to delay the onset or alleviate the effects of oxidative stress in patients undergoing transplantation, particularly in patients undergoing organ transplantation, particularly in patients undergoing lung transplantation. CO preparations can also be used to treat inflammatory lung conditions or inflammation secondary to sepsis or transplant rejection in patients. Although not supported by any theory, low doses of CO are thought to act as an anti-inflammatory agent by inhibiting the production and/or effects of pro-inflammatory cytokines, such as TNF-α, IL-1, IL-6, MIP-1 and/or by inducing or enhancing the effects of the anti-inflammatory cytokines IL-4 and IL-10.

The term "oxidative stress" is used to describe a condition that occurs as a result of an extremely large production of reactive compounds with oxygen, which endogenous antioxidants cannot neutralize. Oxidative stress can lead to permanent tissue damage, which occurs due to the effect of reactive oxygen compounds on the tissue. Physiological manifestations of oxidative stress take the form of or occur during various conditions or diseases such as asthma, emphysema, bronchitis, adult respiratory distress syndrome, sepsis or septic shock, cystic fibrosis, pneumonia, interstitial lung disease, idiopathic lung diseases, other lung diseases including primary pulmonary hypertension, secondary pulmonary hypertension, lung cancer and pulmonary vascular diseases with thrombus formation, such as pulmonary embolism, as well as any other lung disease.

The term "sepsis" is used to describe the presence of various organisms that cause suppuration, as well as other pathogenic organisms or their toxins (generally, lipopolysaccharide or bacterial cell walls with LPS) in the blood. Sepsis often leads to oxidative stress in those tissues exposed to pathogens or their toxins. Sepsis is often manifested by the production of pro-inflammatory cytokines, such as TNF-a, IL-1, IL-6 and MIP-1, the production of which is reduced or stopped after the administration of effective amounts of carbon monoxide.

The subject invention can be used in the treatment of inflammation. The term "inflammation" is used to describe a fundamental pathological process consisting of a dynamic complex of cellular and histological reactions, which occur in the affected blood vessels and surrounding tissues, in response to injury or abdominal stimulation caused by a physical, chemical or biological agent, including local reactions and resulting morphological changes, destruction or removal of material that causes injury and including responses that lead to repair and healing. The term includes different types of inflammation, such as acute, allergic, alterative (degenerative), atrophic, catarrhal (most often in the respiratory tract), croupous, fibrinopurulent, fibrinous, immune, hyperplastic or proliferative, subacute, serous and serofibrinous inflammation. The procedures according to the present invention preferably treat inflammation localized in the liver, heart, skin (e.g. dermatitis, inflammation due to bacterial, fungal or viral infection and/or allergic or autoimmune reactions), spleen, brain, kidney (e.g. bacterial pyelonephritis, interstitial nephritis and/or glomerulonephritis) and the respiratory tract, especially in the lungs, which is associated with sepsis or septic shock.

The term "cancer" is used as a general term to describe any of the different types of neoplasms, most of which invade the surrounding tissues, can metastasize to several places and often return after attempts at removal and cause the death of the patient, if not treated adequately. Cancers that can be treated using the compositions and methods of the present invention include, but are not limited to, for example, gastric, colon, rectal, liver, pancreatic, lung, breast, cervical, ovarian, prostate, testicular, bladder, kidney, brain/CNS, head and neck, pharynx, Hodgkin's disease, non-Hodgkin's leukemia, skin melanoma, various sarcomas, small cell lung cancer, choriocarcinoma, cancer of the oral cavity/pharynx, esophagus, larynx, melanoma, kidney cancer and lymphoma.

The subject invention can be used to treat conditions or diseases affecting the respiratory system, e.g. emphysema, bronchitis, cystic fibrosis, pneumonia and interstitial lung diseases. The term "emphysema" is used here to denote a lung disease characterized by alveolar enlargement. In this condition, the walls of the alveoli are destroyed, leading to loss of structural support of the bronchioles and their collapse during expiration (see, eg, The Merck Manual of Diagnosis and Therapy, 17th ed., Section 6, Chapter 68). The term "bronchitis" means a lung disease characterized by inflammation of the tracheobronchial tree. Bronchitis can occur after infections, e.g. viral infections, e.g. after a common cold; or after bacterial infections, or after exposure to an irritant (see, e.g., The Merck Manual of Diagnosis and Therapy, 17th ed., Section 6, Chapter 69). The term "cystic fibrosis" means a genetic disease of the exocrine glands. This disease primarily affects the gastrointestinal tract and respiratory system, and is usually characterized by chronic obstructive pulmonary disease (COPD) (see, eg, The Merck Manual of Diagnosis and Therapy, 17th ed., Section 19, Chapter 267). The term "pneumonia" means a lung disease that affects the parenchyma and includes, e.g. bacterial, viral, and aspiration pneumonia (see, e.g., The Merck Manual of Diagnosis and Therapy, 17th ed., Section 6, Chapter 73). The term "interstitial lung disease" refers to a group of lung diseases of unknown etiology, which are characterized by diffuse pathological changes that usually involve the interalveolar interstitial tissue (see, e.g., The Merck Manual of Diagnosis and Therapy, 17th ed., Section 6, Chapter 78).

As used herein, the term "arthritis" means a condition characterized by joint inflammation and includes, for example, rheumatoid arthritis (RA) (chronic inflammatory polyarthritis that usually leads to joint destruction), psoriatic arthritis (inflammatory arthritis associated with psoriasis), ankylosing spondylitis (inflammation of the axial skeleton and large peripheral joints), and ankylosis (joint immobility or fusion) (see, e.g., The Merck Manual of Diagnosis and Therapv, 17th ed., Section 5, Chapter 51; Section 5, Chapter 51, and Section 9, Chapter 108, respectively).

The term "Parkinson's disease" refers to "an idiopathic, slowly progressive, degenerative disorder of the CNS, characterized by slow and diminished movements, muscle rigidity, tremors at rest, and postural instability" (The Merck Manual of Diagnosis and Therapy, 17th ed., Section 14, Chapter 179). The term "Alzheimer's disease" means a disease characterized by "progressive, irreversible loss of cognitive function, associated with the accumulation of senile plaques in the cerebral cortex and subcortical gray matter, which also contain |3-amyloid and neurofibrillary tangles composed of tau-protein" (The Merck Manual of Diagnosis and Therapy, 17th edition, Section 14, Chapter 171).

According to the present invention, low doses of CO can also be used to induce HO-1 in patients, preventing or limiting oxidative stress, particularly oxidative stress induced by hyperoxia or sepsis. It is assumed that inducible HO-1 maintains homeostasis in the patient's cells.

Treatment of organs and tissues in order to improve storage stability

The present invention also relates to the use of CO as a preservative in the preservation of organs or tissues used in transplantation. An unexpected result is the finding that injecting low doses of CO into the medium in which organs to be transplanted are stored significantly reduces the likelihood of oxidative damage to organs during storage and significantly increases the storage time during which organs to be transplanted can be safely stored without suffering irreversible oxidative damage. Therefore, in one solution according to the present invention, an effective amount of CO is bubbled through the substrate before, or preferably after placing the organ in the substrate, or shortly thereafter. CO can also be used to increase the storage stability of organs that have been stored on a substrate for some time, but in this case, the oxidative damage may have already become irreversible, limiting the desired effect.

Accordingly, the present invention provides a method for increasing the storage stability of an organ or tissue. Storage stability is increased by exposing organs or tissues to liquid and/or gaseous CO preparations. Exposure of organs or tissues to gaseous CO preparations can be performed in any chamber or space suitable for creating an atmosphere containing appropriate levels of gaseous CO. Such chambers include, for example, incubators, as well as chambers designed to receive organs in a preservation solution. Another example is that a suitable chamber may be a chamber in which the gases are present exclusively in the internal atmosphere of the chamber, so that the CO concentration can be established and maintained at a given level and with a given degree of gas purity, e.g. it is a hermetically sealed chamber. For example, a CO2 incubator can be used to expose organs to a CO preparation, providing a continuous flow of gaseous CO from a vessel containing the gas.

When referring to liquid CO preparations, the exposure may be performed in any chamber or space having sufficient volume to submerge the organ or tissue, wholly or partially, in the CO preparation. In one solution according to the present invention, the organ can be exposed to the preparation with CO by placing it in any suitable container, so that the organ can be flushed and thus exposed to a continuous flow of the preparation with CO. In another solution, the organ is perfused with a CO preparation. The term "perfusion" is a term known in the field that refers to the passage of liquid, e.g. preparations with CO, through blood vessels of organs or tissues. Procedures for organ perfusion ex vivo in situ are well known in the art. An organ can be perfused with a COex vivo preparation, for example, using a continuous hypothermic perfusion machine (see Oxford Textbook of Surgery, Morris & Malt, editors, Oxford University Press (1994)). Optionally, the organ may be perfused with a lavage solution, e.g. UW solution without carbon monoxide, before perfusion with CO preparation, in order to remove donor blood from organs. This procedure can be used to avoid competition of donor hemoglobin for carbon monoxide. Alternatively, the flushing solution may be a CO preparation. As another example, a suitable liquid may be passed through a gas-diffusible tube that passes through an atmosphere containing carbon monoxide (eg, in a chamber, as in the case of an extracorporeal oxygenator membrane), to obtain a liquid CO preparation, which can then be passed through an organ (eg, perfused through an organ by connecting a tube to the organ).

In another example, the organ can be placed, eg, immersed in a medium or solution that does not contain carbon monoxide, and then placed in a chamber where that medium or solution can be converted to a CO preparation by exposure to an atmosphere containing CO, as described herein. In yet another example, the organ can be immersed in a liquid that does not contain carbon monoxide, and the carbon monoxide is bubbled through it.

The subject invention includes any or all of the procedures listed above for exposing organs to liquid CO preparations, e.g. includes flushing, immersion or perfusion, which may be used in a given procedure, e.g. which are used in one of the procedures to improve the stability of the preservation of an organ or tissue.

Carbon monoxide as a diagnostic tool

In addition to the use of CO as a therapeutic agent, determination of CO levels can be a useful diagnostic procedure, e.g. biomarker, when determining whether a patient suffers from oxidative stress or has a condition or disease in which CO plays a role, e.g. in sepsis or septic shock. Generally, a patient suspected of suffering from oxidative stress or at risk for developing oxidative stress is examined to determine whether detectable levels of CO can be measured in exhaled air. If detectable levels of CO (i.e. carbon monoxide of at least 0.01 ppm in the patient's breath) are observed, the prescribing physician or person caring for the patient may administer therapeutic doses of CO to treat oxidative stress or one or more conditions or diseases that result from or lead to oxidative stress.

In one solution, the exhaled air of the patient is analyzed for the presence of CO. The CO content of the patient's breath is measured by a CO sensor (for example, using a Logan LR2000 apparatus), which is sensitive enough to detect CO in the concentration range from 0 to about 1000 ppm (with a sensitivity of 1 ppb). In this procedure, patients exhale slowly from the level of functional FVC into the breath analyzer with a constant flow (5 - 6 l/m) during an interval of 20 - 30 seconds. Two successful recordings are recorded, and their mean value is used for the calculation. The ambient CO level is recorded before each inhalation to determine control or baseline values. Although any increase in CO levels from baseline may indicate an already developed or incipient state of oxidative stress, a CO level of at least 1 ppm provides a clear indication that the patient is or will develop oxidative stress.

Numerous solutions of the subject invention are described. In any case, it should be understood that various modifications can be made without departing from the spirit and scope of protection of the subject invention. Accordingly, other solutions are covered by the scope of protection defined by the following patent claims.

Claims (16)

1. Use of carbon monoxide in the preparation of a drug for the treatment of a patient suffering from or at risk of developing at least one disorder selected from the group consisting of: emphysema, bronchitis, cystic fibrosis, pneumonia, interstitial lung disease, wound healing, arthritis, Parkinson's disease and Alzheimer's disease; or to promote wound healing in a patient. 2. The method according to claim 1, characterized in that said disorder is emphysema. 3. The method according to claim 1, characterized in that said disorder is bronchitis. 4. The method according to claim 1, characterized in that said disorder is cystic fibrosis. 5. The method according to claim 1, characterized in that said disorder is pneumonia. 6. The method according to claim 1, characterized in that said disorder is interstitial lung disease. 7. The method according to claim 1, characterized in that said disorder is arthritis. 8. The method according to claim 1, characterized in that said disorder is Parkinson's disease. 9. The method according to claim 1, characterized in that said disorder is Alzheimer's disease 10. The method according to claim 1, characterized in that carbon monoxide in a gaseous state is used for the preparation of a drug that is used to promote wound healing in a patient. 11. Use of carbon monoxide in a gaseous state for the preparation of a drug used to treat a patient suffering or at risk of local inflammation of at least one organ selected from the group consisting of: kidneys, spleen and skin. 12. The method according to claim 11, characterized in that said organ is a kidney. 13. The method according to claim 11, characterized in that said organ is the spleen. 14. The method according to claim 11, characterized in that said organ is skin. 15. A method for improving stability when storing organs in a medium, indicated by including: providing a medium containing carbon monoxide; and storing the organ in the medium, wherein carbon monoxide is present in the medium in an amount sufficient to improve the storage stability of the organ. 16. A method for determining whether a patient is at risk of developing sepsis or septic shock, characterized in that it includes: measuring the amount of carbon monoxide in the air exhaled by the patient, wherein the amount of carbon monoxide of at least about 1 ppm in the exhaled air is an indication that the patient is at risk of developing sepsis or septic shock.