KR20090097925A - Near-infrared Electromagnetic Variation of Steady-state Membrane Potential of Cells - Google Patents

Abstract

Disclosed herein are systems and methods for applying near-infrared light energy and radiation dose to alter the bioenergy steady-state transmembrane and mitochondrial potential (ΔΨ-steady) of all irradiated cells via optical depolarization effect. The depolarization causes a simultaneous decrease in the absolute value of the transmembrane potential ΔΨ of the irradiated mitochondria and plasma membrane. Many cellular anabolic reactions and drug-resistant mechanisms may be less functional and / or alleviated by a decrease in membrane potential ΔΨ, associated weakening of proton driving force Δp, and associated degradation of phosphorylation potential ΔGp. Within the irradiated exposed area, the decrease in membrane potential ΔΨ will occur simultaneously in bacterial, fungal and mammalian cells. The membrane depolarization provides the ability to potentiate antimicrobial, antifungal and / or antineoplastic drugs only against the targeted undesirable cells.

Description

Near-Infrared Electromagnetic Modification of Cellular Steady-State Membrane Potentials

The present invention generally provides a method and system for generating infrared radiation at selected energy and dosimetry that will modify the bioenergy steady-state transmembrane and mitochondrial potential of irradiated cells via a depolarization effect. And more particularly to methods and systems for membrane depolarization to enhance antimicrobial and antifungal compounds in target bacteria and / or fungi and / or cancer cells.

The global outbreak of bacteria, fungi and other biological contaminants that are resistant to antimicrobial agents poses a serious threat to human existence. Since the discovery of sulfa drugs (sulfanylamide, first used in 1936) and penicillin (1942, Pfizer Pharmaceuticals), the development of resistant contaminants and pathogens through the use of significant amounts of all types of antimicrobials throughout the world An important environment for overgrowth has been created. Certain resistant contaminants have extraordinary mechanical significance because they are prevalent in hospitals and general environments. The widespread use of antibiotics, resistant bacteria, e.g., methicillin-resistant Staphylococcus aureus (Staphylococcus aureus) (MRSA) and vancomycin-as well as stimulating the production of resistant enterococci (VRE), fungal organisms (fungus), for example to produce a favorable environment for the infection of Candida (Candida).

While potent antifungal agents (eg, amphotericin B (AmB)) are present, mortality from candidiasis remains at about 38%. In some cases, in order to treat drug-resistant fungi, high doses of AmB must be administered, which often cause kidney toxicity and other side effects. Moreover, overuse of antimicrobials or antibiotics can lead to bioaccumulation in living organisms, which can also be cytotoxic to mammalian cells. Given the global population growth and the prevalence of drug-resistant bacteria and fungi, the incidence of bacterial or fungal infections is expected to continue without weakening in the near future.

Currently, available therapies for bacterial and fungal infections include the administration of antibacterial and antifungal therapies, or in some cases surgical removal of surgical tissue from the infected area. Since antimicrobial and antifungal therapy alone has little healing power, it is necessary to develop new strategies to treat or prevent microbial infections, particularly in view of the extremely high morbidity of emerging drug-resistant pathogens and surgical therapies that significantly damage their appearance. It's urgent.

Thus, the risk and / or intolerability of biological moieties other than targeted bacteria and fungi (biological contaminants) (eg, mammalian tissue, cells or certain biochemical agents, such as protein preparations) There is a need for a method and system that can reduce the risk of bacterial or fungal infection within / at a given target site without numerous adverse effects.

<Overview of invention>

The present invention reduces the minimum inhibitory concentration (MIC) of antimicrobial molecules (antimicrobial agents) and / or antineoplastic molecules (antineoplastic agents) necessary to attenuate or eliminate microbial and / or neoplastic-related pathological conditions. And a method and system for making a medicament that would otherwise no longer function at safe human doses again useful as adjuvant therapy. In accordance with the method and system of the present invention, near-infrared light radiation (known herein as NIMELS, meaning "near-infrared microbial removal system") at selected energy and radiation doses is used to cause depolarization of all membranes within the irradiated area, which The absolute value of the membrane potential ΔΨ of the irradiated cells will be altered.

Other features and advantages of the invention will be set forth in the detailed description of the following embodiments that will be in part apparent from the description, or may be learned by practice of the invention. Such features and advantages of the invention will be realized and attained by means of the systems, methods and mechanisms particularly pointed out in the written description and claims appended hereto.

Aspects of the invention may be more fully understood from the following description when read in conjunction with the accompanying drawings, which are to be regarded as illustrative in nature and not as restrictive. The drawings are not necessarily drawn to scale, but instead emphasize the principles of the invention.

1 shows a typical phospholipid bilayer.

2 shows the chemical structure of phospholipids.

3 shows the dipole effect (Ψd) in phospholipid bilayer membranes.

4A shows phospholipid bilayers in bacterial plasma membranes, mammalian mitochondrial membranes or fungal mitochondrial membranes with steady-state transmembrane potential prior to NIMELS irradiation. 4B shows the transient-state plasma membrane potential in bacterial plasma membranes, mammalian mitochondrial membranes or fungal mitochondrial membranes after NIMELS irradiation.

5 shows a phospholipid bilayer embedded with a transmembrane protein therein.

6 shows a general depiction of electron transport and proton pumps.

Figure 7 shows a general view of mitochondrial membranes in fungal and mammalian cells corresponding to ΔΨ-mito-fungi or ΔΨ-mito-mam.

8 shows the effect of NIMELS irradiation (at a single dose) on MRSA transmembrane potential measured by green fluorescence emission intensity in control and laser treated samples as a function of time (minutes) after laser treatment. Shows.

9 is a seed measured by percent drop in green fluorescence emission intensity in a laser treated sample compared to a control sample. C. albicans shows the effect of NIMELS irradiation (at various doses) on the transmembrane potential.

10 shows seed measured by red fluorescence emission intensity in a control and laser treated sample. The effect of NIMELS irradiation (at a single dose) on albicans mitochondrial membrane potential; And seed measured as the ratio of red to green fluorescence in the control and laser treated samples. It shows the effect of NIMELS irradiation (at single dose) on albicans mitochondrial membrane potential.

FIG. 11 shows the effect of NIMELS irradiation (at a single dose) on mitochondrial membrane potential of human embryonic kidney cells measured by red fluorescence emission intensity in control and laser treated samples; And NIMELS irradiation (at a single dose) on mitochondrial membrane potential of human embryonic kidney cells measured as the ratio of red to green fluorescence in control and laser treated samples.

12 shows a decrease in total glutathione concentration in MRSA as it correlates with reactive oxygen species (ROS) production in MRSA cells as a result of NIMELS irradiation (at some radiation doses); The decrease in glutathione concentration in the laser treated sample is shown as a percentage relative to the control sample.

Figure 13 shows seeds as a result of NIMELS irradiation (at some radiation doses). Since it correlates with reactive oxygen species (ROS) production in albicans cells. Showing a decrease in total glutathione concentration in albicans; The decrease in glutathione concentration in the laser treated sample is shown as a percentage of the control sample.

14 shows a decrease in total glutathione concentration in human embryonic kidney cells as it correlates with reactive oxygen species (ROS) production in human embryonic kidney cells as a result of NIMELS irradiation (at two different radiation doses); The decrease in glutathione concentration in the laser treated sample is shown as a percentage of the control sample.

15 shows the synergistic effect of NIMELS and methicillin on growth inhibition of MRSA colonies; The data show methicillin is enhanced by sub-lethal NIMELS radiation dose.

16 shows the synergistic effect of NIMELS and Bacitracin on growth inhibition of MRSA colonies; Arrows indicate growth or lack of MRSA colonies in the two samples shown; The image shows that bacitracin is enhanced by NIMELS radiation doses below lethal dose.

17 shows a bar graph depicting synergistic effects as indicated by experimental data of NIMELS with methicillin, penicillin and erythromycin in growth inhibition of MRSA colonies.

FIG. 18 is a composite diagram showing improvement over time in the appearance of toenails of a typical nail fungus patient treated according to the method of the present invention. Panel A shows baseline, infected toenails before treatment, panel B shows toenails 60 days after treatment, panel C shows toenails 80 days after treatment, and panel D shows toenails 100 days after treatment.

19 shows E. coli in lethal dose-less NIMELS irradiation. The detection of reduced membrane potential in E. coli is illustrated.

20 shows E. coli in lethal dose-less NIMELS irradiation. It illustrates the detection of increased glutathione in E. coli.

While specific embodiments are depicted in the drawings and described in connection with it, those skilled in the art will understand that the depicted embodiments are illustrative, variations of what is shown and others described herein can be devised and practiced, and are within the scope of the invention. .

As used herein, the singular indefinite articles "a", "an" and definite articles ("the") also include the plural terms they refer to, unless the content clearly indicates otherwise. For example, “NIMELS wavelength” also includes any wavelength within the described range of NIMELS wavelengths and combinations of such wavelengths.

As used herein, unless specifically indicated otherwise, the word "or" is used in the "inclusive" sense of "and / or" and not in the "exclusive" sense of "one or / or".

The term "about" is used herein to mean approximately, near, roughly or near. The term “about” when used in connection with a numerical range changes the range by extending the boundary above and below the numerical value stated. In general, the term “about” is used herein to change the numerical value above and below the value stated in the 20% variation.

The present invention reduces the minimum inhibitory concentration (MIC) of antimicrobial molecules (antimicrobial agents) and / or antineoplastic molecules (antineoplastic agents) necessary to attenuate or eliminate microbial and / or neoplastic-related pathological conditions. And methods and systems for making antimicrobial agents again useful as adjuvant therapies that would otherwise no longer function at safe human doses. In accordance with the method and system of the present invention, near-infrared light radiation (known herein as NIMELS, meaning "near-infrared microbial removal system") at selected energy and radiation doses is used to cause depolarization of the membrane within the irradiated area, which radiation The absolute value of the membrane potential ΔΨ of the irradiated cells will be altered.

The altered ΔΨ will cause a proton motive force Δp of all affected membranes, and associated weakening of bioenergy. Thus, the effect of NIMELS irradiation (NIMELS effect) can enhance existing antimicrobial molecules against microorganisms that infect human hosts and cause harm. These effects will allow them to function many less functional cellular assimilation reactions (eg, cell wall formation) and drug-resistant mechanisms (eg, efflux pumps) that require chemoosmotic electrochemical energy. Thus, any membrane bound cell resistance mechanism or assimilation reaction using membrane potential ΔΨ, proton driving force Δp, or phosphorylation potential ΔGp for their functional energy needs will be affected by the methods and systems of the present invention.

The methods and systems of the present invention are antimicrobial and / or with a selectivity made possible by the fact that mammalian cells are not generally affected by treatments (using molecules or drugs) intended to damage bacterial or fungal cells. utilizes the optical radiation to enhance only the undesired targeted cells, the anti-fungal drug (e.g., MRSA or Candida infection in the skin).

In an exemplary embodiment, the applied light irradiation used in accordance with the methods and systems of the present invention comprises one or more wavelengths in the range of about 850 nm to about 900 nm at the NIMELS radiation dose as described herein. In one aspect, a wavelength of about 865 nm to about 875 nm is used. In another aspect, the applied radiation has a wavelength from about 905 nm to about 945 nm in the NIMELS radiation dose. In one aspect, the applied light radiation has a wavelength of about 925 nm to about 935 nm. In certain aspects, a wavelength of 930 nm (or narrow wavelength range including it) may be used. In some aspects of the invention, the plurality of wavelength ranges include 870 and 930 nm, respectively.

Microbial pathogens whose bioenergy systems can be affected by the NIMELS according to the present invention include microorganisms such as bacteria, fungi, fungi, mycoplasma, protozoa, and parasites.

In one embodiment, the methods and systems of the present invention are used to treat and / or reduce and / or eliminate infectious entities known for causing skin or wound infections, such as Staphylococcus and enterococci. Staphylococcus and enterococci infections can be involved in most of the skin surfaces on the body, known to cause skin conditions such as boils, large boils, bullous impetigo, and laceration skin syndrome. s. Aureus also causes Staphylococcus food poisoning, enteritis, osteomyelitis, toxic shock syndrome, endocarditis, meningitis, pneumonia, cystitis, sepsis and postoperative wound infections. Staphylococcus infection can be obtained while the patient is in a hospital or long-term care facility. S. With limited population and widespread use of antibiotics. Antibiotic-resistant strains of Aureus have developed. This strain is called methicillin resistant Staphylococcus aureus (MRSA). Infections caused by MRSA are often resistant to a wide variety of antibiotics (particularly β-lactams) and are associated with significantly higher morbidity and mortality, higher costs and longer hospitalization durations than infections caused by non-MRSA microorganisms. Risk factors for MRSA infections in hospitals include nostril colonization, surgery, prior antibiotic therapy, intensive care unit entry, exposure to MRSA-colonized patients or practitioners, staying in the hospital for more than 48 hours, and attracting catheter (indwelling catheter) or other medical equipment that penetrates the skin.

In other embodiments, the methods and systems of the present invention are used to treat and / or reduce and / or eliminate infectious entities known as cutaneous candidiasis. These Candida infections are skin related and can occupy almost any skin surface on the body. However, what most often occurs is within a warm, damp or folded area (eg underarms and groin). Cutaneous candidiasis is very common. Candida is the most common cause of diaper rash and uses the warm, damp conditions inside the diaper. The most common fungus that causes these infections is Candida albicans . Candida infection is also very common in individuals with diabetes and obesity. Candida can also cause infections of the nails, called nail fungus, infections of the skin surrounding the nails (peritonitis), and infections around the edges of the mouth, called corner lipitis.

The term “NIMELS radiation dose” refers to the power density (W / cm 2) and energy density (J), in which the target wavelength according to the invention produces reactive oxygen species (“ROS”), which can reduce the level of biological contaminants at the target site. / Cm 2) (where 1 Watt = 1 Joule / sec). The term also encompasses irradiating cells to increase the susceptibility of biological contaminants through ΔΨ degradation of antimicrobial or antineoplastic agents with simultaneous generation of ROS, otherwise the contaminants are resistant to the agent. The method can be practiced without intolerable risks and / or intolerable side effects on tissues of the host subject other than biological contaminants.

"Enhancing" an antifungal or antimicrobial or antineoplastic agent allows the methods and systems of the invention to inhibit the growth and / or proliferation of fungi, bacteria or cancer at lower concentrations than in the absence of the methods and systems of the invention. Means sufficiently interfere with the resistance mechanisms of fungi, bacteria or cancer. If resistance is essentially complete, i.e., if the agent is ineffective against the cells, enhancement means that the agent will treat the disease state by inhibiting the growth and / or proliferation of pathogenic cells at a therapeutically acceptable dose.

As used herein, the term “microorganism” refers to microscopic organisms and is too small by definition to be invisible to the human eye. For the purposes of the present invention, the microorganism may be a bacterium, fungus, archaea, protozoa and the like. The word 'microbial' is defined as belonging to or relating to a microorganism.

As used herein, the term "cell membrane (or plasma membrane or mitochondrial membrane)" refers to a semipermeable lipid bilayer having a structure common to all living cells. It contains proteins and lipids that are primarily involved in myriad important cellular processes. The target cell membrane of the present invention has a protein / lipid ratio> 1. In other words, the target membrane in the inclusion (or moiety, ie, host tissue) does not contain more than 49.99% lipid by dry weight.

As used herein, the term “mitochondria” refers to an organelle surrounded by a membrane found in most eukaryotic cells (mammal cells and fungi). Mitochondria are "power stations of the cell" because they produce the most eukaryotic cell feed of ATP used as a chemical energy source for cells. Mitochondria include the inner and outer membranes consisting of phospholipid bilayers and proteins. However, the two films have different properties. The mitochondrial outer membrane surrounds the entire organelle, has a similar protein-to-phospholipid ratio to the eukaryotic plasma membrane, and the mitochondrial inner membrane forms an inner compartment known as cristae, and a protein-to-phospholipid similar to the prokaryotic plasma membrane. Have rain. This allows more space for proteins such as cytochromes to function correctly and efficiently. The electron transport system ("ETS") is located on the mitochondrial lining. Within the mitochondrial inner membrane is also a highly controlled transport protein that transports metabolites across the membrane.

As used herein, the term “Fluid Mosaic Model” refers to the widely spread concept of biological membranes as structural and functionally asymmetric lipid-bilayers with larger variety of embedded proteins that aid transmembrane transport. The flow mosaic model is so named because phospholipids move their positions within the membrane with little effort (flow) and all combinations of phospholipids, proteins and glycoproteins present in the membrane provide a mosaic image when viewed from the outside from the cell. Attached. The model is a careful balance of thermodynamic and functional considerations. Changes in membrane thermodynamics affect membrane function.

As used herein, the term "membrane dipole potential Ψd" (as opposed to the transmembrane potential ΔΨ) refers to the potential formed between the highly hydrated lipid head (hydrophilic) and the low polar interior (hydrophobic) of the bilayer at the membrane surface. The lipid bilayer originally has a substantial membrane dipole potential Ψd resulting from the structural organization of the dipole groups and ester linkages of molecules, mainly phospholipids and water. Ψd will be used herein to account for five different dipole potentials, independent of ions at the membrane surface.

1) mammalian plasma membrane dipole potential Ψd-plas-mam;

2) mammalian mitochondrial membrane dipole potential Ψd-mito-mam;

3) fungal plasma membrane dipole translocation Ψd-plas-fungi;

4) fungal mitochondrial membrane dipole potential Ψd-mito-fungi; And

5) bacterial plasma membrane dipole potential Ψd-plas-bact.

As used herein, the term "transmembrane potential" refers to the electrical potential difference between aqueous phases separated by a membrane (dimension mV) and will be represented by the symbol (ΔΨ). ΔΨ depends on ions at the membrane surface and will be used herein to account for three different transmembrane translocations.

1) mammalian transmembrane translocation ΔΨ-plas-mam;

2) fungal plasma transmembrane translocation ΔΨ-plas-fungi;

3) bacterial transmembrane translocation ΔΨ-plas-bact.

As used herein, the term “mitochondrial transmembrane potential” refers to the electrical potential difference between the compartments separated by the mitochondrial inner membrane (dimension mV) and will be used herein to describe two different mitochondrial transmembrane potentials.

1) mammalian mitochondrial transmembrane potential ΔΨ-mito-mam;

2) Fungal mitochondrial transmembrane potential ΔΨ-mito-fungi.

In mitochondria, potential energy from nutrients (eg, glucose) is converted into active energy available for cellular metabolic processes. The energy released during the subsequent redox-reduction reaction pumps protons (H ⁺ ions) from the mitochondrial matrix into the interlumen space. As a result, there is a chemical osmotic electrical potential difference in the mitochondrial membrane when the membrane is polarized (ΔΨ-mito-mam or ΔΨ-mito-fungi). ΔΨ-mito-mam and ΔΨ-mito-fungi are important parameters of mitochondrial functionality and provide direct quantitative values for the energy state (redox state) of the cell.

As used herein, the term “mammal transmembrane translocation (ΔΨ-plas-mam)” refers to the difference in electrical potential within the mammalian cell plasma membrane between aqueous phases. Mammalian plasma membrane translocation is different from bacterial and fungal ΔΨ, which is produced primarily with H ⁺ ions (protons). In mammalian plasma membranes, the main facilitator of ΔΨ is an electrogenerated Na ⁺ / K ⁺ -ATPase pump. ΔΨ-plas-mam is produced by an additional quality of transmembrane K ⁺ diffusion (from inside to outside of the cell) and by an electrogenerated Na ⁺ / K ⁺ -ATPase pump. Mammalian ATP is produced via proton pumps in the mitochondria.

As used herein, the term “fungal transmembrane translocation (ΔΨ-plas-fungi)” refers to the difference in electrical potential within the fungal cell plasma membrane. Fungal plasma membrane translocation is produced by membrane-bound H ⁺ -ATPase (high dose proton pumps requiring ATP to function). The H ⁺ -ATPase pump is necessary for fungal growth and stable cell metabolism and maintenance. Fungal ATP is produced in mitochondria.

As used herein, the term “bacterial transmembrane translocation (ΔΨ-plas-bact)” refers to the difference in electrical potential in bacterial cell plasma membranes. Bacterial plasma membrane translocation is generated by steady-state flow (translocation) of electrons and protons (H ⁺ ) across the bacterial plasma membrane that occur with normal electron transport and oxidative phosphorylation in the bacterial plasma membrane. A common feature of all electron transport circuits is the presence of a proton pump that produces a transmembrane proton gradient. Bacteria are free of mitochondria, but aerobic bacteria perform oxidative phosphorylation (ATP production) by essentially the same processes that occur in eukaryotic mitochondria. As used herein, the term “P-class ion pump” refers to a transmembrane active transport protein assembly containing an ATP-binding site (ie, it requires ATP to function). During the transport process, one of the protein subunits is phosphorylated and the transported ions are thought to migrate through the phosphorylated subunit. Ion pumps of this class include Na ⁺ / K ⁺ -ATPase pumps in mammalian plasma membranes, which maintain the typical Na ⁺ and K ⁺ electrochemical potentials (ΔNa ⁺ / K ⁺ ) and pH gradients of animal cells. Other important members of the P-class ion pump transport protons (H ⁺ ions) out of the cell and K ⁺ ions into the cell.

As used herein, the term "Na ⁺ / K ⁺ ATPase" refers to a P class ion pump that is present in the plasma membrane of all animal cells, which causes hydrolysis of one ATP molecule to cause typical Na ⁺ and K ⁺ electrolysis of animal cells. It is linked to the release of three Na ⁺ ions and the introduction of two K ⁺ ions to maintain chemical potential and pH gradient. Intra-negative membrane potentials in fungal cells (also eukaryotes) are produced by the transport of H ⁺ ions out of the cell by proton pumps powered by different ATPs.

As used herein, the term “ion exchanger and ion channel” refers to a transmembrane protein that is an ATP-independent system and helps establish plasma membrane translocation in mammalian cells.

As used herein, the term “redox (abbreviation for reduction / oxidation reaction)” describes a complex process of oxidation of a sugar, for example, in a cell, through a series of very complex processes involving electron transfer. Redox reactions are chemical reactions in which electrons are transferred from donor molecules to recipient molecules. The term redox is derived from two concepts of reduction and oxidation and can be described in simple terms as follows:

Oxidation describes the loss of electrons by molecules, atoms, or ions.

Reduction describes the acquisition of electrons by molecules, atoms, or ions.

As used herein, the term “redox state” describes the redox environment (or level of oxidative stress) of the cells described.

As used herein, the term "steady-state transmembrane potential (ΔΨ-steady)" refers to mammalian, fungal or bacterial cells prior to irradiation in accordance with the methods and systems of the present invention that will continue in the future in the absence of irradiation. Quantitative plasma membrane translocation is shown.

For example, steady-state flow of electrons and protons across bacterial cell membranes that occur during normal electron transport and oxidative phosphorylation will be steady-state due to the constant flow of conventional redox reactions that occur across the membrane. In contrast, any modification of the redox state will cause a transient-state membrane potential. ΔΨ-steady will be used herein to describe three different steady-state transmembrane translocations based on species:

1) steady-state mammalian transmembrane translocation ΔΨ-steady-mam;

2) steady-state fungal transmembrane translocation ΔΨ-steady-fungi;

3) steady-state bacterial transmembrane translocation ΔΨ-steady-bact.

As used herein, the term “transient-state plasma membrane potential (ΔΨ-tran)” refers to the expression of mammalian, fungal or bacterial cells after irradiation in accordance with the methods and systems of the present invention in which irradiation changes the bioenergy of the plasma membrane. Plasma membrane potential is shown. In bacteria, ΔΨ-tran will also change the redox state of the cell because of the presence of ETS and cytochrome in the plasma membrane. ΔΨ-tran will not occur without radiation using the method of the present invention. ΔΨ-tran will be used herein to describe three different transient-state transmembrane translocations based on species:

1) transient-state mammalian transmembrane translocation ΔΨ-tran-mam;

2) transient-state fungal transmembrane translocation ΔΨ-tran-fungi;

3) transient-state bacterial transmembrane translocation ΔΨ-tran-bact.

As used herein, the term "steady-state mitochondrial membrane potential (ΔΨ-steady-mito)" will continue in the future in the absence of irradiation, quantitatively of mammalian or fungal mitochondria prior to irradiation in accordance with the methods and systems of the present invention. Mitochondrial membrane potential is shown.

For example, the steady-state flow of electrons and protons across the mitochondrial inner membrane that occurs during normal electron transport and oxidative phosphorylation will be steady-state due to the constant flow of conventional redox reactions that occur across the membrane. Any modification of the redox state will cause a transient-state mitochondrial membrane potential. ΔΨ-steady-mito will be used herein to describe two different steady-state mitochondrial membrane potentials based on species:

1) steady-state mitochondrial mammalian translocation ΔΨ-steady-mito-mam;

2) steady-state mitochondrial fungal potential ΔΨ-steady-mito-fungi.

As used herein, the term “transient-state mitochondrial membrane potential (ΔΨ-tran-mito-mam or ΔΨ-tran-mito-fungi)” refers to the methods and systems of the present invention in which irradiation changed the bioenergy of the mitochondrial inner membrane. Shows the membrane potential of the mammalian or fungal cell after irradiation. In mammalian and fungal cells, ΔΨ-tran-mito will also change the redox state of the cell because of the presence of an electron transport system (ETS) and cytochrome in the mitochondrial lining. Since these mitochondrial (H ⁺ ) gradients are generated within the mitochondria to produce ATP suitable for myriad cellular functions, ΔΨ-tran-mito also strongly influences (proton driving forces) Δp-mito-mam and Δp-mito-fungi Can have ΔΨ-tran-mito will not occur without irradiation in accordance with the methods and systems of the present invention. ΔΨ-tran-mito will be used herein to describe two different transient-state mitochondrial membrane potentials based on species:

1) transient-state mitochondrial mammalian translocation ΔΨ-tran-mito-mam;

2) transient-state mitochondrial fungal potential ΔΨ-tran-mito-fungi.

As used herein, the term “cytochrome” refers to a membrane bound heme protein that contains a heme group and performs electron transport. As used herein, the term “electron transport system (ETS)” describes a series of membrane-associated electron carriers (cytochromes) that mediate a biochemical reaction that produces ATP, the energy current of a cell. In prokaryotic cells (bacteria), this occurs at the plasma membrane. In eukaryotic cells (fungal and mammalian cells), this occurs in the mitochondria.

As used herein, the term “pH gradient (ΔpH)” refers to the pH difference between two bulk phases on both sides of the membrane.

As used herein, the term “proton electrochemical gradient (ΔμH ⁺ ) (dimension kJ mol ⁻¹ )” refers to electrical and chemical properties, particularly proton gradients, across a membrane, and the cellular potential available for operation in the cell. Indicates the type of energy. The proton electrochemical potential difference between two sides of the membrane participating in active transport, including a proton pump, is sometimes also referred to as chemoosmotic potential or proton driving force. When ΔμH ⁺ is reduced by any means, it is natural that the cellular assimilation pathway and resistance mechanisms in the affected cells are inhibited. This may be achieved by combining λn and Tn to irradiate only the target site, or may be further enhanced by simultaneous or sequential administration of agents formed and arranged for delivery to the target site (ie, (λn and Tn ) + (Co-targeting of assimilation pathways with drug molecule (s)).

As used herein, the term "ion electrochemical gradient (Δμx ⁺ )" means the electrical and chemical properties across the membrane caused by the concentration gradient of ions (other than H ⁺ ) and are available for operation in the cell. Type of cellular potential energy. In mammalian cells, Na ⁺ ion electrochemical gradients are maintained across the plasma membrane by active transport of Na ⁺ out of the cell. It is a different gradient from the proton electrochemical potential, but is generated from an ATP linked pump, which is produced during oxidative phosphorylation from mammalian mitochondrial proton driving force (Δp-mito-mam). When Δμx ⁺ is reduced by any means, it is natural that cellular assimilation pathways and resistance mechanisms are inhibited in the affected cells. This may be achieved by combining λn and Tn to irradiate only the target site, or may be further enhanced by simultaneous or sequential administration of agents formed and arranged for delivery to the target site (ie, (λn and Tn ) + (Co-targeting of assimilation pathways with drug molecule (s)).

As used herein, the term "co-targeting of bacterial assimilation pathways" (λn and Tn degradation of (ΔμH ⁺ ) and / or (Δμx ⁺ ) of cells at the target site to affect the assimilation pathway) + (same bacteria Drug molecule (s)) can be shown to influence the assimilation pathway and any of the following bacterial assimilation pathways that can be inhibited using the drug molecule:

Here, the targeted assimilation pathway is a peptidoglycan biosynthesis that is co-targeted by an agent that binds to the active site of a bacterial transpeptidase enzyme (penicillin binding protein) that crosslinks with peptidoglycan in the bacterial cell wall. Inhibition of these enzymes ultimately results in cell lysis and death;

Here, the targeted bacterial assimilation pathway binds to the acyl-D-alanyl-D-alanine groups in the cell wall intermediates and thus the peptidom of N-acetylmuramine acid (NAM)-and N-acetylglucosamine (NAG) -peptide subunits. Prevents the integration into the glycan matrix (effectively inhibits peptidoglycan biosynthesis by acting on glycosyltranslation and / or peptide transpeptidation) to prevent the proper formation of peptidoglycans in Gram-positive bacteria Peptidoglycan biosynthesis co-targeted by a medicament to prevent;

Here, the targeted bacteria assimilation path C ₅₅ - iso prenyl fatigue coupled to a phosphate and fatigue phosphatase is C ₅₅ - to prevent interaction with isopropyl prenyl pyrophosphate, building blocks (building block) peptidoglycan of inner glycan Peptidoglycan biosynthesis co-targeted by a medicament that reduces the amount of C ₅₅ -isoprenyl pyrophosphate available for transfer out;

Here, the targeted assimilation pathway binds to 23S rRNA molecules in the subunit 50S subunit of bacterial ribosomes, resulting in the accumulation of peptidyl-tRNA in the cell, thus depleting the free tRNA necessary for the activation of α-amino acids, Bacterial protein biosynthesis co-targeted by an agent that inhibits peptide transfer by causing premature dissociation of peptidyl tRNA from;

Wherein the co-targeted agent can simultaneously bind to two domains of 23S RNA of the 50S bacterial ribosomal subunit, thereby inhibiting the formation of bacterial ribosomal subunits 50S and 30S (ribosome subunit assembly); Here, the co-targeted agent is chlorinated to increase its lipophilia to penetrate into bacterial cells, bind to the 23S portion of the 50S subunit of bacterial ribosomes, and from the aminoacyl site (A-site) to the peptidyl site (P Site) prevents the translocation of peptidyl-tRNA, thereby inhibiting the transpeptidase response, which allows incomplete peptides to be released from the ribosomes;

Wherein the targeted assimilation pathway binds to 30S bacterial ribosomal subunits and prevents the attachment of amino-acyl tRNAs that bind to the recipient site (A-site) of the ribosomes, thereby prolonging codon-anticodone interaction and prolongation of protein synthesis Bacterial protein biosynthesis co-targeted by inhibitors;

Wherein the co-targeted agent binds to bacterial ribosomes more strongly in a different orientation than the conventional subgroups of polyketide antimicrobials having an octahydrotetracene-2-carboxamide backbone, resulting in tet (M) ribosomes and tet (K) S with excretory gene determinant. Becomes active against strains of Aureus;

Wherein the targeted assimilation pathway binds to specific aminoacyl-tRNA synthetases to prevent esterification of a particular amino acid or its precursor to one of its compatible tRNAs, thus preventing the formation of aminoacyl-tRNAs, Bacterial protein biosynthesis co-targeted by a medicament that stops the integration of essential amino acids into the protein;

Here, the targeted assimilation pathway binds to the 50S rRNA through the domain V of the 23S rRNA while interacting with the 16S rRNA of the 30S ribosomal subunit, thereby inhibiting bacterial protein synthesis before beginning, thereby initiating a protein synthesis formyl-methionine initiator ( f-Met-tRNA), and bacterial protein biosynthesis co-targeted by an agent that prevents binding of the 30S ribosomal subunit;

Here, the targeted assimilation pathway interacts with the 50S subunit of bacterial ribosome at protein L3 in the region of the 23S rRNA P site close to the peptidyl transferase center, inhibiting peptidyl transferase activity and peptidyl delivery, Bacterial protein biosynthesis co-targeted with agents that block site interactions and prevent normal formation of active 50S ribosomal subunits;

Wherein the targeted assimilation pathway is DNA replication and transcription co-targeted by an agent that inhibits topoisomerase II (DNA gyrase) and / or topoisomerase IV;

Here, targeted assimilation pathways are DNA replication and translation co-targeted by agents that inhibit DNA polymerase IIIC (an enzyme required for replication of chromosomal DNA in Gram-positive bacteria, but not present in Gram-negative bacteria). to be;

Wherein the targeted assimilation pathway is DNA replication and transcription co-targeted by drug hybrid compounds that inhibit topoisomerase II (DNA garase) and / or topoisomerase IV and / or DNA polymerase IIIC;

Wherein the targeted assimilation pathway is bacterial phospholipid biosynthesis, which is co-targeted by topical agents that act on phosphatidylethanolamine-rich cytoplasmic membranes and work well in combination with other topical synergists;

Here, the targeted assimilation pathway is a bacterium through selective targeting of β-ketoacyl- (acyl-carrying-protein (ACP)) synthase I / II (FabF / B) (an essential enzyme in type II fatty acid synthesis). Bacterial fatty acid biosynthesis co-targeted by agents that inhibit fatty acid biosynthesis;

Here, the targeted assimilation pathway is the maintenance of bacterial transmembrane translocation ΔΨ-plas-bact, and the co-targeting agent does not penetrate into the cell but mainly binds to gram positive cytoplasmic membrane, causing depolarization and loss of membrane potential Destroying many aspects of bacterial cell membrane function on itself by causing inhibition of protein, DNA and RNA synthesis;

Here, co-targeting agents increase the permeability of the bacterial cell wall, thus allowing inorganic cations to move through the wall in a non-limiting manner, disrupting the ionic gradient between the cytoplasm and the extracellular environment;

Wherein the targeted assimilation pathway is maintenance of bacterial membrane selective permeability and bacterial transmembrane translocation ΔΨ-plas-bact, the co-targeting agent is selective for the negatively charged surface of the bacterial membrane relative to the neutral membrane surface of the eukaryotic cell, Cationic antimicrobial peptides that cause prokaryotic membrane permeation and ultimate puncture and / or disruption of bacterial cell membranes, promoting leakage of bacterial cell contents and disruption of transmembrane potentials;

Wherein the co-targeting agent inhibits bacterial protease peptide deformillase that catalyzes the removal of formyl groups from the N-terminus of the newly synthesized bacterial polypeptide;

Here, co-targeting agents inhibit two-component regulatory systems in bacteria, such as their ability to respond to their environment via signal transduction across bacterial plasma membranes (these signal transduction processes are not present in mammalian membranes).

As used herein, the term "co-targeting of the fungal assimilation pathway" (λn and Tn degradation of (ΔμH ⁺ ) and / or (Δμx ⁺ ) of a cell at the target site to influence the assimilation pathway) + (same fungi Agents to influence the assimilation pathway) and any of the following fungal assimilation pathways that can be inhibited using the agent:

Here, the targeted assimilation pathway is phospholipid biosynthesis that destroys the structure of existing phospholipids in fungal cell membranes and is co-targeted by topical agents that work well in combination with other local synergists;

Here, the targeted assimilation pathway inhibits ergosterol biosynthesis in the C-14 demethylation step (part of the 3-step oxidation reaction catalyzed by cytochrome P-450 enzyme 14-a-sterol demethylase), thereby Ergosterol biosynthesis co-targeted by agents that cause ergosterol depletion through disruption of the structure and accumulation of lanosterol and other 14-methylated sterols that inhibit the 'bulk' function of ergosterol as a membrane component;

Wherein the targeted assimilation pathway inhibits the enzyme squalene epoxidase, which in turn is ergosterol biosynthesis, which is co-targeted with a medicament that inhibits ergosterol biosynthesis in fungal cells so that the fungal cell membrane has increased permeability;

Wherein the targeted assimilation pathway is ergosterol biosynthesis co-targeted with a drug that inhibits two enzymes in the ergosterol biosynthetic pathway, ie d14-reductase and d7, d8-isomerase, at separate and distinct points;

Wherein the targeted assimilation pathway inhibits the enzyme (1,3) β-D-glucan synthase, which in turn is fungal cell wall biosynthesis co-targeted with agents that inhibit β-D-glucan synthesis in the fungal cell wall;

Here, the targeted assimilation pathway effectively alters the transition temperature of the cell membrane and causes voids to form in the membrane to form harmful ion channels in the fungal cell membrane (the main sterol to which the co-targeting agent binds) Ergosterol) fungal sterol biosynthesis co-targeted with a medicament;

Wherein the co-targeted agent is formulated for delivery in lipids, liposomes, lipid complexes and / or colloidal dispersions to prevent toxicity from the agent;

Here, the targeted assimilation pathway is taken up into fungal cells by cytosine permease, deaminoated to 5-fluorouracil (5-FU), converted to nucleoside triphosphate, and integrated into RNA Where protein synthesis is co-targeted with a drug that is 5-FC causing miscoding;

Wherein the targeted assimilation pathway is a fungal protein co-targeted with an agent that inhibits fungal elongation factor EF-2 and / or fungal elongation factor 3 (EF-3) not present in mammalian cells that is functionally distinct from its mammalian counterparts. It is synthetic;

Here, the targeted assimilation pathway is fungal chitin co-targeted with agents that inhibit fungal chitin biosynthesis by inhibiting the action of one or more enzymes chitin synthase 2 (an enzyme required for primary septum formation and cell division in fungi). Biosynthesis (β- (1,4) -linked homopolymer of N-acetyl-D-glucosamine);

Wherein the co-targeted agent inhibits the action of enzyme chitin synthase 3 (an enzyme required for the synthesis of chitin during bud appearance and growth, conjugation, and sporulation);

Here, co-targeting agents chelate polyvalent cations (Fe ⁺³ or Al ⁺³ ), resulting in the inhibition of metal-dependent enzymes responsible for mitochondrial electron transport and cellular energy production, and also the inhibition of peroxides in fungal cells. Inhibits normal degradation;

Here, co-targeting agents inhibit two-component control systems in fungi, such as their ability to respond to their environment through signal transduction across fungal plasma membranes.

As used herein, the term "co-targeting of the cancer assimilation pathway" (λn and Tn degradation of (ΔμH ⁺ ) and / or (Δμx ⁺ ) of cells at the target site to influence the assimilation pathway) + (non- Drug to affect the same cancer assimilation pathway to a greater extent than cancerous cells), and any of the following cancer assimilation pathways that can be inhibited using a drug:

Wherein the targeted assimilation pathway is DNA replication co-targeted by an agent that inhibits DNA replication by crosslinking with guanine nucleobases in the DNA double helix strand so that the strand cannot be unwound and separated (which is required for DNA replication);

Wherein the targeted assimilation pathway is DNA replication co-targeted by an agent capable of reacting with two different 7-N-guanine residues in the same strand of DNA or in different strands of DNA;

Wherein the targeted assimilation pathway is DNA replication co-targeted by an agent that acts as an anti-metabolic agent and thereby inhibits DNA replication and cell division;

Wherein the targeted assimilation pathway is cell division that is co-targeted by an agent that inhibits cell division by preventing microtubule function;

Wherein the targeted assimilation pathway is DNA replication co-targeted by agents that inhibit DNA replication and cell division by preventing cells from entering the G1 phase (initiation of DNA replication) and replication of DNA (S phase);

Here, the targeted assimilation pathway is cell division that is co-targeted by an agent that enhances the stability of the microtubules and thus prevents chromosome segregation during anaphase;

Here, targeted assimilation pathways inhibit DNA replication and cell division by inhibition of type I or type II topoisomerase, which will inhibit both transcription and replication of DNA by upsetting suitable DNA supercoiling. DNA replication that is co-targeted by an inhibitor that inhibits it.

As used herein, the term “proton driving force (Δp)” refers to the storage of energy (which acts like a battery) as a combination of proton and voltage gradient across the membrane. The two components of Δp are ΔΨ (membrane potential) and ΔpH (chemical gradient of H ⁺ ). In other words, Δp consists of H ⁺ transmembrane potential ΔΨ (negative charge (acidic) outside) and the transmembrane pH gradient ΔpH (inside alkaline). The potential energy stored in the form of an electrochemical gradient is produced by the pumping of hydrogen ions across a biological membrane (mitochondrial inner membrane or bacterial and fungal plasma membrane) during chemical osmosis. Δp may be used for chemical, osmotic or mechanical work in cells. Proton gradients are generally used in oxidative phosphorylation to drive ATP synthesis and can be used to drive drain pumps in bacterial, fungal or mammalian cells such as cancerous cells. Δp will be used herein to describe four different proton driving forces in the membrane based on species and is mathematically defined as (ΔP = ΔΨ + ΔpH).

1) mammalian mitochondrial proton driving force (Δp-mito-mam);

2) fungal mitochondrial proton driving force (Δp-mito-Fungi);

3) fungal plasma membrane proton driving force (Δp-plas-Fungi);

4) bacterial plasma membrane proton driving force (Δp-plas-Bact).

As used herein, the term "mammal mitochondrial proton driving force (Δp-mito-mam)" refers to the potential energy stored in the form of (H ⁺ ) electrochemical gradient across the mammalian mitochondrial inner membrane. Δp-mito-mam is used in oxidative phosphorylation to drive ATP synthesis in mammalian mitochondria.

As used herein, the term “fungal mitochondrial proton driving force (Δp-mito-fungi)” refers to the potential energy stored in the form of an (H ⁺ ) electrochemical gradient across the fungal mitochondrial inner membrane. Δp-mito-fungi is used in oxidative phosphorylation to drive ATP synthesis in fungal mitochondria.

As used herein, the term “fungal plasma membrane proton driving force (Δp-plas-Fungi)” refers to potential energy stored in the form of an (H ⁺ ) electrochemical gradient across fungal plasma membranes, and membrane-bound H ⁺ -ATPase. By pumping of hydrogen ions across the plasma membrane. The plasma membrane-bound H ⁺ -ATPase is a high dose proton pump that requires ATP to function. ATP for the H ⁺ -ATPase is generated from Δp-mito-Fungi. Δp-plas-Fungi can be used to drive the drain pump in fungal cells.

As used herein, the term “bacterial plasma membrane proton driving force (Δp-plas-Bact)” refers to the potential energy stored in the form of an electrochemical gradient (H ⁺ ) across bacterial plasma membranes and to hydrogen across the plasma membrane during chemical osmosis. It is produced by the pumping of ions. Δp-plas-Bact may be used in oxidative phosphorylation to drive ATP synthesis in bacterial plasma membranes and may be used to drive drain pumps in bacterial cells.

As used herein, the term "assimilation pathway" refers to the cellular metabolic pathway that makes up the molecule from smaller units. These reactions require energy. Many assimilation pathways and processes are powered by adenosine triphosphate (ATP). These processes are simple molecules such as single amino acids and complex molecules such as peptidoglycans, proteins, enzymes, ribosomes, cellular organelles, nucleic acids, DNA, RNA, glucans, chitin, simple fatty acids, complex fatty acids, cholesterol And synthesis of sterols and ergosterols.

As used herein, the term “transduction” refers to a respiratory complex embedded within a membrane that uses an electron transfer reaction to pump protons across a pump membrane and generate an electrochemical potential, also known as a proton electrochemical gradient. Indicates proton transfer.

As used herein, the term "energy conversion" within a cell refers to a chemical bond that is constantly broken and created to enable the exchange and conversion of energy. It is generally said that the conversion of energy from a more concentrated form to a less concentrated form is the driving force of all biological or chemical processes responsible for the respiration of cells.

As used herein, the term “uncoupler” refers to a molecule or equipment that causes the separation of stored energy from the synthesis of ATP into the proton electrochemical gradient (ΔμH ⁺ ) of the membrane.

As used herein, the term “uncoupling” refers to the use of a decoupling agent (molecule or equipment) to cause separation of stored energy into the proton electrochemical gradient (ΔμH ⁺ ) of the membrane from the synthesis of ATP.

As used herein, the term “adenosine 5′-triphosphate (ATP)” refers to a multifunctional nucleotide that functions as the “molecular current” of intracellular energy transfer. ATP transports chemical energy for metabolism in cells and is produced as an energy source during the course of cellular respiration. ATP is consumed by many enzymes and by a series of cellular processes such as biosynthetic reactions, discharge pump function and assimilation cell growth and division.

As used herein, the term “adenosine diphosphate (ADP)” is the product of ATP dephosphorylation by ATPase. ADP is converted back to ATP by ATP synthesis. In aerobic respiratory cells, under physiological conditions, ATP synthase is understood to produce ATP using the proton driving force Δp produced by ETS as an energy source. The whole process of generating energy in this manner is called oxidative phosphorylation. The overall reaction sequence of oxidative phosphorylation is ADP + Pi → ATP. The underlying force that drives a biological reaction is the Gibbs free energy of reactants and products. Gibbs free energy is the ("free") energy available to work, and the term Gibbs free energy change (ΔG) refers to the change in free energy available within the membrane to work. The free energy is a function of enthalpy (ΔH), entropy (ΔS) and temperature (enthalpy and entropy are discussed below).

As used herein, the term “phosphorylation potential (ΔGp)” refers to ΔG for ATP synthesis in any given set of ATP, ADP and Pi concentrations (dimensions: kJ mol ⁻¹ ).

As used herein, the term "CCCP" refers to the carbonyl cyanide m-chlorophenylhydrazone, which is a highly toxic ionophore and decoupling agent of the respiratory chain. CCCP acts as a classical decoupling agent by increasing the conductivity of protons through the membrane, by coupling ATP synthesis from ΔμH ⁺ and dissipating both ΔΨ and ΔpH.

As used herein, the term “depolarization” (de-energization) refers to a decrease in the absolute value of the cell's plasma membrane or mitochondrial membrane potential ΔΨ. It is no wonder that depolarization of any bacterial plasma membrane will result in loss of ATP and increased free radical formation. It is also natural that mitochondrial depolarization of any eukaryotic cells will result in loss of ATP and increased free radical formation.

As used herein, the term “enthalpy change (ΔH)” refers to a change in enthalpy or heat content of a membrane system and is a citation or description of the thermodynamic potential of the membrane system.

As used herein, the term “entropy change (ΔS)” refers to a change in entropy of a membrane system in a more disordered state at the molecular level.

The term “redox stress” refers to a cell state that is different from the standard reduction / oxidation potential (“redox”) state of the cell. Redox stress includes increased levels of ROS, decreased levels of glutathione, and any other situation that alters the redox potential of the cell.

As used herein, the term “reactive oxygen species” refers to one of the following categories:

a) Superoxide ionic radical (O ₂ ⁻ );

b) hydrogen peroxide (non-radical) (H ₂ O ₂ );

c) hydroxyl radicals ( ^* OH);

d) hydroxy ions (OH ⁻ ).

These ROS generally occur through reaction chains:

^{_{O 2 → O 2 - + 2H}} + → H 2 O 2 → OH - + * OH → OH - (e-) (e-) (e-) (e-)

As used herein, the term "singlet oxygen" refers to ("10 ₂ ") and is formed through interaction with a triplet-excited molecule. Singlet oxygen is a non-radical species whose electrons are present in antiparallel spin. Singlet oxygen 1O ₂ has an extremely high oxidizing power, because no spin limits of his electronic, easy film (for example, a polyvalent unsaturated fatty acids or through the PUFA) may be an amino acid residue, attack the protein and DNA.

As used herein, the term "energy stress" refers to a condition that alters ATP levels in a cell. This may be a change in electron transport in mitochondria and / or plasma membranes, and exposure to a decoupling agent or ΔΨs altered radiation.

As used herein, the term “NIMELS effect” refers to the modification of the bioenergy “state” of a cell irradiated at the plasma membrane and mitochondrial membrane levels of the cell from ΔΨ-steady to ΔΨ-trans using the present invention. In particular, NIMELS effects may weaken cellular assimilation pathways or antimicrobial and / or cancer resistance mechanisms that utilize proton driving forces or chemoosmotic potentials for their energy needs.

As used herein, the term “peripheral cytoplasm or periplasm” refers to the space between the plasma membrane and the outer membrane in Gram-negative bacteria and the plasma membrane in Gram-positive bacteria and fungi, such as Candida and Trichophyton species. The space between the cell walls is shown. The periplasmic space is involved in various biochemical pathways, including nutrient acquisition, synthesis of peptidoglycans, electron transport, and alteration of substances that are toxic to cells. In Gram-positive bacteria such as MRSA, the periplasmic space is of great clinical importance because β-lactamase enzymes inactivate the penicillin antibiotics in the periplasmic space.

As used herein, the term “exhaust pump” refers to an active transport protein assembly that releases molecules (eg, antibiotics, antifungals, or poisons) in an energy dependent manner from the cytoplasm or periplasm of a cell to the external environment for removal from the cell. Indicates.

As used herein, the term “exhaust pump inhibitor” refers to a compound or electromagnetic radiation delivery system and method that inhibits the ability of the excretion pump to release molecules from cells. In particular, the excretion pump inhibitors of the present invention utilize the pump's ability to release therapeutic antibiotics, antifungal agents, anti-neoplastic agents and venom from cells through modification of ΔΨ-steady-mam, ΔΨ-steady-fungi or ΔΨ-steady-bact. It is a form of electromagnetic radiation to inhibit.

Cells that "utilize drainage pump resistance mechanisms" allow bacteria or fungi or cancer cells to release antimicrobial and / or antifungal and / or anti-neoplastic agents from their cytoplasm or periplasm to the cell's external environment, thereby reducing the By reducing the concentration below the concentration necessary to inhibit the growth and / or proliferation of the cells.

In the context of cell growth, the term “inhibit” means that the rate of growth and / or proliferation of the cell population is reduced and, if possible, stopped.

In protein chemistry, the primary structure indicates a linear arrangement of amino acids, the secondary structure indicates whether the linear amino acid structure forms a spiral or β-pleated sheet structure, and the tertiary structure of a protein or any other macromolecule Its three-dimensional structure or in other words its spatial organization (including its stereotype) of the whole single-chain molecule; The quaternary structure is an arrangement of a plurality of tertiary structural protein molecules in a multi-subunit complex.

As used herein, the term “protein stress” refers to thermodynamic modifications in tertiary and quaternary structures of proteins, including enzymes and other proteins involved in membrane transport. The term includes denaturation of proteins, misfolding of proteins, crosslinking of proteins, both interchain and intrachain linkages, eg, oxygen-dependent and independent oxidation of disulfide bonds, oxidation of individual amino acids, etc. It is not limited to this.

The term “pH stress” refers to a change in intracellular pH, ie, a decrease in intracellular pH below about 6.0 or an increase in intracellular pH above about 7.5 pH. This can occur, for example, by exposing the cells to the invention described herein and altering cell membrane components or causing changes in steady-state membrane potential ΔΨ-steady.

As used herein, the term “antifungal molecule” refers to a chemical or compound that is fungicidal or fungal. The main efficacy is the ability of the present invention to enhance antifungal molecules by inhibiting assimilation reactions and / or draining pump activity in resistant fungal strains, or by inhibiting other resistance mechanisms that require proton driving force or chemoosmotic potential for energy.

As used herein, the term “antimicrobial molecule (or antimicrobial agent)” refers to a chemical or compound that is bactericidal or bacteriostatic. Another major effect lies in the ability of the present invention to enhance antimicrobial molecules by inhibiting drainage pump activity in resistant bacterial strains, or by inhibiting anabolic reactions and / or resistance mechanisms that require proton driving or chemical osmotic potential for energy. .

As used herein, "inhibition concentration-less than" of an antimicrobial or antifungal molecule refers to a concentration below the concentration required to inhibit the majority of target cells in the population. (In one aspect, the target cell is the cell targeted for treatment, including but not limited to bacterial, fungal and cancer cells). In general, less than the inhibitory concentration refers to a concentration below the minimum inhibitory concentration (MIC), which is defined as the concentration required to reduce the growth or proliferation of the target cell by at least 10% unless specifically stated otherwise.

As used herein, the term “minimum inhibitory concentration” or MIC is defined as the lowest effective or therapeutic concentration that inhibits the growth of a microorganism.

As used herein, the term “therapeutically effective amount” of a pharmaceutical substance or molecule (eg, an antibacterial or antifungal agent), together with NIMELS, refers to a substance that will partially or completely alleviate one or more symptoms caused by the target (pathogenic) cell. Indicates concentration. In particular, the therapeutically effective amount is reduced in combination with NIMELS (1) even if the population of target cells is not removed from the patient's body, or (2) inhibits (ie, slows even if not stopped) the growth of target cells in the patient's body. , (3) slowing down the spread of the infection (ie, slowing it down if not stopped), or (4) reducing the amount of the substance associated with the infection (or eliminating it).

As used herein, the term “coefficient of interaction” is defined as a numerical representation of the magnitude of bacteriostatic / sterile and / or bacteriostatic / fungicidal interactions between a NIMELS laser and / or antimicrobial molecule in a target cell.

Thermodynamics of Energy Conversion in Biological Membranes

The present invention is directed to disturbing cell membrane biological thermodynamics (bioenergy) and consequently reducing the ability of moderately normal energy conversion and energy conversion of irradiated cells.

The method and system of the present invention optically convert Ψd-plas-mam, Ψd-mito-mam, Ψd-plas-fungi, Ψd-mito-fungi and Ψd-plas-bact to induce further alterations of ΔΨ and Δp on the same membrane. To change and modify. This is caused by targeted near-infrared irradiation of C-H covalent bonds in the long chain fatty acids of the lipid bilayer, causing a change in dipole potential Ψd.

To help understand the process of bioenergy transformation, the following description is given of the application of thermodynamics to membrane bioenergy and energy conversion in biological membranes. First, the membrane (lipid bilayer, see FIG. 1) has a significant dipole potential Ψd resulting from the structural association of the dipolar groups and the ester linkage of molecules, mainly phospholipids (FIG. 2) and water. These bipolar groups are oriented such that the hydrocarbon phase becomes positive with respect to the outer membrane zone (FIG. 3). The degree of dipole potential is large, usually hundreds of millivolts. The second major potential (separation of the charge across the film) produces the transmembrane potential ΔΨ. The transmembrane potential is defined as the electrical potential difference between the bulk aqueous phases on two sides of the membrane, resulting from the selective transport of charged molecules across the membrane. Usually, the potential on the cytoplasmic side of the cell membrane is negative for extracellular physiological solution (FIG. 4A).

Dipole potential Ψd constitutes a large and functionally important part of the electrostatic potential of all plasma membranes and mitochondrial membranes. Ψd deforms the electric field inside the film, creating a substantial positive charge at the center of the nonpolar bilayer. As a result of this "positive charge", the lipid membrane exhibits a substantial (eg, six orders of magnitude or less) difference in penetration between positively and negatively charged hydrophobic ions. Ψd also plays an important role in membrane permeability to lipophilic ions.

Certain steps in numerous cellular processes, such as binding and insertion of proteins (enzymes), lateral diffusion of proteins, ligand-receptor recognition, and membrane fusion to endogenous and exogenous molecules, depend heavily on the physical properties Ψd of the membrane bilayer. . Studies in model membrane systems have demonstrated the ability of monovalent and multivalent ions to induce isothermal phase transitions in pure lipids, different phase separations, and distinct clustering of individual components in a mixture. In membranes, such changes as described above are directed to conformational kinetics of membrane-embedded proteins and cytochromes (FIG. 4B), more specifically during their working cycles, which result in large conformational rearrangements in their transmembrane domains. Physical effects may be exerted on the protein (FIG. 5). Most importantly, the change in Ψd is thought to modulate membrane enzyme activity.

Energy conversion

Energy conversion in biological membranes generally involves the following three interrelated mechanisms.

1) Conversion of redox energy to "free energy" stored at the transmembrane ionic electrochemical potential, also called the membrane proton electrochemical gradient ΔμH ⁺ . The proton electrochemical potential difference between the two sides of the membrane that participates in active transport, including a proton pump, is sometimes also referred to as chemical osmotic potential or proton driving force.

2) In mammalian cells, the (Na ⁺ ) ion electrochemical gradient Δμx ⁺ is maintained across the plasma membrane by active transport out of the cell (Na ⁺ ). This is a different gradient from the proton electrochemical potential, but is produced from (pump) via ATP produced during oxidative phosphorylation from mammalian mitochondrial proton driving force Δp-mito-mam.

3) The use of this "free energy" (energy conversion) to generate ATP to drive active transport across the membrane with the simultaneous accumulation of required lysates and metabolites in the cell is called phosphorylation potential ΔGp. That is, ΔGp is ΔG for ATP synthesis at any given set of ATP, ADP and Pi concentrations.

Steady-state Transmembrane  Potential (ΔΨ- steady )

The state of the membrane “system” is equilibrium when the values of its chemical potential gradients ΔμH ⁺ and E (energy) are temporarily independent and there is no flux of energy across the edge of the system. The membrane system parameters of ΔμH ⁺ and E are constant, but if there is a pure flow of energy moving across the system, the membrane system is steady-state and transiently dependent.

The transient dependence of the cells (cells that breathe, grow and divide) focuses on steady-state transmembrane and / or mitochondrial translocation (ΔΨ-steady). The "steady-state" of the flow of electrons and protons, or the flow of N ⁺ / K ⁺ ions across the mitochondria or plasma membrane during normal electron transport and oxidative phosphorylation, probably continues in the future if not disrupted by endogenous or exogenous events Will be. Any exogenous modification of membrane thermodynamics will result in a transient-state transmembrane and / or mitochondrial potential ΔΨ-trans, the above change from ΔΨ-steady to ΔΨ-trans.

The mathematical relationship between the state variables ΔΨ-steady and ΔΨ-trans is called the state equation. In thermodynamics, the state function ( state amount ) is a property or system that depends only on the current state of the system. It depends on how the system gets its particular state. The present invention provides a method of transmembrane and / or mitochondrial potential ΔΨ in a transiently dependent manner to transfer the bioenergy of the membrane from a thermodynamic steady-state condition ΔΨ-steady to a transitional state ΔΨ-trans to one of energy stress and / or redox stress. Facilitate the transition.

This can occur in ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito-mam and ΔΨ-steady-mito-fungi. Without wishing to be bound by theory, the transition is targeted near-infrared irradiation (using a 930 nm wavelength) of CH covalent bonds in the long chain fatty acids of the lipid bilayer causing changes in dipole potential Ψd, and the ΔΨ-steady and redox potential of the membrane. It is thought to be caused by targeted near-infrared irradiation (using λ of 870 nm) of the cytochrome chain, which will simultaneously alter.

First Law and Membrane of Thermodynamics

The principle aspect of the First Law of Thermodynamics (true to membrane systems) is that the energy of the isolated system is conserved, and both heat and work are regarded as equivalent forms of energy. Thus, the energy levels (Ψd and ΔΨ) of the membrane system can increase or decrease the mechanical work exerted by force or pressure action, respectively, over a given distance or within an element of volume; And / or non-destructive heat conducted through a temperature gradient in the membrane.

The law (law of energy conservation) assumes that the total energy of a system isolated from its surroundings does not change. Therefore, the addition of a certain amount of (energy) heat and work to the system must be reflected in the change in the energy of the system.

Absorption of infrared radiation

Individual photons of infrared radiation do not contain enough energy (e.g., as measured by electron-volts) to induce electron transitions (molecules) as seen in photons of ultraviolet radiation. As such, absorption of infrared radiation is limited to compounds with small energy differences in the possible vibrational and rotational states of molecular bonding.

By definition, for membrane bilayers that absorb infrared radiation, vibrations or rotations within the molecular bonds of the lipid bilayers that absorb infrared photons should result in a pure change in the dipole potential of the membrane. If the frequency (wavelength) of the infrared radiation coincides with the oscillation frequency of the absorbing molecule (ie, C-H covalent bond in the long chain fatty acid), the radiation will be absorbed and cause a change in Ψd. This can occur in Ψd-plas-mam, Ψd-mito-mam, Ψd-plas-fungi, Ψd-mito-fungi and Ψd-plas-bact. That is, the methods and systems described herein can be used to cause direct and targeted changes in enthalpy and entropy (ΔH and ΔS) of all cellular lipid bilayers.

The present invention is based on a combination of insights introduced above and derived in part from experimental data, which includes:

Distinct single wavelengths (870 nm and 930 nm) result in the interaction and generation of ROS and toxic singlet oxygen responses to bacterial cells (prokaryotes), for example E. coli. It is understood that E. coli and (eukaryotes) such as Chinese hamster ovary cells (CHO) can be killed (see, eg, US Patent Application 10/649910 (2003), which is hereby incorporated by reference in its entirety). 8/26/106 (filed Feb. 11, 2004). Using the NIMEL system, instead of avoiding individual 870 nm and 930 nm wavelengths, the laser system and process (NIMEL system) of the present invention provides optical capture to advantageously utilize the use of the wavelength for therapeutic laser systems. It has been established to combine wavelengths at a power density (~ 500,000 w / cm 2 smaller power) that is 5 log smaller than typically found in confocal laser microscopy such as those used in traps.

This is a clear purpose of altering, manipulating and depolarizing ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito-mam, and ΔΨ-steady-mito-fungi of all cells within the radiation region. Is done for. This is accomplished in the present invention by targeted near-infrared irradiation (using 930 nm energy) of CH covalent bonds in the long chain fatty acids of the lipid bilayer, thus dipole potentials Ψd-plas-mam, Ψd-mito of all cells in the radiation region. -mam, Ψd-plas-fungi, Ψd-mito-fungi and Ψd-plas-bact. Second, near-infrared irradiation of the cytochrome chain (using 870 nm) will further alter the ΔΨ-steady and redox potentials of membranes with cytochromes (ie bacterial plasma membranes and fungal and mammalian mitochondria) .

Acting as a direct chromophore (CH bonds in cytochrome and long chain fatty acids), there will be a direct enthalpy and entropy change in the molecular kinetics of membrane lipids and cytochromes for all cellular lipid bilayers within the irradiation pathway of the present invention. . This will change each membrane dipole potential Ψ d in all the membranes within the irradiated cells, while at the same time changing the absolute value of the membrane potential ΔΨ.

The change occurs through significantly increased molecular motion (ie, ΔS) of the metallo-protein response centers of lipids and cytochromes when absorbing energy from the NIMEL system in a linear one-photon process. Since even small thermodynamic shifts in the lipid bilayer and / or cytochrome will be sufficient to change the dipole potential Ψ d, the molecular shape (and thus enzymatic reactivity) of the attached electron transport protein or transmembrane protein will be less functional. This will directly affect and modify ΔΨ in all membranes within the irradiated cells.

NIMELS effects occur according to the methods and systems described herein, without significant thermal or ablative mechanical damage to the cell membrane. The combined and targeted low dose approach is a change and improvement that distinguishes it from existing methods that would otherwise cause actual mechanical damage to all films in the light path of energy.

Second Law of Membrane Entropy and Thermodynamics

The conversion of heat to other forms of energy is never perfect and must always be accompanied by an increase in entropy (according to the second law of thermodynamics). Entropy (in the membrane) is a state function that describes the direction of the reaction whose changes in the reaction are due to changes in the (energy) heat input or output and associated molecular rearrangements.

Although thermal and mechanical energy are equivalent in their basic properties (in the form of energy), there is a limit to the ability to convert thermal energy into work, ie too much heat permanently damages the membrane structure and works or works in both directions. Beneficial energy changes can be prevented.

The NIMELS effect will modify the entropy "state" of irradiated cells at the level of the lipid bilayer in a transiently dependent manner. This increase in entropy will alter the Ψd of all irradiated membranes (mitochondria and traits), thus thermodynamically altering the "normal-state" flow of electrons and protons across the cell membrane (FIGS. 6 and 7). This will in turn change the steady-state transmembrane potential ΔΨ-steady to the transient-state membrane potential (ΔΨ-tran). The phenomenon will occur in the following:

1) mammalian transmembrane translocation ΔΨ-plas-mam;

2) fungal plasma transmembrane translocation ΔΨ-plas-fungi;

3) bacterial transmembrane translocation ΔΨ-plas-bact;

4) mammalian mitochondrial transmembrane potential ΔΨ-mito-mam; And

5) Fungal mitochondrial transmembrane potential ΔΨ-mito-fungi.

This is a direct result of targeted enthalpy changes at the level of C-H binding of long chain fatty acids in flow mosaic membranes, causing kinetic disorders (within membranes) that may alter membrane material properties. The flow mosaic increases entropy and can destroy tertiary and quaternary properties of electron transport proteins, leading to redox stress, energy stress and subsequent production of ROS, which further damage the membrane and further bioenergy Will change.

The main function of the electron transport system of respiratory cells is to convert energy under steady-state conditions. The technique according to the invention is used to temporarily machine-optically couple many of the relevant thermodynamic interactions in the conversion process. This can be done with a clear intention to alter the proton electrochemical gradient ΔμH ⁺ and proton driving force of the membrane and the absolute quantitative value of Δp. This phenomenon can especially occur in the following:

1) mammalian mitochondrial proton driving force (Δp-mito-mam);

2) fungal mitochondrial proton driving force (Δp-mito-fungi);

3) fungal plasma membrane proton driving force (Δp-plas-fungi); And

4) bacterial plasma membrane proton driving force (Δp-plas-Bact).

This phenomenon can in turn reduce the Gibbs free energy value ΔG (ΔGp) available for phosphorylation and synthesis of ATP. The present invention seeks to inhibit essential energy dependent anabolic reactions and / or to enhance drug therapy and / or to lower cell resistance mechanisms (for antimicrobial, antifungal and anti-neoplastic molecules) (many of these resistance mechanisms are those molecules). These phenomena are carried out as they use proton driving forces or chemical osmotic potentials for their energy needs to resist and / or discharge.

ΔΨ- steady Glass as a result of deformation of Radical  produce

The action of chemical decouplers for oxidative phosphorylation and other bioenergy operations is believed to depend on the state of charge of the membrane (plasma or mitochondria). In addition, the charged state of the bacterial membrane or eukaryotic mitochondrial inner membrane is thought to be the electrochemical proton gradient ΔμH ⁺ established by primary proton translocation events that occur during cell respiration and electron transport.

Substances that directly dissipate (depolarize) ΔμH ⁺ (eg, by permeating the coupling membrane to the transport of protons or compensation ions) short the energy coupling and cause a decrease in membrane potential ΔΨ-steady Suppress energy work. This will happen while breathing (primary proton translocation) continues quickly.

For example, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a classical decoupling agent of oxidative phosphorylation, leads to a decrease in membrane potential ΔΨ-steady when breathing continues, leading to simultaneous production of ROS. These substances (decoupling agents) cannot generally be used as antimicrobial, antifungal or antineoplastic because their effects are correspondingly toxic to all bacterial, fungal and mammalian cells.

However, in many target cells that are resistant to antimicrobial, antifungal or antineoplastic agents, Δp decouplers (such as CCCP) disrupt the energy gradients required for the discharge pump, thus greatly increasing the intracellular accumulation of these drugs. Appeared. These results clearly show that some resistance mechanisms (eg drug discharge pumps) are driven by proton driving forces. If there is a method of exploiting the effect so that only "target cell" damage is achieved, the selectivity will be a clear improvement on the universal damaging properties of the decoupling agent.

Scientific findings and experimental data of the present invention show that when the membrane is optically depolarized, the production of ROS can also further enhance the depolarization of the affected cells and further enhance the antimicrobial effect of the present invention (see Example VIII). )

NIMELS  Glass by irradiation with laser Radical  And ROS  produce

By modifying ΔΨ-steady and simultaneously mechanically-optically modifying many of the relevant thermodynamic interactions of the membrane energy conversion process, the present invention can act as an optical decoupling agent by lowering ΔμH ⁺ and Δp of the following irradiated film. .

1) mammalian mitochondrial proton driving force (Δp-mito-mam);

2) fungal mitochondrial proton driving force (Δp-mito-Fungi);

3) fungal plasma membrane proton driving force (Δp-plas-Fungi);

4) bacterial plasma membrane proton driving force (Δp-plas-Bact).

The lowered Δp will cause a series of free radicals and radical oxygen species to be produced due to the altered redox state. The production of free radicals and reactive oxygen species has been demonstrated experimentally and is described herein with the change of ΔΨ-steady to ΔΨ-trans in the following (see Example VIII):

1) ΔΨ-steady-mam + (NIMELS treatment) →→ ΔΨ-trans-mam.

2) ΔΨ-steady-fungi + (NIMELS treatment) →→ ΔΨ-trans-fungi.

3) ΔΨ-steady-bact + (NIMELS treatment) →→ ΔΨ-trans-bact.

4) ΔΨ-mito-fungi + (NIMELS treatment) →→ ΔΨ-trans-mito-fungi.

5) ΔΨ-mito-mam + (NIMELS treatment) →→ ΔΨ-trans-mito-mam.

Due to the ΔΨ-steady + (NIMELS treatment) →→ ΔΨ-trans phenomena, altered redox states and the production of free radicals and ROS can cause serious additional damage to biological membranes, such as lipid peroxidation.

Lipid peroxidation

Lipid peroxidation is a major cause of biological cell damage and death in microbial and mammalian systems. In the process, strong oxidants cause destruction of membrane phospholipids containing polyunsaturated fatty acids (PUFAs). The severity of membrane damage can lead to local reduction of membrane fluidity and complete disruption of bilayer integrity.

Peroxidation of mitochondrial membranes (mammal cells and fungi) will have adverse consequences for the respiratory chain, resulting in inadequate production of ATP and disruption of the cellular energy cycle. Peroxidation of plasma membranes (bacteria) impairs membrane permeability, dysfunction of membrane proteins such as porins and excretion pumps, inhibition of signal transduction, and inappropriate cell respiration and ATP formation (i.e. prokaryotes do not have mitochondria In living organisms, the respiratory chain is contained within the plasma membrane).

Glass Radical

Free radicals are defined as atoms or molecules that contain unpaired electrons. Examples of the damage that free radicals can cause in the biological environment include one electron (existing or generated free radical) from a bis-allylic CH bond of polyunsaturated fatty acid (PUFA) that will produce a carbon central free radical. Through) removal.

R ^* + (PUFA) -CH (bis-allyl CH bond) → (PUFA) -C ^* + RH

The reaction may initiate lipid peroxidation damage of the biological membrane. Free radicals can also be added to non-radical molecules to produce free radical products (A ^* + B → AB ^* ) or non-radical products (A ^* + B → AB).

An example thereof may be hydroxylation of an aromatic compound with ^* OH.

Responsive Oxygen species  ( ROS )

Oxygen gas is actually a free radical species. However, because it contains two unpaired electrons in different π-anti-bonding orbitals with parallel spins in the ground state, the (spin limiting) rule generally involves a pair of O ₂ having parallel spins without catalyst. Prevents receiving electrons As a result, O ₂ must receive one electron at a time.

There are many significant donors present in the cells (prokaryotes and eukaryotes) that can stimulate one electron reduction of oxygen, which will produce additional radical species.

These are generally classified as follows:

Superoxide ion radical (O ₂ ⁻ ).

Hydrogen peroxide (non-radical) (H ₂ O ₂ ).

Hydroxyl radical ( ^* OH).

Hydroxy ion (OH ^-).

The reaction chain is as follows:

^{_{O 2 → O 2 - + 2H}} + → H 2 O 2 → OH - + * OH → OH - (e-) (e-) (e-) (e-)

Superoxide

The risk of these molecules on cells is well classified in the literature. For example, superoxides can act as oxidizing or reducing agents.

NADH → NAD ⁺

More importantly for metabolism of organisms, superoxides can reduce cytochrome C. It is generally believed that the reaction rate of lipid (ie, membrane) proteins, and DNA and superoxide (O ₂ ⁻ ) is too slow to have no biological significance. Superoxide hydroperoxyl radicals (HOO ^* ) in protonated form have a lower reduction potential than (O ₂ ⁻ ), but can remove hydrogen atoms from PUFA. Also importantly, the pKa value of (HOO ^* ) is 4.8 and the (acidic) microenvironment around the biological membrane will be beneficial for the formation of hydroperoxyl radicals. In addition, the reaction of any free Fe ⁺³ with superoxide (O ₂ ⁻ ) will produce a “perferryl” intermediate, which can also react with PUFA and induce lipid (membrane) peroxidation.

Hydrogen peroxide

Hydrogen peroxide (H ₂ O ₂ ) is not a good oxidant (alone) and cannot remove hydrogen from PUFA. However, it can cross (somewhat easily) biological membranes, exerting dangerous and detrimental effects in other areas of the cell. For example, (H ₂ O ₂ ) is highly reactive with transition metals (eg Fe ⁺² and Cu ⁺ ) inside the microcellular environment, which can produce hydroxyl radicals ( ^* OH) (Fenton ( Also known as the Fenton reaction). Hydroxyl radicals are one of the most reactive species known in biology.

Hydroxyl Radical

The hydroxyl radical ( ^* OH) will react with almost all kinds of biological molecules. It essentially has a very fast reaction rate controlled by the hydroxyl radical ( ^* OH) diffusion rate and the presence (or absence) of the molecule to react near the site of ( ^* OH) production. Indeed, the standard reduction potential (E0 ′) for hydroxyl radicals ( ^* OH) is (+2.31 V), which is 7x larger than (H ₂ O ₂ ) and classified as the most reactive among biologically relevant free radicals do. Hydroxyl radicals will damage lipids in the biological membrane in addition to damaging proteins and DNA.

PUFA Reactivity resulting from peroxidation of Oxygen species

Moreover, the occurrence of lipid peroxidation (from any source) will produce three different reactive oxygen intermediate molecules from PUFA:

(a) alkyl hydroperoxide (ROOH);

Like H ₂ O ₂ , alkyl hydroperoxides are not technically radical species but are labile in the presence of transition metals such as Fe ⁺² and Cu ⁺ .

(b) alkyl peroxyl groups (ROO ^* ); And

(c) alkoxyl groups (RO ^* ).

Alkyl peroxyl groups and alkoxyl groups are extremely reactive oxygen species and also contribute to the further propagation process of lipid peroxidation. The production of free radicals and ROS due to the altered redox state of the irradiated cells and the ΔΨ-steady + (NIMELS treatment) →→ ΔΨ-trans phenomenon is another subject of the invention. It is intended to further alter cellular bioenergy, inhibit the energy dependent anabolic reactions required, and / or enhance drug therapy, and / or lower cellular resistance mechanisms against antimicrobial, antifungal and antineoplastic molecules. It is an additive effect.

ROS overproduction can damage cellular macromolecules, especially lipids. Lipid oxidation has been shown to modify both the small-scale structural kinetics of biological membranes and also their more macroscopic side tissues and alter the packing density dependent reorientation of the components of the dipole moment Ψd. Oxidative damage of the acyl chain (in the lipid) leads to loss of double bonds, chain shortening, and introduction of hydroperoxy groups. Thus, these changes are believed to affect the structural characteristics and kinetics of the lipid bilayer and the dipole potential Ψd.

Antimicrobial agents  tolerance

Antimicrobial resistance is defined as the ability of a microorganism to survive the effects of an antimicrobial drug or molecule. Antimicrobial resistance can naturally evolve through natural selection, through random mutations, or through genetic engineering. In addition, microorganisms can transfer resistance genes to each other through mechanisms such as plasmid exchange. If a microorganism carries several resistance genes, it is multidrug-resistant or uncognately referred to as a "superbug."

Multidrug-drug resistance in pathogenic bacteria and fungi is a serious problem in the treatment of patients infected with the organism. Currently, creating or developing new antimicrobial drugs that are safe for human use is very expensive and difficult. In addition, there have been evolved resistance mutant organisms that challenge all known antimicrobial classes and mechanisms. Thus, few antimicrobials maintain long-term effectiveness. Most mechanisms of antimicrobial resistance are known.

The four main mechanisms by which microorganisms are resistant to antimicrobial agents are:

a) drug inactivation or modification;

b) alteration of the target site;

c) alteration of metabolic pathways; And

d) reduction in drug accumulation by reduction in drug permeability and / or increase in active excretion on the cell surface.

Resistant microorganisms

Staphylococcus aureus (S. aureus) is one of the major resistant bacterial pathogens currently plaguing human society. The Gram-positive bacteria are found mainly on mucous membranes and skin of nearly half of the world's adult population. s. Aureus is extremely adaptable to pressure from antibiotics of all known classes. s. Aureus is the first bacterium to which penicillin resistance was discovered in 1947. Since then, almost complete resistance to methicillin and oxacillin has been found. "Superbug" MRSA (methicillin resistant Staphylococcus aureus) was first detected in 1961 and is now ubiquitous in hospitals and communities around the world. Today, all S in America. More than half of aureus infections are resistant to penicillin, methicillin, tetracycline and erythromycin. Recently, significant resistance has been reported in the next new class of antibiotics (last resort antimicrobial agents) glycopeptides and oxazolidinones (vancomycin since 1996 and Zyvox since 2003).

The new variant CA-MRSA (community acquired MRSA) has also recently emerged as a pandemic and is responsible for a group of rapidly progressing fatal diseases including necrotizing pneumonia, severe sepsis and necrotizing fasciitis. Outbreaks of community-associated (CA) -MRSA infections are reported daily in correctional facilities, athletic departments, military recruits, neonatal rooms, and among active homosexual men. CA-MRSA infections now appear to be nearly endemic in many urban areas, with most CA-S. Causes aureus infections.

Academia and the medical community have attempted to find existing antimicrobial potentiators and inhibitors of drug resistance systems in bacteria and fungi. Such enhancers and / or inhibitors would be very useful for the treatment of patients infected with pathogenic and drug-resistant microorganisms if they are nontoxic to humans. In the United States, as many as 80% of individuals have S. at some point. It is colonized by Aureus. Most colonize only intermittently, but 20-30% continue to colonize. Healthcare workers, people with diabetes, and dialysis patients all have high colony formation rates. Anterior nostrils are the main site of colony formation in adults; Other possible colony sites include armpits, rectum and perineum.

ΔΨ- in bacteria steady Selective pharmacological alteration of

There is a relatively new class of bactericidal antibiotics called lipopeptides, of which daptomycin is the first FDA approved member. Such antibiotics have been demonstrated (in vitro and in vivo) to rapidly kill virtually all clinically relevant Gram-positive bacteria (eg MRSA) through a mechanism of action that distinguishes them from other currently available antibiotics.

The mechanism of action of daptomycin involves calcium-dependent integration of lipopeptide compounds into bacterial cytoplasmic membranes. At the molecular level, calcium binding between two aspartate residues (in the daptomycin molecule) reduces its net negative charge and allows it to work better with the negatively charged phospholipids usually found in the cytoplasmic membranes of Gram-positive bacteria. . Generally there is no interaction with fungal or mammalian cells at the therapeutic level, and therefore this is a very selective molecule.

The effect of daptomycin has been proposed to result from this calcium-dependent action on bacterial cytoplasmic membranes dissipating the transmembrane membrane electrical potential gradient ΔμH ⁺ . It is only effective for selective chemical depolarization of bacterial membranes. It is well known that maintenance of correctly charged cytoplasmic membranes is essential for the survival and growth of bacterial cells, but nevertheless depolarization (in this way) is not a bacterial lethal by itself and naturally. For example, the antibiotic ballinomycin, which causes depolarization in the presence of potassium ions, is bacteriostatic but not bactericidal as in the case of CCCP.

In contrast, in the absence of the proton driving force Δp whose main component is the transmembrane electrical potential gradient ΔμH ⁺ , the cell cannot make ATP or take the necessary nutrients necessary for growth and reproduction. Disruption of ΔμH ⁺ accounts for dissimilar (harmful) effects produced by daptomycin (eg, inhibition of proteins, RNA, DNA, peptidoglycans, lipotaichoic acid and lipid biosynthesis).

Further studies of the prior art on the drug daptomycin suggest that the addition of gentamicin or minocycline (to daptomycin) enhances its bactericidal activity against MRSA. Both gentamicin and minocycline can be excreted out of MRSA cells via energy dependent pumps and are inhibitors of protein synthesis (assimilation function) at the level of 30S bacterial ribosomes. This indicates that dissipation of the transmembrane electrical potential gradient ΔμH ⁺ by daptomycin may enhance certain antimicrobial drugs. This will occur as a result of the reduction in membrane potential ΔΨ, and the fact that the resistance mechanism is less useful due to the fact that ATP is not available for the assimilation function of protein synthesis (ie, co-lowered ΔGp).

Based on the above, it is possible to optically reduce the activity and / or assimilation of the drug release pump in the target cell by safely reducing the membrane potential ΔΨ (ΔΨ-steady + (NIMELS treatment) →→ ΔΨ-trans) of the cell at a given target region. It would be clearly desirable to be able to suppress it. The method according to the invention achieves this and other processes using infrared wavelengths chosen for example, 870 nm and 930 nm, irrespective of any exogenous chemical, for example daptomycin. This is a clear improvement over the existing prior art methods which require permeable drugs to achieve the same process.

Multi-drug  Resistant discharge pump

Multidrug resistant discharge pumps are now known to be present in Gram-positive bacteria, Gram-negative bacteria, fungi and cancer cells. Ejection pumps generally have multi-specific transporters that confer a wide range of resistance mechanisms. They can enhance the effects of other mechanisms of antimicrobial resistance, for example mutation of antimicrobial targets or enzymatic modification of antimicrobial molecules. Active release for antimicrobials can be clinically relevant for β-lactam antimicrobials, macrolides, fluoroquinolones, tetracyclines and other important antibiotics, along with most antifungal compounds, including itraconazole and terbinafine. .

Using discharge pump resistance, microorganisms have the ability to capture and release antimicrobial agents or toxic compounds to the exterior (environment) of the cell, thereby reducing the intracellular accumulation of the substance. In general, over-expression of one or more of these discharge pumps is considered to prevent intracellular accumulation of material to the threshold required for its inhibitory activity. In microorganisms, the release of drugs is linked to the proton driving force that generates the electrochemical potential and / or the energy (ATP) needed for the needs of these protein pumps. This includes:

1) mammalian mitochondrial proton driving force (Δp-mito-mam);

2) fungal mitochondrial proton driving force (Δp-mito-fungi);

3) fungal plasma membrane proton driving force (Δp-plas-fungi); And

4) bacterial plasma membrane proton driving force (Δp-plas-bact).

Phylogenetically, bacterial antibiotic exhaust pumps belong to five superfamily:

(i) ABC (ATP-binding cassette), which is the main active transporter charged by ATP hydrolysis;

(ii) SMR [small multidrug resistant subfamily of DMT (drug / metabolite transporter) superfamily];

(iii) MATE (MOP (Multi-Antibiotic Extrusion Subfamily of Multidrug / Oligosaccharide-Lipoid / Monosaccharide Flippase) Superfamily);

(iv) MFS (major promoter superfamily); And

(v) RND (resistant / nodulation / split superfamily), which is all secondary active transporters driven by ion gradient.

The method of the present invention for suppressing the discharge pump is an overall deformation of the membrane ΔΨ (optical depolarization) in the irradiated region, which lowers the electrochemical gradient, which is the energy available for the phosphorylation potential ΔGp and the functional energy needs of the pump. Will lower. It is also an object of the present invention to allow the same photobiological mechanism to inhibit many different assimilation and energy driven mechanisms of target cells, including uptake of nutrients for normal growth.

Reduction of discharge pump energy : targeting of driving force of mechanism

Today, there are no drugs belonging to molecules of the "energy-blocker" family that have been developed for clinical use as drain pump inhibitors. There are several molecules that have been found to be "common" inhibitors of the discharge pump. Two such molecules are reserpin and verapamil. These molecules were originally recognized as inhibitors of vascular monoamine transporters and blockers of transmembrane calcium incorporation (or calcium ion antagonists, respectively). Verapamil is known as an inhibitor of the MDR pump in cancer cells and certain parasites and also improves the activity of tobramycin.

Reserpin inhibits the activity of Bmr and NorA (two Gram-positive discharge pumps) by altering the production of membrane proton driving force Δp required for the function of the MDR discharge pump. These molecules can inhibit the ABC transporters involved in the construction of antibiotics (ie tetracyclines), but the concentrations necessary to block the release of bacteria are neurotoxic in humans. To date, no mention is made in the literature of similar experiments with daptomycin. Fungal drug release is mediated primarily by two groups of membrane-bound transport proteins, namely ATP-binding cassette (ABC) transporters and major promoter superfamily (MFS) pumps.

Germ Transmembrane  Potential ΔΨ- plas - bact  And cell wall synthesis

During normal cell metabolism, protons build up through the cytoplasmic membrane to form ΔΨ-plas-bact. This function also acidifies (reduces pH) narrow areas near the bacterial plasma membrane. Gram-positive bacteria Bacillus subtilis subtilis ), the addition of proton conductors has been shown to increase (ie, no longer inhibit) the activity of peptidoglycan autolysin when the electron transport system is blocked. This suggests that ΔΨ-plas-bact and ΔμH ⁺ (regardless of the storage energy for the enzymatic function of the cell) potentially have significant and available effects on cell wall assimilation function and physiology.

In addition, the ΔΨ-plas-bact decoupling agent, together with the accumulation of nucleotide precursors involved in peptidoglycan synthesis, and inhibition of transport of N-acetylglucosamine (GlcNAc) (one of the major biopolymers of peptidoglycan) It has been shown to inhibit doglycan formation.

Also mentioned are antimicrobial compounds called tachyplesin that reduce ΔΨ-plas-bact in gram positive and gram negative pathogens (US Pat. No. 5,610,139, incorporated herein by reference in its entirety; Antimicrobial compositions and pharmaceutical preparations) about). The compound has been shown to have the ability to potentiate the cell wall synthesis inhibitor β-lactam antibiotic ampicillin in MRSA at lethal dose-less concentrations. Linking multiple effects of optically degraded ΔΨ-plas-bact (ie, increased cell wall autolysis, inhibited cell wall synthesis, and cell wall antimicrobial enrichment) to any other related antimicrobial therapies targeting bacterial cell walls. It is preferable. This is particularly relevant in Gram positive bacteria, such as MRSA, which do not have a discharge pump as a resistance mechanism to cell wall inhibitory antimicrobial compounds.

Cell wall inhibitory compounds do not need to be introduced through the membrane in Gram-positive bacteria, as is required in Gram-negative bacteria, to have an effect on the cell wall. Experimental evidence demonstrated that the NIMELS laser and its simultaneous optical ΔΨ-plas-bact degradation were synergistic with cell wall inhibitory antimicrobial agents in MRSA (see Example XII). Since MRSA does not have a bleed pump that functions against peptidoglycan inhibitory antimicrobial agents, it must function through inhibition of anabolic (peripheral cytoplasmic) ATP linked function and does not need to enter the cell to be effective.

ΔΨ- plas - fungi and ΔΨ- mito fungi : Prerequisites for Accurate Cell Function and Antifungal Resistance

During normal cell metabolism, ΔΨ-mito-fungi is produced via an electron transport system within the mitochondria, followed by ATP via the mitochondrial ATP synthase enzyme system. ATP then powers plasma membrane-bound H ⁺ -ATPase to produce ΔΨ-plas-fungi. Fungal mitochondrial ATP synthase has been previously found to be inhibited in a dose-dependent manner by the chemical polygodial (Lunde and Kubo, Antimicrob Agents Chemother 2000 July; 44 (7): 1943-1953). The induced reduction in cytoplasmic ATP concentration was further found to result in the inhibition of plasma membrane-bound H ⁺ -ATPase producing ΔΨ-plas-fungi, which disorder further weakened other cellular activities. In addition, the degradation of ΔΨ-plas-fungi will cause plasma membrane bioenergy and thermodynamic destruction, leading to the influx of protons that disrupt proton driving forces and thus inhibit nutrient intake.

More importantly, ATP is required for the biosynthesis of fungal plasma membrane lipid ergosterol. Ergosterol is a structural lipid targeted by the majority of related commercially antifungal compounds (ie azoles, terbinafine and itraconazole) used in medicine today.

In studies, two antimicrobial peptides (Pep2 and Hst5) have the ability to allow ATP to be excreted out of fungal cells (ie, to deplete intracellular ATP concentrations), and the degraded cytoplasmic ATP is an antifungal ATP-dependent discharge pump. It has been shown to cause inactivation of ABC transporters CDR1 and CDR2.

As a reinforcement for exhaust pump inhibition and assimilation reactions, it is advantageous to use optical methods to depolarize the membranes in fungi and to deplete cellular ATP. Therefore, it would be desirable to optically alter ΔΨ-plas-fungi and / or ΔΨ-mito-fungi to inhibit essential cell function, ATP production, and enhance antifungal compounds.

Thus, one strategy for preventing drug resistance (via the drain pump) is to reduce the level of intracellular ATP, leading to inactivation of the ATP-dependent drain pump. In fungal pathogens, no chemicals were allowed to accomplish the task. However, the NIMELS effect has the ability to optically achieve this goal, and experimental evidence has demonstrated that NIMELS lasers and phenomena are synergistic with antifungal compounds in fungi (see Example XIII).

The NIMELS effect will occur according to the methods and systems described herein without causing thermal or removable mechanical damage to the cell membrane. The combined and targeted low dose approach is a change and improvement that is distinct from all existing methods that would otherwise cause actual mechanical damage to all films in the ray path of energy.

In a first aspect, the present invention provides dipole potentials Ψd (Ψd-plas-mam, Ψd-mito-mam, Ψd-plas-fungi, Ψd-mito-fungi, and Ψd-plas-bact) of all membranes within the path of the NIMELS beam. And a method of operating a cascade of further alterations of ΔΨ and Δp in the same membrane.

Bioenergy steady-state membrane potential ΔΨ-steady (ΔΨ-steady-mam, ΔΨ-steady-fungi, ΔΨ-steady-Bact, ΔΨ-steady-mito-mam and ΔΨ-steady-mito-fungi of all irradiated cells ) Is changed to ΔΨ-trans values (ΔΨ-trans-mam, ΔΨ-trans-fungi, ΔΨ-trans-Bact, ΔΨ-trans-mito-mam and ΔΨ-trans-mito-fungi). This results in quantifiable alterations in simultaneous depolarization and absolute values of Δp (Δp-mito-mam, Δp-mito-Fungi, Δp-plas-Fungi and Δp-plas-Bact) for all irradiated cells.

The phenomenon is caused by irradiating the target site with light radiation at the desired wavelength (s), power density level (s), and / or energy density level (s), so that it is within a given target site other than targeted biological contaminants (bacteria and fungi). / Occurs at an unbearable risk and / or unbearable adverse effects on the biological subject (eg, mammalian tissue, cells or certain biochemical agents, eg protein preparations).

In certain embodiments, the applied light radiation can have a wavelength from about 850 nm to about 900 nm at the NIMELS radiation dose as described herein. In an exemplary embodiment, a wavelength of about 865 nm to about 875 nm is used. In further embodiments, the applied radiation may have a wavelength from about 905 nm to about 945 nm at the NIMELS radiation dose. In certain embodiments, the applied light radiation can have a wavelength from about 925 nm to about 935 nm. In an exemplary non-limiting embodiment illustrated below, the wavelength used is 930 nm.

The bioenergy steady-state membrane potential can be modified in the exemplary embodiment as shown below, and can use multiple wavelength ranges, including the ranges that bind 870 and 930 nm, respectively.

NIMELS  Reinforced Size Scale ( Potentiation Magnitude Scale ; NPMS )

As discussed in more detail above, NIMELS parameters include the average single or additional output power of a laser diode and the wavelength of the diode (870 nm and 930 nm). The information is in combination with the area (cm 2) of the laser beam (s) at the target site, the power output of the laser system and the time of irradiation, of the information that can be used to calculate an effective and safe irradiation protocol according to the invention. Provide a set.

Based on these novel resistance reversal and antimicrobial enhancing interactions available using NIMELS lasers, a quantitative value is needed for the "enhancing effect" that will be effective for each unique antimicrobial and laser radiation dose.

A new set of parameters is defined to consider the performance of any different radiation dose values for NIMELS lasers and any MIC value for a given antimicrobial agent being examined. This can be tailored simply to NIMELS laser systems and methods by generating only a set of variables that quantify the CFU of a pathogenic organism within any given experimental or processing parameter using the NIMELS system.

These parameters generate a measure called the NIMELS Enhanced Size Scale (NPMS), and use power and / or processing time to reverse and / or tolerate tolerance, while also generating a measure of safety for adjacent tissue that is burned and damaged. Or NIMELS laser inherent phenomena to enhance the MIC of antimicrobial drugs. The NPMS measure measures the number of NIMELS effects (N e ) of 1 to 10, wherein the goal is to obtain N e of ≧ 4 in the reduction of the CFU coefficient of the pathogen at any safe combination of antimicrobial concentration and NIMELS radiation dose. CFU coefficients are used herein to quantify pathogenic organisms, but other means of quantification, such as dye detection methods or polymerase chain reaction (PCR) methods, may also be used to obtain values for A, B, and Np parameters. Can be.

The NIMELS effect number N e is an interaction coefficient that indicates to what extent the combined inhibitory / bacterial effect of the antimicrobial drug is synergistic with the NIMELS laser against the pathogen target without harming healthy tissue.

The NIMELS enrichment number (N p ) is a value that indicates whether, at a given concentration, the antimicrobial agent is synergistic or antagonistic to the pathogen target without harming healthy tissue. Thus, within a given set of standard experimental or processing parameters

A = CFU coefficient of the pathogen in NIMELS alone;

B = CFU coefficient of the pathogen in the antimicrobial agent alone;

CFU coefficient of the pathogen at N p = (NIMELS + antimicrobial);

N e = (A + B) / 2N p .

Interpretation of NIMELS effect number N e :

Here, if 2N p <A + B, the (given) antimicrobial agent was successfully enhanced using a NIMELS laser at the concentration and radiation dose used.

Next, if N e = 1, there is no strengthening effect. If N e > 1, there is a strengthening effect. If N e ≧ 2, there is at least a 50% strengthening effect on the antimicrobial agent. If N e ≧ 4, there is at least 75% strengthening effect on the antimicrobial agent. If N e ≧ 10, there is at least a 90% strengthening effect on the antimicrobial agent.

Sample calculation 1 :

A = 110 CFU.

B = 120 CFU.

N p = 75 CFU.

N e = (110 CFU + 120 CFU) / 2 (75) = 1.5.

Sample calculation 2 :

A = 150 CFU.

B = 90 CFU.

N p = 30 CFU.

N e = (150 CFU + 90 CFU) / 2 (30) = 4.

In general, antimicrobials are expensive and are generally associated with undesirable side effects that may include kidney and / or liver damage in the system, so it may be advantageous to use lower doses of antimicrobials when treating microbial infections. Thus, it may be desirable to devise a method of lowering and / or enhancing the MIC of an antimicrobial agent. The present invention provides a system and method for reducing MIC of antimicrobial molecules when the area to be treated is simultaneously treated with a NIMELS laser system.

Reducing the MIC of antimicrobial agents for localized resistant lesion infections (eg, skin, diabetic feet, bedsores) will restore the therapeutic efficacy of many older, cheaper and safer antimicrobials to treat these infections. will be. Thus, reducing the MIC of an antimicrobial agent by addition of a NIMELS laser (e.g., producing a value of N e of> 1 on one side, ≧ 4 on the other side, ≧ 10 on the other side) of the antibiotic It represents a positive step forward in recovering lost treatment efficacy.

Thus, in one aspect, the present invention provides a method for reducing the MIC of antimicrobial molecules required to eradicate or at least attenuate microbial pathogens through depolarization of the membrane within the irradiated region that will reduce the membrane potential ΔΨ of the irradiated cells. And a system. The weakened ΔΨ will cause associated weakening of the proton driving force Δp and associated bioenergy of all affected membranes. It is a further object of the present invention that said "NIMELS effect" enhances existing antimicrobial molecules against microorganisms that infect and harm human hosts.

In certain embodiments, the applied light radiation has a wavelength from about 850 nm to about 900 nm at the NIMELS radiation dose as described herein. In an exemplary embodiment, a wavelength of about 865 nm to about 875 nm is used. In further embodiments, the applied radiation has a wavelength from about 905 nm to about 945 nm at the NIMELS radiation dose. In certain embodiments, the applied light radiation has a wavelength of about 925 nm to about 935 nm. In one aspect, the wavelength used is 930 nm.

Microbial pathogens whose bioenergy systems are affected by the NIMELS laser system according to the present invention include microorganisms such as bacteria, fungi, fungi, mycoplasma, protozoa and parasites. In the exemplary embodiments shown below, multiple wavelength ranges may be used, including the ranges that bind 870 and 930 nm, respectively.

In the method according to one aspect of the invention, the irradiation by the wavelength range under consideration is carried out independently, sequentially, in a mixed ratio, or essentially simultaneously (all of which are pulsed and / or continuous waves ( CW) jobs are available).

Irradiation using NIMELS energy at NIMELS radiation doses to biological contaminants is applied prior to, subsequent to or concurrent with the administration of the antimicrobial agent. However, the NIMELS energy at the NIMELS radiation dose can be administered after reaching the "peak plasma level" in an individual or other mammal infected with the antimicrobial agent. It should be noted that co-administered antimicrobial agents should have antimicrobial activity against any naturally sensitive variant of resistant target contaminants.

The wavelengths irradiated in accordance with the methods and systems of the present invention increase the susceptibility of contaminants to levels of similar non-resistant contaminant strains at concentrations of antimicrobial agents of about 0.01 M or less, or about 0.001 M or less, or about 0.0005 M or less. .

The methods of the present invention slow or eliminate the progression of microbial contaminants within a target site, improve at least some symptomatic or asymptomatic pathology associated with the contaminant, and / or increase the susceptibility of the contaminant to antimicrobial agents. For example, the methods of the present invention reduce the level of microbial contaminants in the target site by increasing the susceptibility of the biological contaminant to the antimicrobial agent to which the biological contaminant has evolved or obtained resistance without a deleterious effect on the biological subject. Or enhance the activity of antimicrobial compounds. The reduction in the level of microbial contaminants can be, for example, at least 10%, 20%, 30%, 50%, 70% or more relative to the pretreatment level. With regard to the sensitivity of biological contaminants to antimicrobial agents, the sensitivity is enhanced by at least 10%.

In another aspect, the present invention provides a system for performing a method according to another aspect of the present invention. The system includes a laser oscillator for generating radiation, a controller for calculating and controlling the dose of radiation, and a delivery assembly (system) for delivering the radiation through the application area to the treatment site. Suitable delivery assemblies / systems include hollow waveguides, optical fibers and / or free space / ray light transmission components. Suitable free space / ray light transmission components include collimating lenses and / or aperture stops.

In one form, the system uses two or more solid state diode lasers to function as dual wavelength near infrared light sources. Two or more diode lasers may be located in a single housing with unified control. The two wavelengths may comprise emission in two ranges from about 850 nm to about 900 nm and from about 905 nm to about 945 nm. The laser oscillator of the present invention is used to emit a single wavelength (or peak value, for example a center wavelength) in one of the ranges disclosed herein. In certain embodiments, the laser is used to emit radiation substantially in the range of about 865-875 nm and about 925-935 nm.

The system according to the invention may comprise a light source suitable for each individual wavelength range to be manufactured. For example, suitable solid state laser diodes, variable ultra-short pulsed laser oscillators, or ion-doped (eg, using suitable rare earth elements) optical fibers or fiber lasers are used. In one form, suitable near infrared lasers include titanium-doped sapphire. Other suitable laser sources, including those with other types of solid state, liquid or gas gain (active) media, can be used within the scope of the present invention.

According to one embodiment of the invention, a treatment system is a light radiation generating system adapted to generate light radiation substantially in a first wavelength range of about 850 nm to about 900 nm, wherein the light radiation is directed through an application zone. And a controller operatively connected to the delivery assembly and to the optical radiation generating equipment for controlling the dose of the delivered radiation through the application zone such that the time integral of the power density and energy density of the delivered radiation per unit area is below a predetermined threshold. It includes. Also within this embodiment is a treatment system that is specifically adapted to generate light radiation substantially in the first wavelength range of about 865 nm to about 875 nm.

According to a further embodiment, the treatment system comprises light radiation generating equipment configured to generate light radiation in a second wavelength range substantially from about 905 nm to about 945 nm; In certain embodiments, the first wavelength range of interest is produced simultaneously or simultaneously / sequentially by the optical radiation generating equipment. Also within the scope of this embodiment is a treatment system employed to produce light radiation, particularly in the first wavelength range of about 925 nm to about 935 nm.

The treatment system includes a delivery assembly (system) for delivering light radiation of a second wavelength range (and, if applicable, the first wavelength range) through the application zone, and substantially the first wavelength range or substantially the second wavelength range or these And may further comprise a controller operable to control the optical radiation generating equipment to selectively generate radiation in any combination of the following.

According to one embodiment, the delivery assembly comprises one or more optical fibers having ends formed and arranged for insertion into patient tissue at a location within the light transmission range of the medical equipment, wherein the radiation is NIMELS to the tissue surrounding the medical equipment. Delivered at the radiation dose. The delivery assembly may further comprise a free ray optical system.

According to a further embodiment, the controller of the treatment system includes a power limiter to control the dose of radiation. The controller may further include a memory for storing the patient's profile, and a radiation dose calculator for calculating the dose required for the particular target site based on information input by the operator. In one aspect, the memory can also be used to store information regarding different types of disease and treatment profiles, eg, patterns of radiation and doses of radiation associated with particular uses. Light radiation can be delivered in different patterns from the treatment system to the site of application. The radiation can be produced and delivered as a continuous wave (CW) or pulsed wave, or a combination of each, for example in a single wavelength pattern or in a multi-wavelength (eg double-wavelength) pattern. For example, radiation of two wavelengths can be multiplexed (optically combined) or delivered simultaneously to the same treatment site. Suitable optical combination techniques include, but are not limited to, the use of polarized light splitters (combiners), and / or superposition of output concentrated from suitable mirrors and / or lenses, or other suitable multitransmission / combination techniques. Can be used. Alternatively, the radiation may be delivered in an alternating pattern, where radiation of two wavelengths is alternately delivered to the same treatment site. The spacing between two or more pulses can be selected as desired in accordance with the NIMELS technique of the present invention. Each process can combine any of these transmission schemes. The intensity distribution of the transmitted light radiation can be selected as desired.

Exemplary embodiments include a top-hat or substantially top-hat (eg, trapezoidal, etc.) intensity distribution. Other intensity distributions such as Gaussian can be used.

As used herein, the term “biological contaminant” refers to a mammal (eg, a recipient) on or near a target site (eg, an infected tissue or organ of a patient) upon direct or indirect contact with the target site. In the case of cells, tissues or organs implanted into or in the case of equipment used on a patient) is intended to mean a contaminant that can cause unwanted and / or deleterious effect (s). Biological contaminants according to the present invention are microorganisms known to those skilled in the art, commonly found at the target site, for example bacteria, fungi, fungi, mycoplasmas, protozoa, parasites.

Those skilled in the art will appreciate that the methods and systems of the present invention can be used in connection with various biological contaminants generally known to those skilled in the art. The following list is provided solely for the purpose of illustrating a wide range of microorganisms that may be targeted according to the methods and apparatus of the present invention and is not intended to limit the scope of the present invention.

Thus, the exemplary non-limiting examples of biological contaminants (pathogens) may be any bacterium, e.g., Escherichia (Escherichia), Enterobacter (Enterobacter), Bacillus, Campylobacter (Campylobacter), Corynebacterium ( Corynebactenum), keulrep when Ella (Klebsiella), Trail Four nematic (Treponema), Vibrio (Vibrio), is not one which limits include Streptococcus (Streptococcus) and Staphylococcus (Staphylococcus).

To further illustrate, the biological contaminants contemplated are any fungi, for example Trichophyton, Microsporum , Epidermophyton , Candida, Scopulariopsis Scopulariopsis brevicaulis), Fusarium (Fusarium) species, Aspergillus (Aspergillus) species, Alterna Liao (Alternaria), Acre monyum (Acremonium), between the desorption pyridinium di MIDI Atum (Scytalidinum dimidiatum ), and cytalinium hyalinum ( Scytahdinium) hyalinum ), but is not limited to such. Parasites such as Trypanosoma and malaria parasites such as Plasmodium species, and fungi; Mycoplasma and prions may also be targeted biological contaminants. Human immunodeficiency virus and other retroviruses, herpes virus, parvovirus, filovirus, circoviruses, paramyxovirus, cytomegalovirus, hepatitis virus (including hepatitis B and hepatitis C), Viruses can also be targeted, including pox viruses, toga viruses, Epstein-Barr viruses, and parvoviruses.

It will be appreciated that the target site to be irradiated does not need to be already infected with biological contaminants. Indeed, the methods of the invention may be used "prophylactically" prior to infection. Further embodiments include medical equipment, such as catheters (eg, IV catheter, central venous catheter, arterial catheter, peripheral catheter, dialysis catheter, peritoneal dialysis catheter, epidural catheter), artificial joint, stent ), External fixator pins, chest tube, gastronomy feeding tube, and the like.

In certain cases, irradiation may be for prevention as well as for relief purposes. Thus, the methods of the present invention are used to irradiate tissue (s) for a therapeutically effective time to treat or alleviate the symptoms of an infection. The expression “treating or alleviating” means reducing and / or preventing and / or reversing the symptoms of an individual treated in accordance with the present invention relative to the symptoms of an untreated individual.

Those skilled in the art will appreciate that the present invention is useful in connection with various diseases caused or associated by any microbial, fungal and viral infection (Harrison's, Principles , which is hereby incorporated by reference in its entirety). of Internal Medicine , 13 ^th Ed, McGraw Hill, New York (1994). In certain embodiments, the methods and systems according to the present invention are conventional treatment methods available in the art for treating infections by administration of known antimicrobial compositions (e.g., incorporated herein by reference in its entirety). And Goodman and Gilman's, The Pharmacological Basis of Therapeutics, 8th ed, 1990, Pergmon Press. The terms “antimicrobial composition”, “antimicrobial agent” refer to compounds and combinations thereof that are administered to animals, including humans, and that inhibit the growth of microbial infections (eg, antimicrobial, antifungal and antiviral).

The wide range of uses contemplated includes, for example, a variety of dermatology, podiatry, pediatrics and general medicine, with a few mentions. The interaction between the target site to be treated and the energy imparted is wavelength (s); Chemical and physical properties of the target site; Power density or radiation of the light beam; Whether continuous wave (CW) or pulsed irradiation is used; Laser beam spot size; It is defined by many parameters, including exposure time, energy density, and any change in the physical properties of the target site as a result of laser irradiation using any of these parameters. In addition, the physical properties of the target site (eg, absorption and scattering coefficients, scattering anisotropy, thermal conductivity, thermal capacity, and mechanical strength) can also affect the overall effect and results.

NIMELS radiation doses may cause the target wavelength to produce ROS, reducing the level of biological contaminants at the target site and / or being intolerable for biological moieties other than biological contaminants (eg, mammalian cells, tissues, or organs). The power density at which the contaminant can be irradiated to increase the susceptibility of the biological contaminant to antimicrobials to which the contaminant is tolerated through the reduction of ΔΨ with simultaneous generation of ROS without risk and / or unbearable side effects (W / Cm 2) and energy density (J / cm 2; here 1 watt = 1 joule / sec).

See Boulnois, 1986, Lasers Med., Which is hereby incorporated by reference in its entirety. Sci. 1: 47-66 (1986), at low power densities (also referred to as radiation) and / or energy, laser-tissue interactions can be described purely as optical (photochemistry), while At high power densities, light-thermal interactions occur. In certain embodiments exemplified below, the NIMELS radiation dose parameters lie between known photochemical and photo-thermal parameters in areas traditionally used for photodynamic therapy with exogenous drugs, dyes and / or chromophores, but with exogenous drugs, It can function in the field of photodynamic therapy without requiring dyes and / or chromophores.

The energy density (also expressed as fluence, or the product (or integral) of particle or radiation flow and time) for medical laser applications is usually about 1 J / cm 2 to about 10, OOO J / cm 2 (of order 5). Power density (radiation) varies from about 1 × 10 ⁻³ W / cm 2 to about 10 ¹² W / cm 2 (15 orders of magnitude). Given the inverse correlation between power density and irradiation exposure time, one can observe that approximately the same energy density is required for any intended specific laser-tissue interaction. As a result, laser exposure duration (radiation irradiation time) is a key parameter that determines the nature and safety of laser-tissue interactions. For example, when mathematically exploring thermal evaporation (non-removable) of tissues in vivo (see Boulnois 1986), to generate an energy density of 1000 J / cm 2 (see Table 1), It can be seen that the following radiation dose parameters can be used:

Example of Derived Values Based on the Boulnois Table Power density time Energy density 1x10 ⁵ W / ㎠ 0.01 sec 1000 J / ㎠ 1x10 ⁴ W / ㎠ 0.10 sec 1000 J / ㎠ 1x10 ³ W / ㎠ 1.00 sec 1000 J / ㎠

The sequence describes a suitable method or basic algorithm that can be used for NIMELS interaction with biological contaminants in tissues. That is, the mathematical relationship is inversely correlated to achieve the laser-tissue interaction phenomenon. This fundamental principle can be used as a basis for calculating radiation dose for the observed antimicrobial phenomena imparted by NIMELS energy with the insertion of NIMELS experimental data in energy density and time and power parameters.

Based on specific interactions at the target site to be irradiated (eg, chemical and physical properties of the target site; whether continuous wave (CW) or pulsed irradiation is used; laser beam spot size; using any of these parameters) Any changes in the physical properties of the target site as a result of laser irradiation, such as absorption and scattering coefficients, scattering anisotropy, thermal conductivity, thermal capacity, and mechanical strength), and those skilled in the art Density and time can be adjusted.

The examples provided herein show this relationship in the context of both in vitro and in vivo treatments. Thus, for example, in the context of treating onychomycosis or infected wounds for spot sizes 1-4 cm in diameter, safe and non-injured / minimal levels well below the "degeneration" and "tissue overheat" levels. The power density values varied from about 0.5 W / cm 2 to about 5 W / cm 2 to lie within the damaging thermal laser-tissue interaction. Other suitable spot sizes may be used. In the inverse correlation, the threshold energy density required for NIMELS interaction using these wavelengths can be maintained regardless of the point size at which the desired energy is delivered. In an exemplary embodiment, light energy is delivered to the tissue through a uniform geometric distribution (eg, flat-top or top-hat propagation). Using this technique, a suitable NIMELS radiation dose (NIMELS effect) sufficient to generate ROS is required to reduce the level of biological contaminants and / or increase the susceptibility of biological contaminants to antimicrobial agents to which the contaminants are resistant. But can be calculated to reach threshold energy densities below the "degeneration" and "tissue overheat" levels.

The NIMELS radiation dose (eg, nail fungus) exemplified herein for targeting microorganisms in vivo is from about 200 J / cm 2 to about 700 J / cm 2 for about 100 to 700 seconds. These power values do not approach power values associated with photoremoval or photothermal (laser / tissue) interactions.

The intensity distribution of the aimed laser beam is given by the power density of the beam and is defined as the ratio of the area of the circle (cm 2) in the laser output power to energy and the spatial distribution pattern of energy. Thus, the illumination pattern of a 1.5 cm irradiation spot with an incident Gaussian light pattern of 1.77 cm 2 area can produce at least six different power density values within the 1.77 cm 2 irradiation area. These varying power densities increase in intensity (or concentration of power) over the surface area of points from 1 (on the outer periphery) to 6 at the center point. In certain embodiments of the invention, a ray pattern is provided that overcomes the inherent errors associated with traditional laser ray emission. The NIMELS parameter can be calculated as a function of processing time (Tn) as follows: Tn = energy density / power density.

In certain embodiments (see, eg, in vitro experiments below), Tn is about 50 to about 300 seconds; In other embodiments, Tn is about 75 to about 200 seconds; In another embodiment, Tn is about 100 to about 150 seconds. In an in vivo embodiment, Tn is about 100 to about 1200 seconds.

Using this relationship and the desired optical intensity distribution, for example, the flat top illumination geometry described herein, a series of in vivo energy parameters have been experimentally demonstrated as effective for NIMELS microbial decontamination therapy in vitro. Thus, a key parameter for a given target site has been shown to be the energy density required for NIMELS therapy at various different spot sizes and power densities.

“NIMELS radiation dose” is such that there is an unbearable adverse risk or effect from the first threshold point to the second endpoint, and / or on the biological moiety, in which the target wavelength according to the invention can optically reduce ΔΨ in the target site. Range of power density and / or energy density until the generation of ROS just prior to the value detected (eg, thermal damage, eg perforation) to increase the susceptibility of the biological contaminant to the antimicrobial to which the contaminant is resistant. It includes. Those skilled in the art will understand that under certain circumstances, adverse effects and / or risks at target sites (eg, mammalian cells, tissues, or organs) may be acceptable in view of the inherent advantages arising from the methods of the present invention. . Thus, the stopping point considered is a point where the deleterious effect is significant and therefore undesirable (eg, cell death, protein denaturation, DNA damage, morbidity or mortality).

In certain embodiments, for example, for in vivo use, the power density range contemplated herein is from about 0.25 to about 40 W / cm 2. In other embodiments, the power density ranges from about 0.5 W / cm 2 to about 25 W / cm 2.

In further embodiments, the power density range may comprise a value from about 0.5 W / cm 2 to about 10 W / cm 2. Power densities exemplified herein are about 0.5 W / cm 2 to about 5 W / cm 2. In vivo power densities of about 1.5 to about 2.5 W / cm 2 have been shown to be effective against a variety of microorganisms.

Experimental data appear to indicate that higher power density values are generally used when targeting biological contaminants in in vitro settings (eg plates) rather than in vivo (eg claws).

In certain embodiments (see in vitro examples below), the energy density range contemplated herein is greater than 50 J / cm 2 but less than about 25,000 J / cm 2. In other embodiments, the energy density ranges from about 750 J / cm 2 to about 7,000 J / cm 2. In another embodiment, the energy density range is about 1,500 J / cm 2 depending on whether the biological contaminant should be targeted in an in vitro setting (eg, plate) or in vivo (eg, toenail or surrounding medical device). To about 6,000 J / cm 2. In certain embodiments (see in vivo examples below), the energy density is between about 100 J / cm 2 and about 500 J / cm 2. In another in vivo embodiment, the energy density is about 175 J / cm 2 to about 300 J / cm 2. In yet another embodiment, the energy density is between about 200 J / cm 2 and about 250 J / cm 2. In some embodiments, the energy density is about 300 J / cm 2 to about 700 J / cm 2. In some other embodiments, the energy density is about 300 J / cm 2 to about 500 J / cm 2. In another embodiment, the energy density is between about 300 J / cm 2 and about 450 J / cm 2.

The power density tested empirically for various in vitro treatments of microbial species is from about 1 W / cm 2 to about 10 W / cm 2.

Those skilled in the art will appreciate that identification of particularly suitable NIMELS radiation dose values within the power and energy densities contemplated herein for a given situation can be made empirically through routine experimentation. One skilled in the art (e.g., a dentist) using near infrared energy in conjunction with periodontal treatment routinely adjusts power density and energy density based on the requirements associated with each given patient (e.g., tissue color, tissue structure and Adjust the parameter as a function of the depth of pathogen invasion). As an example, laser treatment of periodontal infection in brightly colored tissue (eg, melanin deficient patients) will have greater thermal safety parameters than darker tissue, which will allow darker tissue to absorb near infrared energy more efficiently. Therefore, they convert these near-infrared energy into heat faster in the tissue. Thus, the ability of those skilled in the art is clearly needed to identify a number of different NIMELS dose values for different treatment protocols.

As illustrated below, it has been found that antibiotic resistant bacteria can be effectively treated according to the present invention. It has also been found that the methods of the present invention can be used to augment traditional approaches, in combination with, or instead of, or even as continuous effective treatment options. Thus, the present invention can be combined with antibiotic therapy. The term “antibiotic” refers to β-lactams, penicillins and cephalosporins, vancomycins, bacitracins, macrolides (erythromycins), ketolides (tellrismycins, lincosamides (clindomycins), chloramphenicols, Tetracycline, aminoglycosides (gentamicins), amphotericin, anilinouracil, cefazoline, clindamycin, mupyrosine, sulfonamides and trimethoprim, rifamcillin, metronidazole, quinolone, novobiocin, polymyxin, oxa Zolidinone class (e.g. linezolid), glycylcycline (e.g. tigecycline), cyclic lipopeptide (e.g. daptomycin), fluromutylin (e.g. retafaline ), And any salt or variant thereof, including gramicidine, etc. Tetracycline is immunocycline, chlortetracycline, oxytetracycline, demeclocycline, metasa It is also understood that within the scope of the present invention, including but not limited to, clean, doxycycline, minocycline, etc. The aminoglycoside antibiotics include, but are not limited to, gentamicin, amikacin, neomycin and the like. It is understood to be within the scope of.

As illustrated below, it has been found that antifungal agent resistant fungi can be effectively treated according to the methods of the present invention. It has also been found that the methods of the present invention can be used to augment traditional approaches, in combination with, or instead of, or even as continuous effective treatment options. Thus, the present invention can be combined with antifungal therapy. The term "antifungal agent" refers to polyenes, azoles, imidazoles, triazoles, allylamines, echinocandines, sicopyrox, flucitosine, gliofulvin, amololpin, sodarin and combinations thereof (including salts thereof) Including but not limited to.

As illustrated below, it is believed that anti-neoplastic agent resistant cancers can be effectively treated according to the methods of the present invention. It has also been found that the methods of the present invention can be used in combination with traditional therapies, instead or even as continuous effective treatment methods, to augment traditional approaches. Thus, the present invention may be combined with antineoplastic agents. The term “antineoplastic agent” includes, but is not limited to, actinomycin, anthracycline, bleomycin, plicamycin, mitomycin, taxanes, etoposide, teniposide, and combinations thereof, including salts thereof. .

A common principle of prior art attempts to find inhibitors of drug resistance systems, or enhancers of antimicrobial agents, in bacteria and fungi is that the material must always be nontoxic to infected mammalian tissue to have any inherent value. In addition, it has always been the fact that antimicrobials affect bacterial or fungal cell processes that are not common to mammalian hosts and are therefore generally safe and therapeutic in nature and design. In the prior art, antimicrobials, potentiators and / or resistant reversal entities also could not be used safely as therapeutic agents if they also affected mammalian cells in the same way as damaging the pathogen.

In the present invention, experimental data (see, eg, Example IX) supports the universal alteration of ΔΨ and Δp between all cell types, so that the electro-mechanical as well as the electro-mechanical aspects of all cell membranes are also appropriately It derives the concept of having different characteristics that can be separated. This indicates that not only pathogenic cells (undesired cells) but also all cells in the path of light are affected by depolarization.

By reconfirming that the photobiological and cellular energetics data of the NIMELS system have already been illuminated (ie, all membrane energies are affected in the same way across prokaryotic and eukaryotic species), the technique according to the present invention is directed to antimicrobial molecules. The universal optical depolarization effect is utilized to be added to the treatment plan and to be utilized independently in undesirable cells by enhancing the molecule only in undesirable cells. Such targeted therapeutic outcomes can take advantage of the universal depolarization effects of NIMELS lasers, which can be more graded and transient for mammalian cells within the path of treatment than for bacteria and fungi. Thus, as experimental data suggests, the transient and energetic robustness measurements of mammalian cells should be greater in terms of optical depolarization and ROS production than those seen in bacterial or fungal cells.

The examples below provide experimental evidence demonstrating the concept of universal optical membrane depolarization associated with preservation of thermodynamics as applied to our present understanding of photobiology and cell energyology and as applied to cellular processes.

The following examples are included to illustrate exemplary embodiments of the invention and are not intended to limit the scope of the invention. Those skilled in the art will understand that many changes can be made in particular embodiments, and that similar results can continue to be made without departing from the spirit and scope of the invention.

Example  I

Sensitive, moderately reactive and resistant S. a. MIC value for Aureus Minimum Inhibitory Concentration (MIC) Descriptive Standards for Staphylococcus Species (µg / ml) Antimicrobial agents sensibility Intermediate reactivity tolerance penicillin ≤0.12 - ≥0.25 Methicillin ≤8 - ≥16 Aminoglycosides Gentamicin ≤4 8 ≥16 Kanamycin ≤16 32 ≥64 Macroroll Erythromycin ≤0.5 1-4 ≥8 Tetracycline Tetracycline ≤4 8 ≥16 Fluoroquinolone Ciprofloxacin ≤1 2 ≥4 Folate pathway inhibitor Trimethoprim ≤8 - ≥16 Ansamycin Rifampin ≤1 2 ≥4

Example II

Germ way: In vitro MRSA  For the experiment NIMELS  Treatment parameters

The following parameters illustrate the general bacterial method according to the invention applied to MRSA for in vitro experiments V and VIII-XII.

A. Experimental Materials and Methods for MRSA :

CFU counting method Time (hours) task FTE (hours) T-18 Inoculate overnight cultures 50 ml directly from glycerol stock One T-4 Set up starter cultures Three dilutions in LB medium 1:50, 1: 125, 1: 250 One OD ₆₀₀ monitoring of starter culture 4 T0 Preparation of Plating Cultures. At 10:00 AM, the culture with OD ₆₀₀ = 1.0 is diluted 1: 300 in PBS (final volume 50 ml) and stored for 1 hour at RT. (Room temperature should be ~ 25 ℃) One T +1 Inoculation of 24-well plates. 2 ml aliquots are dispensed into pre-designated wells in 24-well plates and transferred to NOMIR. One T +2 to +8 Dilution of processed sample. After laser treatment, 100 μl from each well is serially diluted with a final dilution of 1: 1000 in PBS. 4 Plating of processed samples. 100 μl final dilution is plated (5 ×) in triplicate on TSB agar with and without antibiotics (10 TSB plates per well). 2 Plates are incubated at 37 ° C. for 18-24 hours. T +24 Count colonies on each plate. 6

this. Coli and seeds. Similar cell culture and kinematics protocols were performed for all NIMELS irradiations using the Albicans in vitro test. Thus, for example, Mr. Albicans ATCC 14053 liquid cultures were grown at 37 ° C. in YM medium (21 g / L, Difco). Normalized suspensions were aliquoted into selected wells in 24-well tissue culture plates. After laser treatment, 100 μl was removed from each well and serially diluted 1: 1000 to obtain a final dilution of 1: 5 × 10 ⁶ of the initial culture. An aliquot of each final dilution was diffused onto a separate plate. The plate was then incubated at 37 ° C. for about 16-20 hours. Manual colony counts were performed and recorded.

ΔΨ and ROS Analysis Methods Time (hours) task FTE (hours) T-18 Inoculate overnight cultures 50 ml directly from glycerol stock One T-4 Set up starter cultures Three dilutions in LB medium 1:50, 1: 125, 1: 250 One OD ₆₀₀ monitoring of starter culture 4 T0 Preparation of Plating Cultures. At 10:00 AM, the culture with OD ₆₀₀ = 1.0 is diluted 1: 300 in PBS (final volume 50 ml) and stored for 1 hour at RT. (Room temperature should be ~ 25 ℃) One T +1 Inoculation of 24-well plates for analysis. 2 ml aliquots are dispensed into pre-designated wells in 24-well plates and transferred to NOMIR. One T +2 to +8 Dilution of processed sample. After laser treatment, each control and laser treated sample was processed according to the instructions of the individual assay. 4

Again, this. Coli and seeds. Similar cell culture and kinematics protocols were performed for all NIMELS irradiations using the Albicans in vitro assay. Thus, for example, Mr. Albicans ATCC 14053 liquid cultures were grown at 37 ° C. in YM medium (21 g / L, Difco). Normalized suspensions were aliquoted into selected wells in 24-well tissue culture plates. After laser treatment, each laser treatment and control sample was processed according to the instructions of the individual assay.

Example III

Mammalian Cell Method: In vitro HEK293  (Human Embryonic Kidney Cells) for Experiments NIMELS  Processing parameters

The following parameters illustrate the general bacterial method according to the invention, applied to HEK293 cells for in vitro experiments.

A. HEK29S  Experimental Materials and Methods for Cells

HEK293 cells were seeded in Freestyle medium (Invitrogen) at a density of 1 × 10 ⁵ cells / ml (0.7 ml total volume) into appropriate wells of a 24-well plate. Cells were incubated for about 48 hours in 8% CO ₂ at 37 ° C. before experiments in a humidified incubator. The cells showed about 90% fusion at the time of experiment equivalent to approximately 3 × 10 ⁵ total cells. Immediately before treatment, cells were washed in pre-warmed phosphate buffered saline (PBS) and covered 2 ml of PBS during treatment.

After laser treatment, cells were mechanically removed from the wells and transferred to a 15 ml centrifuge tube. Mitochondrial membrane potential and total glutathione were determined according to the kit manufacturer's instructions.

Example IV

CRT + (Yellow) and CRT - (White) s . In Aureus  About NIMELS In vitro  exam

We genetically engineered to remove the crt gene (yellow carotenoid pigment). Experiments were carried out using the crt- (white) mutants of Aureus, and these mutants were wild type (yellow) S. aureus. Treatment with a previously determined non-lethal dose of NIMELS laser for Aureus. The purpose of this experiment is to test for the phenomenon of radical oxygen species (ROS) generation and / or singlet oxygen production using a NIMELS laser. In the academic literature, Liu et al. Found yellow S. neutrophils. A similar model was previously used to test the antioxidant protective activity of aureus ( ^* carotenoid) pigments (Liu et al, Staphylococcus aureus golden pigment impairs neutrophil killing and promotes virulence through its antioxidant activity, Vol. 202, No 2, July 18, 2005, 209-215).

s. The gold color in the aureus was previously measured by a carotenoid (antioxidant) pigment that can protect the organism from singlet oxygen, and when a mutant that does not produce the carotenoid pigment is isolated (crt-), Mutant colonies are “white” in appearance, more sensitive to oxidative lethality, and have impaired neutrophil survival.

Non-lethal radiation doses of NIMELS lasers (for wild type S. aureus) consistently kill up to 90% of mutant "white" cells and normal S. aureus. It was found not to kill Aureus. s. The only genetic difference between the two strains of Aureus is the lack of antioxidant pigments in the mutants. The experimental data indicate that the endogenous production of radical oxygen species and / or singlet oxygen is "white" S. Strongly suggests killing Aureus.

Example  V

MRSA , Seed. Albicans  And this. Coli  For changes in ΔΨ NIMELS In vitro  exam

There are selected fluorescent dyes that can be taken up by intact cells and accumulate in intact cells within 15 to 30 minutes without significant staining of other plasma components. These dye markers of membrane potential have been available for many years and have been used to study cell physiology. Since their spectral fluorescence properties are responsive to changes in the value of the transmembrane potential ΔΨ-steady, the fluorescence intensity of these dyes can be easily monitored.

These dyes generally work by translocation-dependent distribution between extracellular medium and the membrane or cytoplasm of the membrane. This is caused by the redistribution of the dye through the interaction of the voltage potential with the ionic charge on the dye. The fluorescence can be removed within about 5 minutes by protonated carbonyl cyanide m-chlorophenylhydrazone (CCCP), which is dependent on the internal-negative transmembrane potential where maintenance of dye concentration is maintained by functional ETS and Δp. Indicates that it is dependent.

Hypothesis test:

The null hypothesis is μ ₁ -μ ₂ = 0:

μ ₁ is the fluorescence intensity in control cell culture (without laser) treated with carbocyanine dye.

μ ₂ is the fluorescence intensity in the same cell culture pre-radiated with a lethal dose-less radiation dose from a NIMELS laser.

The data indicate that the fluorescence of the cells is dissipated by pre-treatment (of the cells) using a NIMELS laser system (less than a control group of non-irradiated or “laser treated” cells), which means that the NIMELS lasers pass through the plasma membrane It interacts with the respiratory process and oxidative phosphorylation.

μ ₁ -μ ₂ = 0 will support that lethal dose-less NIMEL irradiation on cell culture shows no effect on ΔΨ-steady.

μ ₁ -μ ₂ > 0 will support that lethal dose-less NIMEL irradiation on cell culture has a dissipating or depolarizing effect on ΔΨ-steady.

Substances and Methods:

BacLight ™ Bacterial Membrane Translocation Kit (B3495Q, Invitrogen). The BacLight ™ Bacterial Membrane Translocation Kit contains carbocyanine dye DiOC2 (3) (3,3'-diethyloxacarbocyanine iodide, component A) and CCCP (carbonyl cyanide 3-chlorophenylhydrazone, component B ) Are both provided in DMSO and 1 x PBS solution (component C).

DiOC2 (3) shows green fluorescence in all bacterial cells, but the fluorescence shift towards red emission as the dye molecule itself is associated at higher cytoplasmic concentrations caused by larger membrane potentials. Proton ion carriers, such as CCCP, remove the proton gradient, causing higher green fluorescence to disrupt membrane potential.

MRSA Of membrane potential ΔΨ in

Green fluorescence emission was calculated using population average fluorescence intensity for control and laser treated samples at lethal dose-less radiation doses.

MRSA radiation dose progression First laser treatment procedure: both 870 and 930 Second laser treatment procedure: 930 only parameter Output power (W) Radial point (cm) Area of point (㎠) Time in seconds 870 @ 4.25 W and 930 @ 4.25 W for 16 minutes, then 8.5 1.5 1.77 960 930 @ 8.5 W for 7 minutes 8.5 1.5 1.77 420

The data shows μ ₁ − μ ₂ > 0 because laser treated cells have less “green fluorescence” as shown in FIG. 8. These MRSA samples showed a clear change and degradation of ΔΨ-steady-bact to one of ΔΨ-trans-bact using lethal dose-less NIMELS radiation dose.

Seed. At albicans  Detection of membrane potential ΔΨ

Green fluorescence emission was calculated using population average fluorescence intensity for the control and laser treated samples at lethal doses below the radiation doses listed in Table 7 below.

First laser treatment procedure: both 870 and 930 Second laser treatment procedure: 930 only parameter Output power (W) Radial point (cm) Area of the point (㎠) Time in seconds Laser # 1 Test (H-1) 870 at 4 W and 930 at 4 W for 18 minutes, then 8.0 1.5 1.77 1080 Test (H-1) 930 at 8 W for 8 minutes 8.0 1.5 1.77 480 Laser # 2 Test (H-2) 870 at 4.25 W and 930 at 4.25 W for 18 minutes, then 8.5 1.5 1.77 1080 Test (H-2) 930 at 8.5 W for 8 minutes 8.5 1.5 1.77 480 Laser # 3 Test (H-3) 870 at 4 W and 930 at 4 W for 20 minutes, then 8.0 1.5 1.77 1200 Test (H-3) 930 at 8 W for 10 minutes 8.0 1.5 1.77 600

Data is laser treated seeds. Since albicans cells have less "green fluorescence" as shown in Figure 9, μ ₁ -μ ₂ > 0 is shown. These seeds. Albicans samples showed a clear change and degradation to one of the ΔΨ-trans-fungi of ΔΨ-steady-fungi using lethal dose-less NIMELS radiation dose with increasing (less lethal dose-less) NIMELS dose.

this. In Coli  Detection of membrane potential ΔΨ

Red / green ratios were calculated using population average fluorescence intensity for the control and laser treated samples at lethal dose-less radiation doses.

The data shows μ ₁ − μ ₂ > 0 because laser treated cells have less “green fluorescence” as shown in FIG. 19. These teeth. E. coli samples showed a clear alteration and degradation of ΔΨ-steady-bact to one of ΔΨ-trans-bact using lethal dose-less NIMELS radiation dose.

Example VI

Seed using lethal doses-less than laser radiation dose. Albicans  ΔΨ- in mito For NIMELS In vitro  exam

Hypothesis test:

The null hypothesis is μ ₁ -μ ₂ = 0:

a) μ ₁ is the fluorescence intensity in the control cell culture mitochondria treated with the mitochondrial membrane potential detection kit.

b) μ ₂ is the fluorescence intensity in the same cell culture pre-radiated with a lethal dose-less radiation dose from a NIMELS laser and treated with a mitochondrial membrane potential detection kit.

The data indicate that the fluorescence of the mitochondria was dissipated (less than the control untreated cells) by pre-treatment (of cells) using the NIMELS laser system, and the results indicate that the NIMELS laser cells in the mitochondria of fungal and mammalian cells. It interacts with the respiratory process and oxidative phosphorylation.

μ ₁ -μ ₂ = 0 will support that lethal dose-less NIMEL irradiation on cell culture mitochondria shows no effect on ΔΨ-steady.

μ ₁ -μ ₂ > 0 will support that lethal dose-less NIMEL irradiation on cell culture has a dissipating or depolarizing effect on ΔΨ-steady-mito.

Substances and Methods:

Mitochondrial Membrane Potential Detection Kit (APO LOGIX JC-1) (Cell Technology Inc, Mountview Suite D Lenstov Avenue 950, CA 94043, USA).

Loss of mitochondrial membrane potential (ΔΨ) is an indicator of apoptosis. The APO LOGIX JC-1 Assay Kit measures mitochondrial membrane potential in cells.

In non-apoptotic cells, JC-1 (5,5 ', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanin iodide) is cytoplasmic ( Green as a monomer and accumulate as aggregates in mitochondria that are colored red. On the other hand, in apoptotic and necrotic cells, JC-1 is present in monomeric form and stains the cytoplasm green.

Candida albicans radiation dose table First laser treatment procedure: both 870 and 930 Second laser treatment procedure: 930 only exam parameter Output power (W) Radial point (cm) Area of point (㎠) Time in seconds Candida Mito 1 Test (H-3) 870 at 4.25 W and 930 at 4.25 W for 16 minutes, then 8.5 1.5 1.77 960 Test (H-3) 930 at 8.5 W for 10 minutes 8.5 1.5 1.77 600

(APO LOGIX JC-1) kit measures membrane potential by conversion of green fluorescence to red fluorescence. In FIG. 10A, the appearance of the red color was measured and plotted, which would only occur in cells with an intact membrane and is shown in FIG. 10B for both control and laser treated samples of green to red ratio.

Obviously in this test, red fluorescence decreased in the laser treated sample, while the ratio of green to red increased, indicating depolarization. These results are identical to the transmembrane ΔΨ test (ie, both data show depolarization).

These results also show that μ ₁ -μ ₂ > 0 and that lethal dose-less NIMEL irradiation on cellular mitochondria has a dissipating or depolarizing effect on ΔΨ-steady-mito, which is Candida albicans ΔΨ-steady The apparent reduction of -mito-fungi to ΔΨ-trans-mito-fungi is shown.

Example VII

ΔΨ- using lethal dose-less laser radiation dose mito  NIMELS for human embryonic kidney cells In vitro  exam

Hypothesis test:

The null hypothesis is μ ₁ -μ ₂ = 0:

a) μ ₁ is the fluorescence intensity in the mammalian control cell culture mitochondria (no laser) treated with the mitochondrial membrane potential detection kit.

b) μ ₂ is the fluorescence intensity in the same mammalian cell culture pre-irradiated with a lethal dose-less radiation dose from a NIMELS laser and treated with a mitochondrial membrane potential detection kit.

The data indicate that the fluorescence of the mitochondria is dissipated (less than cells without control laser) by pre-treatment (of cells) using the NIMELS laser system, and the results indicate that the NIMELS laser respirates cells within the mitochondria of mammalian cells. It interacts with the process and oxidative phosphorylation.

μ ₁ -μ ₂ = 0 will support that lethal dose-less NIMEL irradiation on mammalian cell culture mitochondria shows no effect on ΔΨ-steady-mito-mam.

μ ₁ -μ ₂ > 0 will support that lethal dose-less NIMEL irradiation on mammalian cell culture has a dissipating or depolarizing effect on ΔΨ-steady-mito-mam.

Substances and Methods:

Mitochondrial Membrane Potential Detection Kit (APO LOGIX JC-1) (Cell Technology Inc .; Mountain View 950 Lenstov Avenue Suit D, 94043, USA).

Loss of mitochondrial membrane potential (ΔΨ) is an indicator of apoptosis. The APO LOGIX JC-1 Assay Kit measures mitochondrial membrane potential in cells.

In non-apoptotic cells, JC-1 (5,5 ', 6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolylcarbocyanin iodide) is cytoplasmic ( Green as a monomer and accumulate as aggregates in mitochondria that are colored red. On the other hand, in apoptotic and necrotic cells, JC-1 is present in monomeric form and stains the cytoplasm green.

Mammalian Cell Radiation Dose First laser treatment procedure: both 870 and 930 Second laser treatment procedure: 930 only parameter Output power (W) Radial point (cm) Area of point (㎠) Time in seconds Test (H-2) 870 at 4.25 W and 930 at 4.25 W for 18 minutes, then 8.5 1.5 1.77 1080 Test (H-2) 930 at 8.5 W for 10 minutes 8.5 1.5 1.77 600

HEK-293 (human embryonic kidney cell) ΔΨ-mito test:

(APO LOGIX JC-1) kit measures membrane potential by conversion of green fluorescence to red fluorescence. In FIG. 11A, the appearance of the red color was measured and plotted, which would only occur in cells with an intact membrane and is shown in FIG. 11B for both control and laser treated samples of green to red ratio.

Obviously in this test, red fluorescence decreased in the laser treated sample, while the ratio of green to red increased, indicating depolarization. These results show that μ ₁ -μ ₂ > 0, and that lethal dose-less NIMEL irradiation on mammalian cell mitochondria has a dissipating or depolarizing effect on ΔΨ-steady-mito-mam, which is mammalian ΔΨ -steady-mito-mam shows a clear decrease to ΔΨ-trans-mito-mam.

Example VIII

Responsive Oxygen species  ( ROS For) NIMELS In vitro  exam

ΔΨ-trans- of ΔΨ-steady-mito-fungi to ΔΨ-trans-bact of bacterial transmembrane ΔΨ-steady-bact using the lethal dose-less laser radiation dose equivalent to that used in the ΔΨ test in the preceding example. The in vitro test for the generation of reactive oxygen species (ROS) was performed after laser change to mito-fungi and to ΔΨ-trans-mito-mam of ΔΨ-steady-mito-mam.

Substances and Methods:

Total Glutathione Quantification Kit (Dojindo Laboratories, Tashima Kumamoto Techno Research Park, 2025-5, Kashimaki-gun, Mashiki-machi, 861-2202, Kumamoto, Japan).

Glutathione (GSH) is the most abundant thiol (SH) compound in animal tissues, plant tissues, bacteria and yeast. GSH can play many different roles, such as protection against reactive oxygen species and maintenance of protein SH groups. During these reactions, GSH is converted to glutathione disulfide (GSSG: oxidized form of GSH). Since GSSG is enzymatically reduced by glutathione reductase, GSH is the dominant form in the organism. DTNB (5,5'-dithiobis (2-nitrobenzoic acid)), also known as Elman reagent, was developed for the detection of thiol compounds. In 1985, it was proposed that the glutathione circulation system by DTNB and glutathione reductase produces a highly sensitive glutathione detection method. DTNB and glutathione (GSH) react to produce 2-nitro-5-thiobenzoic acid and glutathione disulfide (GSSG). Since 2-nitro-5-thiobenzoic acid is a yellow product, the GSH concentration in the sample solution can be determined by absorbance measurements at 412 nm. GSH is produced by glutathione reductase from GSSG and reacts again with DTNB to produce 2-nitro-5-thiobenzoic acid. Thus, the circulatory reaction improves the sensitivity of total glutathione detection.

At significant concentrations, ROS will react rapidly and specifically with the target by components of the glutathione antioxidant system (catalase, peroxidase, GSH) at rates exceeding their reduction rate.

ΔΨ- steady - bact ΔΨ- trans - bact  At a lethal dose of less than NIMELS MRSA  Of glutathione in the stomach

The results shown in FIG. 12 clearly show a decrease in total glutathione in MRSA at lethal dose-less NIMELS radiation that changes ΔΨ-steady-bact to one of ΔΨ-trans-bact, indicating that transmembrane ΔΨ-steady-bact Evidence of the generation of ROS with lethal dose-less alteration to one of ΔΨ-trans-bact.

Transmembrane  ΔΨ- steady ΔΨ- trans  Changing from one of the lethal doses to less than NIMELS radiation dose. Coli  Of glutathione in the stomach

The results shown in FIG. 20 show that E. coli at lethal dose-less NIMELS radiation changes ΔΨ-steady-bact to one of ΔΨ-trans-bact. The decrease in total glutathione in E. coli is clearly shown, which is evidence of the production of ROS with lethal dose-less alteration of the transmembrane ΔΨ-steady-bact to one of the ΔΨ-trans-bact.

Seed in lethal dose-less NIMELS, changing ΔΨ-steady-mito-fungi to ΔΨ-trans-mito-fungi and subsequently ΔΨ-steady-fungi to one of ΔΨ-trans-fungi. Detection of Glutathione in Albicans

ΔΨ- steady - mito - fungi ΔΨ- trans - mito - fungi in, Subsequently  ΔΨ- steady ΔΨ- fungi trans - fungi  Seeds from lethal dose-less NIMELS radiation dose changed to one of the following. Albicans  Of glutathione in the stomach

The results shown in FIG. 13 show that at lethal doses of less than NIMELS radiation which changes ΔΨ-steady-mito-fungi to ΔΨ-trans-mito-fungi and subsequently changes ΔΨ-steady-fungi to one of ΔΨ-trans-fungi. Seed. The decrease in total glutathione in the albicans is clearly shown, which is the ΔΨ-trans-mito-fungi of the transmembrane ΔΨ-steady-mito-fungi, and subsequently the lethal dose to one of the ΔΨ-trans-fungi of ΔΨ-steady-fungi -Evidence of the generation of ROS with less than alteration.

Detection of glutathione in HEK 293 (human embryonic kidney cells) at lethal dose-less NIMELS radiation doses that change ΔΨ- steady - mito - mam to ΔΨ- trans - mito - mam

The results shown in FIG. 14 clearly show a decrease in total glutathione in HEK-293 (human embryonic kidney cells) at lethal dose-less NIMELS radiation doses that change ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam. This is evidence of the generation of ROS with NIMELS-mediated lethal dose-changes of transmembrane ΔΨ-steady-mito-mam to ΔΨ-trans-mito-mam.

Example IX

Erythromycin  And Trimethoprim  together MRSA Lethal dose for NIMELS  Assessment of the impact of the laser

In this example, it was determined whether lethal dose-less dose NIMEL lasers would enhance the effects of antibiotic erythromycin over MR trimethoprim in MRSA. The discharge pump serves as a major factor in erythromycin resistance. Gram-positive S. No mechanism of trimethoprim discharge pump resistance in Aureus has been reported.

Background: Erythromycin is a macrolide antibiotic with a very similar antimicrobial activity range to β-lactam penicillin. In the past, it has been effective in the treatment of a wide variety of Gram-positive bacterial infections affecting the skin and airways and has been considered one of the safest antibiotics to use. In the past, erythromycin has been used in people allergic to penicillin. The mechanism of action of erythromycin is to prevent bacterial growth and replication by blocking bacterial protein synthesis. This is accomplished by erythromycin binding to 23S rRNA molecules in the 50S of bacterial ribosomes, blocking the release of growing peptide chains and inhibiting the translocation of peptides. Erythromycin resistance (as in other macrolides) is widespread and widespread, and is achieved through two significant resistance systems:

A) Modification of 23S rRNA in 50S ribosomal subunit to be insensitive.

B) Exhaustion of the drug out of the cell.

Trimethoprim is an antibiotic used historically in the treatment of urinary tract infections. It is a member of the class of antimicrobial agents known as dehydrofolate reductase inhibitors. Since trimethoprim is an analog of dihydrofolic acid, the mechanism of action of trimethoprim is to inhibit the system of bacterial dihydrofolate reductase (DHFR). This leads to competitive inhibition of DHFR due to affinity 1000 times greater than the natural substrate for the enzyme.

Thus, trimethoprim inhibits the synthesis of molecular tetrahydrofolic acid. Tetrahydrofolic acid is an essential precursor in de novo synthesis of DNA nucleotide thymidylate. Bacteria cannot take folic acid from the environment (ie, infectious host) and are therefore dependent on the de novo synthesis of their own tetrahydrofolic acid. Inhibition of enzymes ultimately prevents DNA replication.

Trimethoprim resistance generally results from overproduction of normal chromosome DHFR, or drug resistant DHFR enzymes. Trimethoprim-resistant S. The reporting literature for aureus showed that the resistance was a chromosomal mediated type or encoded on a large plasmid. Some strains have been reported to exhibit both chromosome and plasmid-mediated trimethoprim resistance. Gram-positive pathogen S. In Aureus, resistance to trimethoprim is due to genetic mutations, and active release of trimethoprim out of cells has not been reported.

Discharge pump in bacteria

The main route of drug resistance in bacteria and fungi is the active release (emission) of antibiotics out of the cell such that therapeutic concentrations are not achieved in the cytoplasm of the cell.

Active release of antibiotics (and other harmful molecules) is mediated by a series of transmembrane proteins in the cytoplasmic membrane of Gram-positive bacteria and in the outer membrane of Gram-negative bacteria.

In the clinic, antibiotic resistance mediated through the discharge pump is most relevant in Gram positive bacteria against macrolides, tetracyclines and fluoroquinolones. In Gram-negative bacteria, β-lactam release mediated resistance is also clinically relevant.

Hypothesis test:

The null hypothesis is μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ = 0:

a) μ ₁ is the lethal dose-less radiation dose from the NIMEL laser system for MRSA as a control;

b) μ ₂ is the same lethal dose-less radiation dose from the NIMEL laser system for MRSA with trimethoprim at resistant MIC just below the effective level;

c) μ ₃ is the same lethal dose-less radiation dose from the NIMEL laser system for MRSA with erythromycin at the resistant MIC just below the effective level.

The data show that addition of antibiotics trimethoprim or erythromycin reduces the growth of these MRSA colonies after lethal dose-less irradiation as follows:

μ ₁ -μ ₂ = 0 will support that addition of trimethoprim does not have a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₂ > 0 will support that addition of trimetoprim has a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ − μ ₃ = 0 will support that the addition of erythromycin does not have a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₃ > 0 will support that the addition of erythromycin has a detrimental effect on the normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

result:

This experiment clearly showed that under the lethality-less laser parameters using the NIMELS system, μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ ≥ 0. This indicates that the drain pump is inhibited and the resistance to erythromycin is reversed by the NIMELS effect on ΔΨ-steady-bact of MRSA.

Example  X

Tetracycline  And Rifampin and  together MRSA Lethal dose for NIMELS  Assessment of the impact of the laser

The purpose of this experiment was to observe whether lethal dose-less dose NIMEL lasers would enhance the effects of antibiotic tetracycline over antibiotic rifampin in MRSA. Exhaust pumps are well studied and function as major factors in tetracycline resistance. However, Gram-positive S. No rifampin drain pump resistance mechanism has been reported in Aureus.

This experiment was also performed earlier using erythromycin and trimetapririm, and the data show that the NIMELS effect can compromise the discharge pump resistance mechanism for erythromycin.

Tetracycline :

Tetracycline is considered to be bacteriostatic antibiotic, meaning that it inhibits the growth of bacteria by inhibiting protein synthesis. Tetracycline achieves this by inhibiting the action of bacterial 30S ribosomes through the binding of the enzyme aminoacyl-tRNA. Tetracycline resistance is often due to the acquisition of new genes encoding energy-dependent release of tetracycline or encoding proteins that protect bacterial ribosomes from the action of tetracycline.

Rifampin :

Rifampin is a bacterial RNA synthase inhibitor and functions by directly blocking the extension of RNA. Rifampicin is commonly used to treat mycobacterial infections, but is also effective in the treatment of methicillin-resistant Staphylococcus aureus (MRSA) in combination with fusidic acid (a bacteriostatic protein synthesis inhibitor). Rifampin resistance through the drain pump in MRSA has not been reported.

theory:

The null hypothesis is μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ = 0:

a) μ ₁ is the lethal dose-less radiation dose from the NIMEL laser system for MRSA as a control;

b) μ ₂ is the same lethal dose-less radiation dose from the NIMEL laser system for MRSA with tetracycline at the resistant MIC just below the effective level;

c) μ ₃ is the same lethal dose-less radiation dose from the NIMEL laser system for MRSA with rifampin added at resistant MIC just below the effective level.

The data show that addition of the antibiotic tetracycline or rifampin reduces the growth of these MRSA colonies after lethal dose-less irradiation as follows:

μ ₁ -μ ₂ = 0 will support that addition of tetracycline does not show a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₂ > 0 will support that addition of tetracycline has a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₃ = 0 will support that the addition of rifampin does not have a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₃ > 0 will support that addition of rifampin has a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

result:

This experiment clearly showed that under the lethality-less laser parameters using the NIMELS system, μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ ≥ 0. This indicates that the drain pump is inhibited and the resistance to tetracycline is reversed by the NIMELS effect on ΔΨ-steady-bact of MRSA.

Example XI

With methicillin MRSA Lethal dose for NIMELS  ΔΨ- of laser effect assessment and cell wall synthesis plas - bact  control

Methicillin :

Methicillin is resistant to Gram-positive bacteria, in particular β-lactamase-producing organisms, for example most penicillins. Β-lactam previously used to treat infections caused by Aureus, but is no longer used in the clinic. The term methicillin-resistant S. Aureus (MRSA) is resistant to all penicillins. Continued use to describe the Aureus strain.

Action Mechanism :

Like other β-lactam antibiotics, methicillin acts by inhibiting the synthesis of peptidoglycans (bacterial cell walls).

In gram positive bacterium Bacillus subtilis, the activity of peptidoglycan autolysis was shown to be increased (ie, no longer inhibited) when ETS is blocked by adding proton conductors. This suggests that ΔΨ-plas-bact and ΔμH ⁺ (regardless of the storage energy for the enzymatic function of the cell) potentially have significant and available effects on cell wall assimilation function and physiology.

In addition, the ΔΨ-plas-bact decoupling agent, along with the inhibition of the accumulation of nucleotide precursors involved in peptidoglycan synthesis and the transport of N-acetylglucosamine (GlcNAc) (one of the major biopolymers of peptidoglycan) It has been reported to inhibit peptidoglycan formation.

Hypothesis test:

Bacitracin will enhance the multiple effects of optically degraded ΔΨ-plas-bact on the growing cell wall (ie increased cell wall autolysis, inhibited cell wall synthesis). This is particularly relevant in Gram positive bacteria, such as MRSA, which do not have a discharge pump as a resistance mechanism to cell wall inhibitory antimicrobial compounds.

The null hypothesis is μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ = 0:

a) μ ₁ is the lethal dose-less radiation dose from the NIMEL laser system for MRSA as a control;

b) μ ₂ is the same lethal dose-less radiation dose from the NIMEL laser system for MRSA with methicillin at resistant MIC just below the effective level.

μ ₁ -μ ₂ = 0 will support that the addition of methicillin does not show a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₂ > 0 will support the addition of methicillin to deleterious effects on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

result:

As shown in FIG. 15, this experiment clearly showed that under a lethal dose-less laser parameter using the NIMELS system, μ ₁ -μ ₂ ≥ 0, indicating that the addition of methicillin was CFU after lethal dose-less NIMEL irradiation. As indicated by the coefficient, it is meant to have a deleterious effect on normal growth of MRSA colonies. This suggests that methicillin (regardless of drain pump) is enhanced by the NIMELS effect on ΔΨ-steady-bact of MRSA.

Thus, NIMELS lasers and their simultaneous optical ΔΨ-plas-bact degradation are synergistic with cell wall inhibitory antimicrobial agents in MRSA. Without wishing to be bound by theory, since MRSA does not have a discharge pump for methicillin, it will function through inhibition of assimilation (peripheral cytoplasmic) ATP linked function.

Example XII

With bacitracin MRSA Lethal dose for NIMELS  ΔΨ- of laser effect assessment and cell wall synthesis plas - bact  control

Bacitracin is a mixture of cyclic polypeptides produced by Bacillus subtilis. Since it is a toxic and difficult to use antibiotic, bacitracin is generally not used orally but is used topically.

Mechanism of Action:

Bacitracin inhibits dephosphorylation of C ₅₅ -isoprenyl pyrophosphate (the molecule that moves the building blocks of the peptidoglycan bacterial cell wall to the inner membrane in gram negative organisms and out of the plasma membrane in gram positive organisms).

In gram positive bacterium Bacillus subtilis, the activity of peptidoglycan autolysis was shown to be increased (ie, no longer inhibited) when ETS is blocked by adding proton conductors. This indicates that ΔΨ-plas-bact and ΔμH ⁺ (regardless of the storage energy for the enzymatic function of the cell) potentially have significant and available effects on cell wall assimilation function and physiology.

In addition, the ΔΨ-plas-bact decoupling agent, along with the inhibition of the accumulation of nucleotide precursors involved in peptidoglycan synthesis and the transport of N-acetylglucosamine (GlcNAc) (one of the major biopolymers of peptidoglycan) It has been reported to inhibit peptidoglycan formation.

Hypothesis test:

Bacitracin enhances multiple effects of optically degraded ΔΨ-plas-bact (ie increased cell wall autolysis, inhibited cell wall synthesis) on growing cell walls. This is particularly relevant in Gram positive bacteria, such as MRSA, which do not have a discharge pump as a resistance mechanism to cell wall inhibitory antimicrobial compounds.

The null hypothesis is μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ = 0:

a) μ ₁ is the lethal dose-less radiation dose from the NIMEL laser system for MRSA as a control;

b) μ ₂ is the same lethal dose-less radiation dose from the NIMEL laser system for MRSA added bacitracin at resistant MIC just below the effective level.

μ ₁ -μ ₂ = 0 will support that addition of bacitracin does not show a deleterious effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

μ ₁ -μ ₂ > 0 will support that addition of bacitracin has a detrimental effect on normal growth of MRSA colonies after lethal dose-less NIMEL irradiation.

result:

As shown in FIG. 16, this experiment clearly showed that under a lethal dose-less laser parameter using the NIMELS system, μ ₁ -μ ₂ ≥ 0, indicating that the addition of bacitracin was MRSA after lethal dose-less NIMEL irradiation. It has a detrimental effect on the normal growth of colonies. In FIG. 16, arrows indicate MRSA growth or deficiency in the two samples shown. This suggests that bacitracin (regardless of drain pump) is enhanced by the NIMELS effect on ΔΨ-steady-bact of MRSA.

Thus, NIMELS lasers and their simultaneous optical ΔΨ-plas-bact degradation are synergistic with cell wall inhibitory antimicrobial agents in MRSA. While not wishing to be bound by theory, since MRSA does not have a discharge pump for bacitracin, it probably functions through inhibition of assimilation (peripheral cytoplasmic) ATP linked function.

Example XIII

Lamisil  ( Lamisil ) And Spornox  ( Sporanox Mr.) Albicans  For lethal doses-under dose NIMELS  Assessment of the impact of the laser

The objective of this experiment was to have a lethal-less dose NIMEL laser. The purpose of this study is to observe whether or not to enhance the effects of the antifungal compounds lamisil and / or sporanox in albicans.

Introduction:

Reduction of cytoplasmic ATP concentrations in fungal cells has been shown to result in inhibition of plasma membrane-bound H ⁺ -ATPase producing ΔΨp-fungi, and the disorder has been shown to weaken other cellular activities. In addition, the degradation of ΔΨp-fungi causes plasma membrane bioenergy and thermodynamic destruction, leading to the influx of protons that disrupt proton driving forces and thus inhibit nutrient intake. More importantly, ATP is required for the biosynthesis of fungal plasma membrane lipid ergosterol. Ergosterol is a structural lipid that is targeted by the majority of related commercially antifungal compounds (ie, azoles, terbinafine and itraconazole) used in medicine today, including lamisil and sporanox (and their generic counterparts).

In addition, recently, two novel antimicrobial peptides (Pep2 and Hst5) have the ability to allow ATP to be excreted out of fungal cells (ie, to deplete intracellular ATP concentrations), and the degraded cytoplasmic ATP is the ATP of an antifungal agent. It has been shown to cause inactivation of the dependent transport pumps ABC Transporter CDR1 and CDR2.

Lamisil :

Lamisil (like other allylamines) inhibits ergosterol synthesis by inhibiting squalene epoxidase (an enzyme that is part of the fungal cell wall synthesis pathway).

Sporanox :

The mechanism of action of itraconazole (Sporanox) is the same as other azole antifungal agents; Inhibits fungal cytochrome P450 oxidase-mediated synthesis of ergosterol.

theory:

Seed. At lethal dose-less radiation doses to albicans, NIMELS lasers enhance lamisil and sporox because of optically degraded ΔΨ-plas-fungi and / or ΔΨ-mito-fungi by depolarizing the membrane in fungi and depleting cellular ATP.

The null hypothesis is μ ₁ -μ ₂ = 0 and μ ₁ -μ ₃ = 0:

a) μ ₁ is the control. Dose-less radiation dose from the NIMEL laser system for albicans;

b) μ ₂ is the seed added with sporanox at resistant MIC just below the effective level. The same lethal dose-less radiation dose from the NIMEL laser system for albicans;

c) μ ₃ is the seed with lamisil added at resistant MIC just below the effective level. The same lethal dose-less radiation dose from the NIMEL laser system for albicans.

The data indicate that the addition of the antifungal agent Lamisil and / or Sporanox was followed by these seeds after fatality-less radiation. Decreases growth of albicans colonies:

μ ₁ -μ ₂ = 0, the addition of Sporanox seeded after lethal-less NIMEL irradiation. Will support no adverse effects on normal growth of albicans colonies.

μ ₁ − μ ₂ > 0, the addition of Sporanox seeded after lethal dose-less NIMEL irradiation. Will support deleterious effects on normal growth of albicans colonies.

μ ₁ − μ ₃ = 0 the addition of lamisil seeded after lethal dose-less NIMEL irradiation. Will support no adverse effects on normal growth of albicans colonies.

μ ₁ − μ ₃ > 0, the addition of lamisil seeded after lethal dose-less NIMEL irradiation. Will support deleterious effects on normal growth of albicans colonies.

Candida albicans NIMELS radiation dose table First laser treatment procedure: both 870 and 930 Second laser treatment procedure: 930 only exam parameter Output power (W) Radial point (cm) Area of point (㎠) Time in seconds AF-8 Test (H-1) 870 at 4.25 W and 930 at 4.25 W for 18 minutes, then 8.0 1.5 1.77 AF-8 Test (H-1) 930 at 8.5 W for 12 minutes 8.0 1.5 1.77

result:

This experiment clearly showed that under the lethality-less laser parameters using the NIMELS system, μ ₁ -μ ₂ > 0 and μ ₁ -μ ₃ > 0, which indicated that the addition of lamisil was after lethal dose-less NIMEL irradiation. It has a detrimental effect on the normal growth of albicans colonies. This suggests that ergosterol biosynthesis inhibitors (ramisil and sporanox) are enhanced by lethal dose-less radiation irradiation of the NIMELS laser system.

Example XIV

NIMELS  Calculation of radiation dose

The following examples illustrate selected experiments depicting the ability of the NIMELS solution to affect the viability of various commonly found microorganisms at the wavelengths described herein. Illustrated microorganisms are E. coli. E. coli K-12, multidrug-drug resistant. E. coli, Staphylococcus aureus, methicillin-resistant S. Aureus, Candida albicans, and Trichophyton rubrum ).

As discussed in more detail above, the NIMELS parameters include the average single or additional output power of the laser diode, and the wavelength (870 nm and 930 nm) of the diode. The information, in combination with the area (cm 2) of the laser beam (s) at the target site, provides an initial set of information that can be used to calculate an effective and safe irradiation protocol in accordance with the present invention.

The power density of a given laser measures the potential effect of NIMELS at the target site. Power density is a function of any given laser output power and light ray area, and can be calculated using the following equation.

For a single wavelength:

1) Power Density (W / cm2) = Laser Output Power / Ray Diameter (cm2)

For dual wavelength processing:

2) Power density (W / cm 2) = laser (1) output power / ray diameter (cm 2) + laser (2) output power / ray diameter (cm 2)

The ray area can be calculated by the following equation:

3) ray area (cm 2) = diameter (cm) ² * 0.7854 or

Ray area (cm 2) = Pi * radius (cm) ² .

The total photon energy delivered into the tissue by one NIMELS laser diode system operating at a specific output power over a period of time is measured in joules and calculated as follows:

4) Total energy (joules) = laser output power (watts) * time (seconds)

The total photon energy delivered into tissue at the same time by two NIMELS laser diode systems (two wavelengths) at a given output power over a period of time is measured in joules and calculated as follows:

5) Total Energy (Joules) = [Laser (1) Output Power (Watts) * Time (Sec)] + [Laser (2) Output Power (Watts) * Time (Sec)]

In practice, it is useful (but not necessary) to determine the distribution and allocation of total energy over the radiation treatment area to accurately measure the dose for the maximum NIMELS beneficial response. The total energy distribution can be measured as energy density in joules / cm 2. As discussed below, for light of a given wavelength, energy density is the most important factor in determining tissue response. The energy density for one NIMELS wavelength can be derived as follows:

6) Energy Density (Joules / cm 2) = {Laser Output Power (Watts) * Time (Seconds)} / Ray Area (㎠)

7) Energy Density (Joules / ㎠) = Power Density (W / ㎠) * Time (seconds)

When two NIMELS wavelengths are used, the energy density can be derived as follows:

8) Energy Density (Joules / cm 2) = {Laser (1) Output Power (Watts) * Time (Seconds)} / Ray Area (cm 2) + {Laser (2) Output Power (Watts) * Time (Seconds)} / Ray area (cm 2), or

9) Energy Density (Joules / ㎠) = Power Density (1) (W / ㎠) * Time (sec) + Power Density (2) (W / ㎠) * Time (sec)

To calculate the treatment time for a particular capacity, one skilled in the art can use the output power (W) and the ray area (cm 2) as well as the energy density (J / cm 2) or energy (J) using one of the following equations:

10) Treatment time (seconds) = energy density (joules / cm 2) / output power density (W / cm 2)

11) Processing time (seconds) = energy (joules) / laser output power (watts)

Since radiation dose calculations as illustrated in this example can be cumbersome, the treatment system can also include a computer database that stores all studied therapeutic possibilities and radiation doses. The computer in the controller (radiation dose and parameter calculator) is preprogrammed using an algorithm based on the equations described above so that any operator can easily retrieve data and parameters on the screen and add additional necessary data (e.g., spot size). Total energy required, time and pulse width of each wavelength, irradiated tissue, irradiated bacteria) can be entered along with any other necessary information, so that any and all algorithms needed for an advantageous therapeutic outcome and The calculation is generated by the radiation dose and parameter calculator and can thus be executed by the laser.

In summary, in the examples below, when bacterial cultures are exposed to a NIMELS laser, bacterial mortality (measured by counting colony forming units (CFU) on culture plates after treatment) is 93.7% (multidrug-drug resistance E. coli). E. coli) to 100% (all other bacteria and fungi).

Example XV

Germ way: In vitro  this. Coli Targeting  for NIMELS  Processing parameters

The following parameters are at a final temperature well below the temperature indicated in the literature as being associated with thermal damage. Illustrates the method according to the invention applied to E. coli

A. a. Experimental materials and methods for E. coli K-12 :

this. E. coli K12 liquid cultures were grown in Luria Bertani (LB) medium (25 g / L). The plate contained 35 mL of LB plate medium (25 g / L LB, 15 g / L bacterial agar). Culture dilution was performed using PBS. All protocols and manipulations were performed using aseptic technique.

B. Growth Kinematics

Multiple 50 mL LB cultures taken from the inoculation culture were inoculated and grown overnight at 37 ° C. The next morning, the healthiest cultures were selected and used to inoculate 5% into 50 mL LB at 37 ° C. and measurements were taken every 30 to 45 minutes until the cultures were stationary to monitor OD ₆₀₀ over time.

C. Stock Master (Master Stock ) Production

Using log phase culture (OD ₆₀₀ approximately 0.75) as starting material, add 10 mL of 50% glycerol at 4 ° C. in 10 mL of this culture, aliquot into 20 cryovials, and rapidly freeze in liquid nitrogen It was. The cryovials were then stored at -80 ° C.

D. Liquid Culture

this. Liquid culture of E. coli K12 was configured as described above. 100 μl aliquots were removed from the subculture and serially diluted 1: 1200 in PBS. The dilutions were incubated at room temperature for about 2 hours or until no further increase in OD ₆₀₀ was observed, ensuring that the cells in the PBS suspension reached static state (growth) without significant doubling and a relatively consistent number of The cells can be further aliquoted for testing.

Once the K12 dilution was determined to be static, 2 mL of this suspension was aliquoted into selected wells in a 24-well tissue culture plate for the selected NIMELS experiment at a given radiation dose parameter. Plates were incubated at room temperature until ready for use (about 2 hours).

After laser treatment, 100 μl was removed from each well and serially diluted 1: 1000 to obtain a final dilution of the first K12 culture of 1: 12 × 10 ⁵ . Aliquots of 3 × 200 L of each final dilution were tripled onto separate plates. The plate was then incubated at 37 ° C. for about 16 hours. Manual colony counts were performed and recorded. Digital photographs of each plate were also taken. s. Aureus and mr. Similar cell culture and kinematics protocols were performed for all NIMELS irradiation tests using the Albicans in vitro test. For example, Mr. Albicans ATCC 14053 liquid cultures were grown at 37 ° C. in YM medium (21 g / L, Difco). Normalized suspensions were aliquoted into selected wells in 24-well tissue culture plates. After laser treatment, 100 μl was removed from each well and serially diluted 1: 1000 to obtain a final dilution of the initial culture of 1: ⁵ × 10 ⁵ . 3 × 100 μl of each final dilution was spread onto a separate plate. The plate was then incubated at 37 ° C. for about 16-20 hours. Manual colony counts were performed and recorded. Digital photographs of each plate were also taken.

Tricophyton rubrum ATCC 52022 liquid cultures were grown in peptone-dextrose (PD) medium at 37 ° C. Normalized suspensions were aliquoted into selected wells in 24-well tissue culture plates. After laser treatment, aliquots were removed from each well and diffused onto separate plates. The plate was then incubated at 37 ° C. for about 91 hours. Manual colony counts were performed and recorded after 66 and 91 hours incubation. All organisms grew in control wells, whereas 100% of the laser-treated wells did not grow as described herein. Digital photographs of each plate were also taken.

Thermal tests were performed starting at room temperature for PBS solution. 10 watts of NIMELS laser energy was available for use in the 12 minute laser treatment cycle, after which the temperature of the system was raised close to the threshold threshold of 44 ° C.

Example XVI

this. Coli In vitro Targeting  for NIMELS  Laser wavelength 930 NM Radiation dose for

This experiment demonstrates that NIMELS single wavelength λ = 930 nm in vitro results within safe thermal parameters for mammalian tissue. To quantifiable antimicrobial efficacy against E. coli.

In vitro experimental data demonstrates that 100% antimicrobial efficacy is still achievable when the threshold of total energy into a system using 930 nm alone of 5400 J and an energy density of 3056 J / cm 2 meet 25% less time.

In vitro experimental data also showed that treatments using a single energy of λ = 930 nm resulted in bacterial species S. aureus within safe thermal parameters for mammalian tissue. Demonstrates having in vitro antimicrobial efficacy against Aureus.

In addition, a threshold of total energy into the system of 5400 J and an energy density of 3056 J / cm 2 was obtained. It is contemplated that 100% antimicrobial efficacy will still be achieved when encountering Aureus and other bacterial species in 25% less time.

In vitro experimental data also showed that NIMELS single wavelength of λ = 930 nm was found in vitro in the safe thermal parameters for mammalian tissue. It has been shown to have antifungal efficacy against albicans.

In addition, if the threshold of total energy into the system of 5400 J and the energy density of 3056 J / cm 2 meet 25% less time, it is believed that 100% antifungal efficacy is still achieved.

Example XVII

In vitro NIMELS  Laser wavelength 870 nm Radiation dose for  value

In vitro experimental data are also available. Using a wavelength of 870 nm alone for E. coli demonstrates that no significant mortality was achieved up to a total energy of 7200 J and an energy density of 4074 J / cm 2 and a power density of 5660 W / cm 2.

s. Comparable results were also observed using the radiation of λ = 870 nm alone in Aureus.

Example XVIII

870 nm With 930 nm  Among the light energy Of NIMELS  specific Alternate  Synergistic effect

In vitro experimental data also demonstrate that when alternating (870 nm and then 930 nm), there is an additive effect between the two NIMELS wavelengths (λ = 870 nm and 930 nm). The presence of the 870 nm NIMELS wavelength as the first emissivity was found to enhance the effect of the antimicrobial efficacy of the second 930 nm NIMELS wavelength emissivity.

In vitro experimental data demonstrate that the synergistic effect (combining 870 nm wavelength to 930 nm wavelength) allows for reduction of 930 nm light energy. As shown herein, when the (870 nm and 930 nm) wavelengths are combined in an alternating manner, the light energy is NIMELS 100%. It was reduced to about one third of the total energy and energy density required for E. coli antimicrobial efficacy.

In vitro experimental data also showed that due to the synergistic mechanism, adding 930 nm energy at 20% higher power density after adding the same amount of 870 nm light energy to the system would result in NIMELS of 930 nm light energy (total energy and energy density). 100% Yi. It is possible to reduce to about half of the total energy density required for E. coli antimicrobial efficacy.

Teeth from alternating NIMELS wavelengths. E. coli data Output power (W) Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) this. E. coli lethal% 8W / 8W 1.5 540/180 12 minutes 4320/1440 = 5760 2445/815 = 3529 4.53 / 4.53 100.0% 10W / 10W 1.5 240/240 8 minutes 2400/2400 = 4800 1358/1358 = 2716 5.66 / 5.66 100.0%

Since 930 nm light energy heats the system at a faster rate than 870 nm light energy, this synergistic capacity is important for human tissue safety, and it is beneficial for mammalian systems to generate as little heat as possible during processing.

If NIMELS light energy (870 nm and 930 nm) is altered in this way in other bacterial species, it is believed that the 100% antimicrobial effect will also be essentially the same.

In vitro experimental data also demonstrate that there is also an additive effect between the two NIMELS wavelengths (870 nm and 930 nm) when alternating during irradiation of fungi (930 nm after 870 nm). The presence of the 870 nm NIMELS wavelength as the first emissivity mathematically enhances the effect of the antifungal efficacy of the second 930 nm NIMELS wavelength emissivity.

In vitro experimental data (see table below) showed that synergistic mechanism was achieved by adding an equal amount of 870 nm light energy to the system and then adding 930 nm energy at 20% higher power density than required for bacterial species antimicrobial efficacy. This demonstrates that 930 nm light energy (total energy and energy density) can be reduced to about one half of the total energy density required for NIMELS 100% antifungal efficacy.

Seeds from alternating NIMELS wavelengths. Albicans data Output power (W) Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) Seed. Albicans lethal% 10W / 10W 1.5 240/240 8 minutes 2400/2400 = 4800 1358/1358 = 2716 5.66 / 5.66 100.0% ^*

Since 930 nm light energy heats the system at a faster rate than 870 nm light energy, this synergistic capacity is important for human tissue safety, and it is beneficial for mammalian systems to generate as little heat as possible during processing.

If NIMELS light energy (870 nm and 930 nm) is altered in this way in other fungal species, it is believed that the 100% antimicrobial effect will also be essentially the same.

Example XIX

λ = 870 nm And λ = 930 nm  Among the light energy NIMELS  Unique Simultaneous Synergy

In vitro experimental data also demonstrates that an additive effect exists between the two NIMELS wavelengths (870 nm and 930 nm) when used simultaneously (combining 870 nm with 930 nm). The presence of 870 nm NIMELS wavelengths and 930 nm NIMELS wavelengths as simultaneous radioactivity absolutely improves the effect of the antimicrobial efficacy of the NIMELS system.

In vitro experimental data (e.g., see Tables IX and X below) combine λ = 870 nm and λ = 930 nm (used simultaneously in this example) to yield 930 nm light energy and density according to the invention. It demonstrates effective reduction to about half of the total energy and energy density required when using the treatment.

From the combined NIMEL wavelength. E. coli data Output power (W) 870 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) this. E. coli lethal% 5W + 5W = 10W 1.5 720 3600 (x2) = 7200 2037 (x2) = 4074 5.66 100.0%

S. from the combined NIMEL wavelength. Aureus Data Output power (W) 870 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) s. Aureus Lethal% 5W + 5W = 10W 1.5 720 3600 (x2) = 7200 2037 (x2) = 4074 5.66 98.5% 5.5W + 5.5W = 11W 1.5 720 3960 (x2) = 7920 2241 (x2) = 4482 6.22 100%

Since 930 nm light energy heats the system at a faster rate than 870 nm light energy, this simultaneous synergistic capacity is important for human tissue safety, and it is beneficial for mammalian systems to generate as little heat as possible during treatment.

If NIMELS light energy (870 nm and 930 nm) is used simultaneously in this manner in different bacterial species, the 100% antimicrobial effect is also considered essentially the same (see Figures 17, 18 and 19).

In vitro experimental data also demonstrate that there is an additive effect between the two NIMELS wavelengths (870 nm and 930 nm) when used simultaneously against fungi. The presence of 870 nm NIMELS wavelengths and 930 nm NIMELS wavelengths as simultaneous radioactivity has been found to enhance the effect of antifungal efficacy of the NIMELS system.

In vitro experimental data show that 930 nm light energy is NIMELS 100% due to the synergistic effect (coupling 870 nm wavelength to 930 nm wavelength for simultaneous irradiation) when combining wavelengths in combination (870 nm and 930 nm). Seed. It is possible to reduce to about half of the total energy and energy density required for albicans antifungal efficacy.

Candida albicans from combined NIMELS wavelengths Output power (W) 870 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) Seed. Albicans lethal% 5W + 5W = 10W 1.5 720 3600 (x2) = 7200 2037 (x2) = 4074 5.66 100%

Thus, the NIMELS wavelengths (λ = 870 nm and 930 nm) can be used in alternating fashion or simultaneously or in any combination of the above to achieve antimicrobial and antifungal efficacy, such that at λ = 930 associated with a minimized temperature increase. Reduce exposure.

In vitro experimental data are also available. When E. coli was irradiated with (control) wavelength alone of λ = 830 nm, in the following parameters, the control 830 nm laser showed zero antimicrobial efficacy during the 12 minute irradiation cycle and at the same parameters for the minimum NIMELS radiation dose. It is demonstrated that it is associated with 100% antibacterial and antifungal efficacy when using λ = 930 nm radiation.

this. E. coli single wavelength λ = 830 nm Output power (W) Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) 8.0 1.5 720 5760 3259 4.53 9.0 1.5 720 6480 3667 5.09

In vitro experimental data also demonstrate that when applied at a safe thermal radiation dose, there is little additive effect when using a λ = 830 nm ore in combination with λ = 930 nm. The presence of the 830 nm control wavelength as the first emissivity is much inferior to the enhancement effect of the 870 nm NIMELS wavelength in producing the second 930 nm NIMELS wavelength and synergistic antimicrobial efficacy.

E. from substituted alternating 830 nm control wavelengths. E. coli data Output power (W) 830 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) this. E. coli lethal% 8W / 8W 1.5 540/180 12 minutes 4320/1440 = 5760 2445/815 = 3529 4.53 / 4.53 0% 10W / 10W 1.5 240/240 8 minutes 2400/2400 = 4800 1358/1358 = 2716 5.66 / 5.66 65%

In vitro experimental data also demonstrate that when applied at safe thermal radiation doses, the additive effect at 830 nm wavelength and NIMELS 930 nm wavelength at the same time is less. Indeed, the in vitro experimental data is 100% when 870 nm is combined with 930 nm simultaneously compared to the commercial 830 nm. It is demonstrated that 17% less total energy, 17% less energy density, and 17% less power density are required to achieve E. coli antibacterial efficacy. This substantially reduces the heat and harm to in vivo systems that are again treated with NIMELS wavelengths.

E. from substituted simultaneous 830 nm control wavelength. E. coli data Output power (W) 830 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) this. E. coli lethal% 5W + 5W = 10W 1.5 720 3600 (x2) = 7200 2037 (x2) = 4074 5.66 91% 5.5W + 5.5W = 11W 1.5 720 3960 (x2) = 7920 2250 (x2) = 4500 6.25 90% 6W + 6W = 12W 1.5 720 3960 (x2) = 8640 ^* 2454 (x2) = 4909 ^* 6.81 ^* 100%

Amount of lethal bacteria:

In vitro data also showed that in vitro, the NIMELS laser system was capable of producing 2,000,000 (2 × 10 ⁶ ) colony forming units (CFU). Coli and s. It has been shown to be effective (within the heat resistance range) against a solution of bacteria containing aureus. This is a 2x increase than would normally be seen in a 1 g sample of infected human ulcer tissue. Brown et al. Reported that in 75% of diabetic patients tested, all of the microbial cells were at least 100,000 CFU / g, and in 37.5% of the patients the amount of microbial cells was greater than 1,000,000 (1 × 10 ⁶ ) CFU (in its entirety herein). See Brown et al, Ostomy Wound Management, 401: 47, issue 10, (2001), incorporated herein by reference.

Thermal parameters:

In vitro experimental data also demonstrates that the NIMELS laser system can achieve 100% antimicrobial and antifungal efficacy within a range of safe heat resistance to human tissues.

Example XX

NIMELS Effect of temperature drop on

Cooling of Bacterial Species:

In vitro experimental data also showed that optical antimicrobial efficacy was not achieved at any currently reproducible antibacterial energy using the NIMELS laser system by substantially lowering the starting temperature of the bacterial sample to 4 ° C. in PBS for 2 hours before the laser treatment cycle. Proved.

Example XXI

Trichophyton In the room  About NIMELS Effect

This example shows the effect of NIMELS wavelengths (870 nm and 930 nm) when used in an alternating or simultaneous manner.

NIMELS Trichophyton Lubrication Test-Shift Wavelength Experiment Output power (W) 870 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) One 8W / 8W 1.5 540/180 12 minutes 4320/1440 = 5760 2445/815 = 3529 4.53 / 4.53 2 10W / 10W 1.5 240/240 8 minutes 2400/2400 = 4800 1358/1358 = 2716 5.66 / 5.66 Experiment 1 = Minimal Effect Experiment 2 = 100% Lethality on All Plates

NIMELS Trichophyton Lubrication-Simultaneous Wavelength Experiment Output power (W) 870 nm / 930 nm Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) 3 5W + 5W = 10W 1.5 720 12 minutes 3600 (x2) = 7200 2037 (x2) = 4074 5.66 4 5.5W + 5.5W = 11W 1.5 720 3960 (x2) = 7920 2250 (x2) = 4500 6.25 5 6W + 6W = 12W 1.5 720 3960 (x2) = 8640 2454 (x2) = 4909 6.81 Experiments 3, 4 and 5 = 100% lethality on all plates

NIMELS Trichophyton Rubrum-Single Wavelength Experiment λ = 930 Output power (W) Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) 6 8.0 1.5 720 5760 3259 4.53 7 9.0 1.5 720 6840 3681 5.11 Experiments 6 and 7 = 100% lethality on all plates

Contrast Tricophytone Lubrication-830 nm / 930 nm Shift Experiment λ = 830 & λ = 930 Output power (W) Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) 8 8W / 8W 1.5 540/180 12 minutes 4320/1440 = 5760 2445/815 = 3529 4.53 / 4.53 9 10W / 10W 1.5 240/240 8 minutes 2400/2400 = 4800 1358/1358 = 2716 5.66 / 5.66 Experiment 8 = No Effect Experiment 9 = 100% Lethal

Treatments as described in Table XVIII above resulted in 100% lethality.

In vitro targeting of Trichophyton rubrum with λ = 830 nm and 930 nm Output power (W) Radial point (cm) Time in seconds Total energy line Energy density (J / ㎠) Power density (W / ㎠) 5 + 5 = 10 1.5 720 3600 (x2) = 7200 2037 (x2) = 4074 5.66

Example XXII

MRSA Of Antimicrobial agents  reinforce

This example shows the use of NIMELS wavelengths (λ = 830 nm and 930 nm) in the in vitro targeting of MRSA to increase antimicrobial susceptibility to methicillin. Four separate experiments were performed. Data sets for these four experiments are presented in the table below. See also FIG. 17, which shows (a) the synergistic effect of NIMELS with methicillin, penicillin and erythromycin in growth inhibition of MRSA colonies. (The data shows that penicillin and methicillin drive bacterial plasma membrane proton driving force -plas-Bact) to inhibit the peptidoglycan synthetic assimilation process co-targeted with the drug, thereby enhancing it by the lethal dose-less NIMELS radiation dose), and (b) the NIMELS effect is enhanced by the protein synthesis assimilation process and Erythromycin is shown to be enhanced to a greater extent because it inhibits bacterial plasma membrane proton driving force (Δp-plas-Bact), which supplies energy for erythromycin resistant discharge pumps.

matter:

Germ ATCC® Number: Top of Form Bottom of Form BAA-43 ™ price: organism Staphylococcus aureus subspecies Aureus Rosenbach; Deposited as Staphylococcus aureus Rosenbach. designation HSJ216 Isolation: 1998 Lisbon Hospital, Portugal [ 51476 ] Depositor H De Lencastre Biosafety level : 2 shipping: Freeze drying Growth conditions: ATCC medium 260 : Trypticase soy agar containing defibrated sheep blood Growth conditions: aerobic temperature: 37.0C Authorization / Form: In addition to the MTA mentioned above, other ATCC and / or regulatory permits may be required for the transport of the ATCC material. The person purchasing the ATCC substance is ultimately responsible for obtaining the license. Click here for information on specific requirements for shipment to your location. Related Product Remark: MRSA's Brazilian clone [ 12386 ] apply: Resistance to methicillin [ 51476 ] Reference: 51476: de Sousa MA, et al. Intercontinental spread of a multidrug-resistant methicillin-resistant Staphylococcus aureus clone. J. Clin. Microbiol. 36: 2590-2596, 1998. PubMed: 9705398 12386: Herminia De Lencastre, personal communication, the entire teachings of which are incorporated herein by reference.

General method for CFU counting:

Time (hours) task FTE (hours) -18 Inoculate overnight cultures 50 ml directly from glycerol stock -4 Starter culture setting 3 dilutions 1:50, 1: 125, 1: 250 OD ₆₀₀ monitoring of starter culture 0 Preparation of Plating Cultures. At 10:00 AM, the culture with OD ₆₀₀ = 1.0 is diluted 1: 300 in PBS (final volume 50 ml) and stored for 1 hour at RT. (Room temperature should be ~ 25 ℃) +1 Inoculation of 24-well plates. 2 ml aliquots are dispensed into pre-designated wells in 24-well plates and transferred to NOMIR (8 24-well plates in total). +2 to +8 Dilution of processed sample. After laser treatment, 100 μl from each well is serially diluted with a final dilution of 1: 1000 in PBS. Plating of processed samples. 100 μl of final dilution is plated in triplicate on TSB agar with and without 30 μg / ml methicillin (6 TSB plates per well). Plates are incubated at 37 ° C. for 18-24 hours. +24 Count colonies on each plate (96 plates in total).

Independent Report on MRSA Studies, November 7, 2006

(MRSA Data Progress 11-07-06 Experiment # 1)

Experiment 1-Design:

Eight different laser doses were used to treat saline-suspension of MRSA growing in log phase and labeled A1 to H1.

Treated and control untreated suspensions were diluted and plated in triplicate on tryptic soybean agar with or without 30 μg / ml methicillin.

After 24 hr growth at 37 ° C., colonies were counted.

CFU (colony forming units) were compared between plates with and without methicillin for both control (untreated) and treated MRSA.

Experiment 1-Results:

Conditions D1 to H1 showed a similar decrease in CFU on methicillin plates in treated and untreated samples.

Conditions A1, B1 and C1 showed 30%, 33% or 67% less CFU in the laser treated samples, respectively, compared to the untreated control.

This indicates that treatments performed on samples A1, B1 and C1 sensitized MRSA to the effect of methicillin.

Independent Report on MRSA Studies, 8 November 2006

(MRSA Data Progress 11-08-06 Experiment # 2)

Experiment 2-Design:

Eight different laser doses based on the effective doses established above and in Experiment 1 were treated with saline-suspensions of MRSA growing on log and labeled A2 to H2.

Treated and control untreated suspensions were diluted and plated in triplicate on tryptic soybean agar with or without 30 μg / ml methicillin.

After 24 hr growth at 37 ° C., colonies were counted.

Experiment 2-Results:

Comparison of CFU on plates with and without methicillin showed a significant increase in the effectiveness of methicillin in all laser treated samples except A2 and B2. The data is summarized in table form below in Table 37.

Schematic Protocol for the NOMIR MRSA Study-November 9, 2006 (11-09-06 Experiment # 3) Method: Time (hours) task FTE (hours) T-18 Inoculate overnight cultures 50 ml directly from glycerol stock One T-4 Starter culture setting 3 dilutions 1:50, 1: 125, 1: 250 One OD ₆₀₀ monitoring of starter culture 4 T0 Preparation of Plating Cultures. At 10:00 AM, the culture with OD ₆₀₀ = 1.0 is diluted 1: 300 in PBS (final volume 50 ml) and stored for 1 hour at RT. (Room temperature should be ~ 25 ℃) One T +1 Inoculation of 24-well plates (8 plates in total). 2 ml aliquots are dispensed into pre-designated wells in 24-well plates and transferred to NOMIR (8 24-well plates in total). One T +2 to +8 Dilution of processed sample. After laser treatment, 100 μl from each well is serially diluted with a final dilution of 1: 1000 in PBS. 4 Plating of processed samples. 100 μl of the final dilution is plated in triplicate ( 5 ×) on TSB agar with and without 30 μg / ml methicillin (10 TSB plates per well). 2 Plates are incubated at 37 ° C. for 18-24 hours. T +24 Colonies are counted on each plate (160 plates in total). 6

Independent Report on MRSA Studies, 9-10 November 2006

MRSA Data Progression 11-10-06 Experiment # 3.

Experiment 3-Design:

Eight different laser doses based on the effective doses established above and in Experiments 1 and 2 were used to treat saline-suspensions of MRSA growing on log and labeled with A3 to H3.

Treated and control untreated suspensions were diluted and plated in triplicate on tryptic soybean agar with or without 30 μg / ml methicillin.

 After 24 hr growth at 37 ° C., colonies were counted.

Experiment 3-Results:

Comparison of CFU on plates with and without methicillin showed a significant increase in the effectiveness of methicillin in all laser treated samples. The data is summarized in the table below.

Schematic Protocol for the NOMIR MRSA Study-November 10, 2006 Method: Time (hours) task FTE (hours) T-18 Inoculate overnight cultures 50 ml directly from glycerol stock One T-4 Starter culture setting 3 dilutions 1:50, 1: 125, 1: 250 One OD ₆₀₀ monitoring of starter culture 4 T0 Preparation of Plating Cultures. At 10:00 AM, the culture with OD ₆₀₀ = 1.0 is diluted 1: 300 in PBS (final volume 50 ml) and stored for 1 hour at RT. (Room temperature should be ~ 25 ℃) One T +1 Inoculation of 24-well plates (6 plates in total). 2 ml aliquots are dispensed into pre-designated wells in 24-well plates and transferred to NOMIR (6 total 24-well plates). One T +2 to +8 Dilution of processed sample. After laser treatment, 100 μl from each well is serially diluted with a final dilution of 1: 1000 in PBS. 4 Plating of processed samples. 100 μl of final dilution is plated (5 ×) onto TSB agar in 5-fold in the following manner: 24 well plates # 1 and 2 with 30 μg / ml methicillin and without methicillin. 24 well plates # 3 and 4 were with and without μg / ml penicillin. 24 well plates # 5 and 6 were with and without erythromycin in μg / ml erythromycin. (10 TSB plates per well). 2 Plates are incubated at 37 ° C. for 18-24 hours. T +24 Colonies are counted on each plate (120 plates total). 6

Independent Report on MRSA Studies, November 10-11, 2006

(MRSA Data Progress 11-10-06 Experiment # 4)

Experiment 4-Design:

Two different laser doses based on the effective doses established in the previous experiments were used to treat saline-suspensions of MRSA growing in log phase and labeled with A4 to F4.

Treated and untreated suspensions of control were diluted and 30 μg / ml methicillin (groups A4 and B4), 0.5 μg / ml penicillin G (groups C4 and D4) or 4 μg / ml erythromycin (group E4) And triplicate on tripty soybean agar with or without F4).

After 24 hr growth at 37 ° C., colonies were counted.

Experiment 4-Results:

Laser treatment increases the sensitivity of MRSA several times for each antibiotic tested. The data is summarized below.

series drug A4 Methicillin B4 Methicillin C4 penicillin D4 penicillin E4 Erythromycin F4 Erythromycin

Example XXIII

In vivo  Safety Test-Human Patient

After in vitro fibroblast studies, the inventors titrated themselves to ensure a safe, maximum level of energy and exposure time that can be delivered to human skin tissue without burning or otherwise damaging the irradiated tissue. Was performed.

The method we used was to irradiate the big toe with different lengths of time and power using a NIMELS laser. The results of this self-exposure experiment are described below.

Time / temperature evaluations were tabulated to ensure the thermal safety of these laser energies on human skin tissue (data not shown). In one laser procedure, the toe surface temperature was measured using a laser infrared thermometer, with the big toe exposed at 870 nm and 930 nm up to 233 seconds. At a surface temperature of 37.5 ° C., it was found that using the radiation dose using 870 nm and 930 nm together at a combined power density of 1.70 W / cm 2, pain was generated and the laser was turned off.

In the second laser procedure, the toe surface temperature was measured again using a laser infrared thermometer, with the big toe exposed at 930 nm for 142 seconds. At a surface temperature of 36 ° C., using 930 nm alone at a power density of 1.70 W / cm 2, it was found that pain occurred and the laser was turned off.

Example XXIV

In vivo  Safety Trial-Limited Clinical Pilot ( PILOT ) Research

Following the experiment, additional patients with nail fungus on the feet were treated. All of these patients were unpaid volunteers and signed informed consent. The primary goal of this limited pilot study is to achieve in vivo the same level of fungal decontamination as achieved in vitro using a NIMELS laser device. We also decided to apply the maximum time exposure and temperature limits that we endured during the self-exposure experiment. In a highly controlled and monitored environment, three to five laser exposure procedures were performed for each subject. Four subjects were recruited and treated. We did not compensate the person who signed the informed consent form and told us that we could cancel it at any time during the procedure.

The radiation dose used to treat the first subject was the same as that used during the self-exposure of the present inventors (as presented above). The temperature parameter on the surface of the toenail was also equivalent to the temperature we found upon self-exposure.

Treated toenails significantly reduced foot ringworm and abutment surrounding the toenail layer, indicating decontamination of the toenail serving as a reservoir for fungi. Control claws were scraped off with a cross-cut tissue bur, and debris was obtained and plated on mycological media. Treated toenails were scraped and plated in exactly the same manner.

To incubate claw debris, Sabouraud dextrose agar (2% dextrose) medium was prepared by adding: Chloramphenicol (0.04 mg / ml) for the overall fungal test; Chloramphenicol (0.04 mg / ml) and cycloheximide (0.4 g / ml) (selective for skin fungi); Chloramphenicol (0.04 mg / ml) and griseofulvin (20 μg / ml) (to serve as negative controls for fungal growth).

The 9-day fungal results for Treatment # 1 and Treatment # 2 (performed after 3 days of Treatment # 1) were the same, ie dermatophytes grew on control toenails and did not grow on treated toenails. The treated plate showed no growth, whereas the untreated control culture plate showed significant growth.

The first subject participated for 120 days and received four treatments under the same protocol. FIG. 18 shows a comparison of claws before treatment (A), 60 days after treatment (B), 80 days after treatment (C) and 120 days after treatment (D). In particular, healthy uninfected claw plates covered 50% of the claw area and grew from healthy cuticles after 120 days.

While particular embodiments have been described herein, those skilled in the art will appreciate that the methods, systems, and apparatus of the present invention may be embodied in other specific forms without departing from the spirit thereof. Accordingly, embodiments of the present invention should be regarded as illustrative in all aspects and not restrictive. It is understood that human claws act as refractory lenses and disperse and / or reflect some of the NIMELS infrared energy. Therefore, pig skin dose / tolerance studies were performed to titrate the maximum NIMELS radiation dose without burning / damaging tissue. Pork skin was used as a model for human skin. These studies were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals in accordance with the provisions of the Animal Protection Act. These tests are shown below.

Pig Skin Dose / Tolerance Study

Example XXV

specific heat NIMELS  Interaction

Evidence for Non-Column NIMELS Interactions:

Experiments (in vitro bath studies) have demonstrated that the temperatures reached in in vitro NIMELS experiments were not originally high enough by themselves to neutralize pathogens.

In the table below, simple teeth. Coli. It can be clearly seen that the bacteria achieve 91% growth of colonies when inoculated in vitro in a water bath at 47.5 ° C. for 8 minutes in succession. Thus, in essence, the NIMELS reaction has been shown to be photochemical in nature and to take place in the absence of exogenous drugs and / or dyes.

Claims (75)

An effective amount of a suitable drug, and NIMELS light generating lasers formed and arranged to produce an output of NIMELS radiation λn at the NIMELS radiation dose Tn to irradiate the target site Wherein the output is capable of altering the membrane dipole potential Ψd of the irradiated cell membrane to produce a NIMELS effect, wherein the NIMELS effect enhances the drug. The therapeutic system of claim 1, wherein the system is formed and arranged to avoid undesirable damage to a biological moiety at the target site. The treatment system of claim 1, wherein the NIMELS light generating laser is formed and arranged to produce the output of NIMELS radiation using a wavelength of 870 nm or 930 nm or both. The treatment system of claim 1, wherein the NIMELS light generating laser is formed and arranged to irradiate (Tn) the target site for a time of about 50 to about 1200 seconds. The treatment system of claim 1, wherein the NIMELS radiation dose provides an energy density of about 100 J / cm 2 to about 2000 J / cm 2 at the target site. The treatment system of claim 1, further comprising a light dispersion tip formed and arranged to scatter the output at the target site. The treatment system of claim 1, further comprising a light delivery subsystem configured and arranged to produce an output having a geometric flat top intensity distribution. 8. The treatment system of claim 7, wherein the light delivery subsystem comprises a flat top lens. The treatment system of claim 1, further comprising a light delivery system comprising an optical fiber. The method of claim 1, based on the calculation of total energy or power or output time at the target site to produce a NIMELS effect that can change the steady-state transmembrane potential ΔΨ-steady to the transient-state transmembrane potential ΔΨ-trans. And a radiation dose controller configured and arranged to adjust the energy output. The system of claim 1, wherein the medicament is selected from the group consisting of antimicrobial agents, antifungal agents, antineoplastic agents, and combinations thereof. The method of claim 11, wherein the antimicrobial agent is β-lactam, glycopeptide, cyclic polypeptide, macrolide, ketolide, anilinouracil, lincosamide, chloramphenicol, tetracycline, aminoglycoside, bacitracin, cefazoline Cephalosporins, pyrosine, nitroimidazole, quinolones and fluoroquinolones, novobiocins, polymyxins, cationic detergent antibiotics, oxazolidinones or other heterocyclic organic compounds, glycylcyclines, lipopeptides, cy Click lipopeptides, fluomutillins, and gramicidines, daptomycin, lineezolide, ansamycins, carvacesfem, carbapenems, monobactams, platensimacins, streptogramine, tinidazoles, and their And any combination thereof, including any salt. The method of claim 11, wherein the antifungal agent is selected from polyenes, azoles, imidazoles, triazoles, allylamines, echinodines, cyclopyrox, flucitocin, griseofulvin, amolol, pin, and any of these. And a combination thereof, including salts. The treatment according to claim 11, wherein the anti-neoplastic agent is selected from the group consisting of actinomycin, anthracycline, bleomycin, plicamycin, mitomycin, taxane, etoposide, teniposide and combinations thereof. system. An effective therapeutic amount of a medicament; A NIMELS light generating laser formed and arranged to produce NIMELS radiation λ n at a NIMELS radiation dose T n for irradiation of a target site for selectively irradiating C-H covalent bonds in the long chain fatty acid of the lipid bilayer; And Light delivery system suitable for delivering the NIMELS radiation λ n to a target site at a NIMELS radiation dose Tn Wherein the targeted irradiation of the covalent bonds is carried out to alter the membrane dipole potential Ψd of the irradiated cell membrane, the combination of λn and Tn (i) irradiates a cytochrome chain, and (ii) Suitable for changing the bioenergy of the membrane from thermodynamic steady-state conditions to one of transitional energy stress and / or redox stress, and the light delivery system provides the NIMELS radiation dose to the target site without damaging the desired host cell. And to form and arrange to deliver the NIMELS radiation λn at Tn. The system of claim 15, wherein the NIMELS light generating laser is formed and arranged to irradiate (Tn) the target site for a time of about 50 to about 1200 seconds. The system of claim 15, wherein the NIMELS radiation dose provides an energy density of about 100 J / cm 2 to about 2000 J / cm 2 at the target site. The treatment system of claim 15, wherein the medicament is selected from the group consisting of antimicrobial, antifungal, anti-neoplastic and combinations thereof. The system of claim 18, wherein the anti-neoplastic agent is selected from the group consisting of actinomycin, anthracycline, bleomycin, plicamycin, mitomycin, and combinations thereof. 19. The method of claim 18, wherein the antimicrobial agent is β-lactam, glycopeptide, cyclic polypeptide, macrolide, ketolide, anilinouracil, lincosamide, chloramphenicol, tetracycline, aminoglycoside, bacitracin, cefazoline Cephalosporins, pyrosine, nitroimidazole, quinolones and fluoroquinolones, novobiocins, polymyxins, cationic detergent antibiotics, oxazolidinones or other heterocyclic organic compounds, glycylcyclines, lipopeptides, cy Click lipopeptides, fluomutillins, and gramicidines, daptomycin, lineezolide, ansamycins, carvacesfem, carbapenems, monobactams, platensimacins, streptogramine, tinidazoles, and their And any combination thereof, including any salt. 19. The method of claim 18, wherein the antifungal agent is selected from polyenes, azoles, imidazoles, triazoles, allylamines, echinodines, cyclopyrox, flucitocin, griseofulvin, amorrolopine, sodarin, and any of these. And a combination thereof, including salts. The system of claim 15, wherein the system is formed and arranged to alter proton motive force Δp, wherein cells in the biological moiety or biological contaminant are proteins, RNA, DNA, in the biological moiety or biological contaminant. A therapeutic system in which the ingestion of essential nutrients necessary for proper growth and reproduction, including the synthesis of peptidoglycan, lipoteichoic acid, and lipids, or the manufacture of appropriate ATPs is prevented. 23. The method of claim 22, wherein the system enhances the drug by lowering the ATP available through the modified Gibbs free energy value, ΔGp, to impair the energy dependent release of the drug associated with the proton driving force Δp. A treatment system that is formed and arranged. The treatment according to claim 15, wherein the system is formed and arranged to produce a NIMELS effect (Ne) at a value of 2 to 10 representing an antimicrobial enrichment level in any tissue, moiety, or solution against a microbial pathogen. system. The treatment system of claim 15, wherein the light delivery system comprises a flat top lens, an optical fiber, or a dispersing tip. Combining λn and Tn to irradiate the target site; Simultaneously decreasing Δp-mito-mam and / or Δp-plas-Bact at the target site; And Simultaneously or sequentially administering an antimicrobial agent to the target site Wherein the inhibition of one or more cellular assimilation pathways is achieved at the target site, wherein ΔμH ⁺ or Δμx ^{+ is applied} to the cells at the target site to inhibit the cellular assimilation pathway and attenuate cellular resistance mechanisms against antimicrobial molecules. How to reduce. The method of claim 26, wherein the targeted assimilation pathway is peptidoglycan biosynthesis co-targeted by the antimicrobial agent that binds to the active site of the bacterial transpeptidase enzyme. The method of claim 26, wherein the targeted bacterial assimilation pathway binds to an acyl-D-alanyl-D-alanine group in the cell wall intermediate to N-acetylmuramic acid (NAM)-and N-acetylglucosamine (NAG). -A peptidoglycan biosynthesis co-targeted by the antimicrobial agent which prevents the proper formation of peptidoglycan in gram positive bacteria by preventing the integration of the peptidoglycan matrix into the peptidoglycan matrix. 27. The method of claim 26, wherein the targeted bacteria assimilation path, C ₅₅ - iso prenyl to fatigue coupled to a phosphate fatigue phosphatase is C ₅₅ - by preventing interaction with isopropyl prenyl pyrophosphate building blocks (building block) peptides A peptidoglycan biosynthesis co-targeted by the antimicrobial agent which reduces the amount of C ₅₅ -isoprenyl pyrophosphate available for transferring doglycans out of the lining. 27. The method of claim 26, wherein the targeted assimilation pathway binds to 23S rRNA molecules in subunit 50S subunit of bacterial ribosomes to accumulate peptidyl-tRNA in cells, thereby depleting the free tRNA required for activation of α-amino acids. And bacterial protein biosynthesis co-targeted by the antimicrobial agent which inhibits peptide transfer by causing early dissociation of peptidyl tRNA from ribosomes. The method of claim 30, wherein the antimicrobial agent simultaneously binds to two domains of 23S RNA of the 50S bacterial ribosomal subunit to inhibit the formation of bacterial ribosomal subunits 50S and 30S. 31. The method of claim 30, wherein the antimicrobial agent is chlorinated to increase its lipophilic properties, binds to the 23S portion of the 50S subunit of bacterial ribosomes, and from the active site (A-site) to the peptidyl site (P-site). Inhibiting the transpeptidase response by preventing the translocation of peptidyl-tRNA. The method of claim 30, wherein the targeted assimilation pathway binds to the 30S bacterial ribosomal subunit to block binding of amino-acyl tRNA to the recipient site (A-site) of the ribosome, thereby codon-anticodone interactions and And bacterial protein biosynthesis co-targeted by the antimicrobial agent that inhibits the prolongation of protein synthesis. 34. The method of claim 33, wherein said antimicrobial agent binds to said bacterial ribosome more strongly in an orientation different from a conventional subgroup of polyketide antimicrobial agents having an octahydrotetracene-2-carboxamide backbone. The method of claim 26, wherein the targeted assimilation pathway binds to a specific aminoacyl-tRNA synthetase to prevent esterification of one of its intercompatible tRNAs with a particular amino acid or precursor thereof, thereby preventing aminoacyl-tRNA A bacterial protein biosynthesis co-targeted by the antimicrobial agent preventing the formation of The protein synthesis formyl of claim 26, wherein the targeted assimilation pathway binds to the 50S rRNA via domain V of the 23S rRNA while interacting with the 16S rRNA of the 30S ribosomal subunit, thereby inhibiting bacterial protein synthesis prior to initiation. A bacterial protein biosynthesis co-targeted by the antimicrobial agent which prevents binding of methionine initiator (f-Met-tRNA) and 30S ribosomal subunit. 27. The peptidyl transferase activity and peptidyl delivery of claim 26, wherein the targeted assimilation pathway interacts with the 50S subunit of bacterial ribosome at protein L3 in the region of the 23S rRNA P site close to the peptidyl transferase center. And bacterial protein biosynthesis co-targeted by the antimicrobial agent that inhibits, blocks P-site interactions, and prevents the normal formation of active 50S ribosomal subunits. 27. The method of claim 26, wherein the targeted assimilation pathway is DNA replication and transcription co-targeted by the antimicrobial agent that inhibits topoisomerase II (DNA gyrase) and / or topoisomerase IV. Way. 27. The method of claim 26, wherein the targeted assimilation pathway is co-regulated by the antimicrobial agent that inhibits DNA polymerase IIIC, an enzyme required for replication of chromosomal DNA in Gram-positive bacteria but not present in Gram-negative bacteria. The method is targeted DNA replication and translation. The DNA of claim 26, wherein the targeted assimilation pathway is co-targeted by the antimicrobial agent that inhibits topoisomerase II (DNA gyase) and / or topoisomerase IV and / or DNA polymerase IIIC. Methods that are replication and transcription. The method of claim 26, wherein the targeted assimilation pathway is bacterial phospholipid biosynthesis co-targeted by the antimicrobial agent acting on phosphatidylethanolamine-rich cytoplasmic membrane. 27. The method of claim 26, wherein the targeted assimilation pathway of β-ketoacyl- (acyl-carrying-protein (ACP)) synthase I / II (FabF / B) is an essential enzyme of type II fatty acid synthesis. A bacterial fatty acid biosynthesis co-targeted by the antimicrobial agent which inhibits bacterial fatty acid biosynthesis via selective targeting. 27. The method of claim 26, wherein the targeted assimilation pathway is maintenance of bacterial transmembrane translocation ΔΨ-plas-bact, and the antimicrobial agent binds to Gram positive cytoplasmic membranes, causing depolarization and loss of membrane potential, thereby Causing inhibition of RNA synthesis and disrupting bacterial cell membrane function. 27. The method of claim 26, wherein the antimicrobial agent increases the permeability of the bacterial cell wall, disrupting the ion gradient between the cytoplasm and the extracellular environment by allowing inorganic cations to move through the wall in a non-limiting manner. The surface of claim 26, wherein the targeted assimilation pathway is bacterial membrane selective permeability and maintenance of bacterial transmembrane translocation ΔΨ-plas-bact, and wherein the antimicrobial agent has a negative charge on the bacterial membrane relative to the neutral membrane surface of the eukaryotic cell. A cationic antimicrobial peptide that is selective for and promotes membrane permeation and ultimate perforation and / or disruption of bacterial cell membranes to promote leakage of bacterial cell contents and disruption of transmembrane potentials. The method of claim 26, wherein the antimicrobial agent inhibits bacterial protease peptide deformillase that catalyzes the removal of formyl groups from the N-terminus of the newly synthesized bacterial polypeptide. 27. The method of claim 26, wherein the antimicrobial agent inhibits a two-component regulatory system in bacteria, which regulates its ability to respond to their environment through signal transduction across bacterial plasma membranes, a process not present in mammalian membranes. Which method. 27. The method of claim 26, wherein the antimicrobial agent attenuates or inactivates the antimicrobial agent and / or acts as a discharge pump inhibitor, such that any targeted bacteria produces as a resistance mechanism to help restore the effects of the antimicrobial agent. In combination with or delivered in conjunction with a second molecule that acts as a competitive inhibitor for the protein or enzyme of. Combining λn and Tn to irradiate the target site; Simultaneously decreasing Δp-mito-mam and / or Δp-plas-Bact at the target site; And Administering multiple antimicrobials simultaneously or sequentially to the target site Wherein the inhibition of one or more cellular assimilation pathways is achieved at the target site, wherein ΔμH ⁺ or Δμx ⁺ is reduced in cells of the target site to inhibit the cellular assimilation pathway and attenuate cellular resistance mechanisms against antimicrobial molecules. How to let. 50. The bacterium of claim 49, wherein at least one of the antimicrobial agents targets as a resistance mechanism to attenuate or inactivate one of the antimicrobial agents and / or act as a discharge pump inhibitor to aid in the recovery of the effect of the antimicrobial agent. In combination with a second molecule that acts as a competitive inhibitor for any protein or enzyme it produces. Combining λn and Tn to irradiate the target site; Simultaneously decreasing Δp-mito-mam, Δp-mito-Fungi, Δp-plas-Fungi at the target site; And Simultaneously or sequentially administering an antifungal agent to the target site Wherein the inhibition of one or more cellular assimilation pathways is achieved at the target site, wherein ΔμH ⁺ or Δμx ⁺ is applied at the cells of the target site to inhibit the cellular assimilation pathway and attenuate cellular resistance mechanisms against antifungal molecules. How to reduce. The method of claim 51, wherein the targeted assimilation pathway is phospholipid biosynthesis co-targeted by the antifungal agent that disrupts the structure of existing phospholipids in the fungal cell membrane. The lanosterol of claim 51, wherein the targeted assimilation pathway inhibits ergosterol biosynthesis in the C-14 demethylation step, thereby inhibiting ergosterol depletion and the function of ergosterol as a membrane component through disruption of the plasma membrane structure. And ergosterol biosynthesis co-targeted by the antifungal agent causing the accumulation of other 14-methylated sterols. The ergosterol biosynthesis of claim 51, wherein the targeted assimilation pathway inhibits squalene epoxidase, which in turn is co-targeted with the antifungal agent to inhibit ergosterol biosynthesis in fungal cells such that the fungal cell membrane has increased permeability. How to be. The method of claim 51, wherein the targeted assimilation pathway is ergosterol biosynthesis co-targeted with the antifungal agent that inhibits d14-reductase and d7, d8-isomerase. The method of claim 51, wherein the targeted assimilation pathway is co-targeted with the antifungal agent that inhibits (1,3) β-D-glucan synthase, which in turn inhibits β-D-glucan synthesis in the fungal cell wall. The method is fungal cell wall biosynthesis. 52. The sterol in the fungal cell membrane of claim 51, wherein the targeted assimilation pathway effectively changes the transition temperature of the cell membrane to allow voids to form in the membrane to form deleterious ion channels in the fungal cell membrane (main sterol is ergosterol). Fungal sterol biosynthesis co-targeted with the antifungal agent in combination with The method of claim 57, wherein the antifungal agent is formulated for delivery to lipids, liposomes, lipid complexes and / or colloidal dispersions to prevent toxicity from the medicament. The method of claim 51, wherein the targeted assimilation pathway is protein synthesis, and the antifungal agent is taken up into fungal cells by cytosine permease, deaminoated with 5-fluoro racil (5-FU), and 5FC, which is converted to cleoside triphosphate and integrated into RNA, where miscoding occurs. The method of claim 51, wherein the targeted assimilation pathway is fungal protein synthesis co-targeted with the antifungal agent that inhibits fungal elongation factor EF-2. The method of claim 51, wherein the targeted assimilation pathway is fungal chitin biosynthesis co-targeted with the antifungal agent that inhibits fungal chitin biosynthesis by inhibiting the action of one or more enzymes chitin synthase 2. 62. The method of claim 61, wherein said antifungal agent inhibits the action of chitin synthase 3, an enzyme required for the synthesis of chitin during emergence and growth, conjugation and sporulation. 52. The method of claim 51, wherein the antifungal agent chelates the polyvalent cation Fe ⁺³ or Al ⁺³ , resulting in the inhibition of metal-dependent enzymes responsible for mitochondrial electron transport and cellular energy production, and also the inhibition of peroxides in fungal cells. To inhibit normal degradation. The method of claim 51, wherein the antifungal agent inhibits a two-component regulatory system in the fungus and the regulatory system responds to the environment through signal transduction across the fungal plasma membrane. 53. The method of claim 51, wherein the antifungal agent acts on any protein or enzyme produced by the targeted fungus as a resistance mechanism to attenuate or inactivate the antifungal agent and act as an excretion pump inhibitor, thereby helping to restore the effects of the antifungal agent. In combination with a second molecule that is a competitive inhibitor. Combining λn and Tn to irradiate the target site; Simultaneously decreasing Δp-mito-mam, and / or Δp-mito-fungi, and / or Δp-plas-fungi in the cell at the target site; And Administering a plurality of antifungal agents simultaneously or sequentially to the target site Wherein the inhibition of one or more cellular assimilation pathways is achieved at the target site, wherein the ΔμH ⁺ or Δμx ⁺ is reduced in cells of the target site to inhibit the cellular assimilation pathway and attenuate cellular resistance mechanisms against antifungal molecules. How to let. 67. The protein of claim 66, wherein at least one of the antifungal agents weakens or inactivates one of the antifungal agents and acts as an evacuation pump inhibitor, such that any protein produced by the targeted fungus as a resistance mechanism to assist in the recovery of the effects of the antifungal agent. In combination with a second molecule that is a competitive inhibitor for the vagina or enzyme. Combining λn and Tn to irradiate the target site; Reducing Δp-mito-mam and / or mammalian transmembrane translocation ΔΨ-plas-mam; And Simultaneous or sequential administration of anti-neoplastic agents to the target site Wherein the inhibition of one or more cellular assimilation pathways is achieved at the target site, wherein ΔμH ⁺ or Δμx ⁺ is expressed in the cells at the target site to inhibit the cellular assimilation pathway and attenuate cellular resistance mechanisms against anti-neoplastic agents. How to reduce. 69. The method of claim 68, wherein said targeted assimilation pathway is cross-linked with guanine nucleobases in the DNA, thereby inhibiting DNA replication by disabling and separating DNA strands, a process necessary for DNA replication. And DNA replication co-targeted by. The method of claim 68, wherein the targeted assimilation pathway is DNA replication co-targeted by the anti-neoplastic agent that reacts with two different 7-N-guanine residues in the same strand of DNA or in different strands of DNA. 69. The method of claim 68, wherein the targeted assimilation pathway is DNA replication co-targeted by the anti-neoplastic agent that acts as an anti-metabolite to inhibit DNA replication and cell division. The method of claim 68, wherein the targeted assimilation pathway is cell division co-targeted by the anti-neoplastic agent that inhibits cell division by preventing microtubule function. The method of claim 68, wherein the targeted assimilation pathway is DNA replication co-targeted by the anti-neoplastic agent that inhibits DNA replication and cell division by preventing cells from entering replication of G1 and DNA. The method of claim 68, wherein the targeted assimilation pathway is cell division that is co-targeted by the anti-neoplastic agent that enhances the stability of the microtubules and prevents the separation of chromosomes during anaphase. 69. The method of claim 68, wherein the targeted assimilation pathway inhibits DNA replication and cells by inhibition of type I or type II topoisomerase, which inhibits both transcription and replication of DNA by disrupting suitable DNA supercoiling. And DNA replication co-targeted by the antineoplastic agent that inhibits cleavage.

Images