MXPA99007701A - Methods and compositions for the protection of mitochondria - Google Patents

Abstract

Protection of mitochondria from oxidative damage due to natural or disease processes as well as by the effects of exogenous factors such as incident sunlight, exposure via inhalation to oxidative environmental toxins, consumption of dietary oxidants, and oxidative-stress-inducing pharmaceuticals, exposure to radiation including radiation therapy, among others, is provided by a composition comprising L-ergothioneine. L-ergothioneine may be prepared in a pharmaceutically-acceptable carrier to form an agent for topical application to the skin, and for orally or parenteral administration. Effective application and delivery of L-ergothioneine is enhanced by encapsulation in a liposome, a preferred embodiment. Diagnostic methods for determining exposure and susceptibility to radiation, radical, and reactive oxygen species in mammals is also provided.

Description

METHODS AND COMPOSITIONS FOR THE PROTECTION OF THE MITOCHONDRIA BACKGROUND OF THE INVENTION Mitochondria are subcellular organelles present in all organisms that use oxygen, where energy is generated in the form of adenosine triphosphate (ATP), and oxygen is reduced to water. Ninety percent of the oxygen taken is consumed in the mitochondria. A substantial by-product of this generation of ATP is the formation of potentially toxic oxygen radicals. For example, it is estimated that 1-2% of all reduced oxygen produces superoxide (O2-) and hydrogen peroxide (H2O2). Other reactive oxygen species (ROS) that are formed are singlet oxygen (1O2) and hydroxyl radical (OH »). Under voltage conditions in the cell this can increase up to 10% of all the oxygen consumed. The membranes of the mitochondria are sensitive to the peroxidation and depolarization of lipids that result from these ROS. Damage to the mitochondria is also the result of exposure to sunlight, which forms ROS as indicated above. Because it is believed that damage to mitochondria may be the cause or the most important factor in some diseases, such as cancer, diabetes, cataracts, neurodegenerative diseases, porphyrias, cadiovascular disease, and also contributes to the complications of aging, it is necessary a method to protect mitochondria from said damage and repair said damage. The cellular damage of burns to the skin and lungs through contact or exposure to fire and other sources of intense heat is mediated through radical damage. In addition, exposure to unfavorable environmental factors, including industrial pollutants in the air, and oil and tobacco combustion products, can contribute to the oxidative damage of lung tissues and other body tissues. Also, various therapeutic regimens such as chemotherapeutic drugs and radiation therapy for the treatment of disproportionate diseases induce important side effects related to oxidative stress, such as cardiotoxicity. The present invention relates to applied agents that protect the mitochondria from said damage. L-ergothioneine is an amino acid that contains sulfur and is found in several tissues of mammals, however it is not synthesized endogenously and must be consumed in the diet. Although it exists in some tissues in millimolar quantities, its exact role is uncertain (see: Melville, 1959, Vitamins and Hormones 7: 155-204). It is normally seen as an antioxidant, however the results cause conflict. Some see it as a hydrogen peroxide scavenger (see: Hartman, 1190, Methods in Enzymology 186: 310-318), while others claim that it does not react with hydrogen peroxide but that it cleans the hydroxyl radical (see: Akamnu et al. , 1991, Arch. Biochem. Biophys., 298: 10-16, 1991). Although previous in vitro studies have shown their ability to protect DNA and proteins against phototoxic drug-induced binding UV radiation (eg, Van den Broeke and others, 1993, J. Photochem, Photobiol B 17: 279-286), and to protect bacteriophages against gamma radiation (Hartman et al., 1988, Radiation Research 114: 319-330 ), in vivo results are not as promising. Although it is claimed that L-ergothioneine is useful in topical formulations for purifying radicals and ultraviolet light protectants for damage to hair and skin (for example, WO 9404129), Van den Broeke et al. (1193, Int. J. Radiat, Biol. 63: 493-500), it was not found that topically applied L-ergothioneine was effective in an UV-induced phototoxic drug binding animal model that binds to epidermal biomolecules. Other proposed in vivo uses include decreasing circulating levels of lipoprotein (a) (U.S. Patent 5,272,166), and inhibition of skin pigmentation., for example, remove dark spots and freckles (JP 63008335 and JP 61155302). As described above, numerous disease processes are attributed to the unfavorable reaction of the body to the presence of high levels of reactive oxygen species (ROS). In the eye, cataracts, macular degeneration and degenerative damage to the retina are attributed to ROS. Other organs and their diseases related to ROS include: lung cancer induced by tobacco and asbestos combustion products; accelerated aging and its manifestations, damage to the skin; atherosclerosis; ischemia and reperfusion injury, diseases of the nervous system such as Parkinson's disease, Alzheimer's disease, muscular dystrophy, multiple sclerosis; others Lung diseases include emphysema and bronchopulmonary dysphasia; excess iron diseases such as hemochromatosis and thalassemia; pancreatitis; diabetes; kidney diseases that include autoimmune nephrotic syndrome and heavy metal-induced nephrotoxicity; and radiation injuries. Some antineoplastic drugs such as adriamycin and bleomycin induce severe oxidative damage, especially to the heart, limiting the patient's exposure to the drug. Active redox metals such as iron induce oxidative damage to tissues; Industrial chemicals and ethanol, by exposure and consumption, induce a series of injuries related to oxidative damage, such as cardiomyopathy and liver damage. Industrial and petrochemical pollutants in the air, such as ozone, nitric oxide, radioactive particulate compounds and halogenated hydrocarbons, induce oxidative damage to the lungs, gastrointestinal tract, and other organs. Radiation poisoning from industrial sources, including runoff from nuclear reactors and exposure to nuclear weapons, are other sources of radiation and radical damage. Other exposure routes can occur when living or working near sources of electromagnetic radiation, such as power plants and high-voltage power lines, x-ray machines, particle accelerators, radar antennas, radio antennas, and the like. , as well as other electronic products and devices that emit electromagnetic radiation such as cell phones, and television and computer monitors. It is convenient to protect the mitochondria of all these etiological agents.

BRIEF DESCRIPTION OF THE INVENTION An object of the present invention is to provide means to protect mitochondria from damage by means of methods and compositions, by which the membranes of mammalian mitochondria are protected. Another object of the present invention is to provide methods and compositions by means of which mammalian mitochondria can be protected from oxidative damage. A further object of the invention is to provide methods and compositions that protect mammalian mitochondria from damage caused by the effects of incident sunlight as well as other radiation injuries. Another object of the invention is to provide methods and compositions that protect the mitochondria of mammals from damage caused by the effects of oxidizing toxins in the air, such as those that occur in industrial pollutants and in petrochemical and tobacco combustion products. It is a further object of the invention to provide methods and compositions that protect mammalian mitochondria from damage caused by the effects of elevated levels of oxidizing compounds and reactive oxygen species that occur in various processes of mammalian mitochondria. diseases, as well as those induced by different therapeutic agents and regimens. A further objective of the present invention is to provide diagnostic tests to determine, in a mammal, the extent and susceptibility to damage of mitochondria by radiation, radicals and reactive oxygen species. Mitochondria are injured by oxidative damage due to natural processes and diseases as well as by other effects of exogenous factors such as incident sunlight, inhalation exposure to environmental oxidizing toxins, electromagnetic radiation, consumption of food oxidants, and pharmaceuticals that induce stress oxidant, among others. In the present invention, the pretreatment or treatment of cells with L-ergothioneine protects the mitochondria from said damage and reduces the damage caused to the mitochondria by sunlight and by the presence of oxygen radicals. In a non-limiting example, L-ergothioneine is combined in a pharmaceutically acceptable carrier to form an agent for topical application to the skin. The invention includes methods of treatment using L-ergothioneine administered orally or parenterally. In a preferred embodiment, the effective application and delivery of L-ergothioneine are improved by encapsulation in a liposome. In one example, a liposome composed of phosphatidylcholine, phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate is used. L-ergothionein encapsulated in liposome it is also combined with a pharmaceutically acceptable vehicle for topical application.

DETAILED DESCRIPTION OF THE INVENTION The inhibition of oxidative damage to mitochondria in various tissues of mammalian bodies is of therapeutic benefit for the prophylaxis and treatment of many pathological conditions ranging from those responsible for mortality and significant pathology, such as atherosclerosis and cancer, to those less pathological but with an unfavorable psychological component, such as unpleasant changes in the skin as a result of long-term photoaging. In various diseases such as cancer, diabetes, atherosclerosis, cataracts and certain neurological diseases, among others, reactive oxygen species (ROS) are implicated in the pathophysiology of the disease. Ischemia, where tissues are deprived of blood and oxygen flow as occurs during an embolism and myocardial infarction, followed by reperfusion of ischemic tissue, initiates significant ROS damage to tissue that does not die directly during the infarction. Chemotherapeutic agents against cancer, such as adriamycin and bleomycin, induce oxidative damage, as well as radiation therapy (eg, x-rays) against cancer. As critical subcellular organelles are involved in aerobic energy metabolism and therefore oxidative reactions, Mitochondria are sensitive to endogenous and exogenous influences and can be easily damaged or destroyed. The metabolism of dysfunctional energy and, more severely, damaged mitochondria, can lead to cellular senescence and death, descending tissue, and organ damage or dysfunction. In the skin, increased oxidative damage as a result of exposure to ultraviolet light can damage the cellular structure of the skin resulting in premature and psychologically debilitating changes related to aging, such as thinning of the skin, wrinkle formation and abnormal pigmentation. Exposure of the lungs to environmental oxidants can induce mitochondrial damage and cell damage that results in chronic obstructive airways disorders. Exposure to electromagnetic and nuclear radiation also induces oxidative damage. In accordance with the present invention, mitochondrial protection is provided through the application or administration of a composition containing L-ergothioneine (L-ET). Administration to the target cells, tissue or organ may be parenterally; transmucosally, for example, orally, nasally, rectally; or transdermal. Parenteral administration is via intravenous injection and also includes, but is not limited to, intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal, and intracranial administration. For example, the composition of the present invention can be infused directly into a tissue or organ that has suffered a heart attack, such as the brain or heart after an embolism or heart attack, in order to protect the mitochondria in cells of the ischemic penumbra, those outside the immediate area of infarction that do not die during the cessation of blood flow, but suffer extensive ROS-mediated damage when the blood flow is restored. L-ET can be prepared as a tablet or capsule formulation for oral administration. For topical delivery, a solution of L-ET in water, regulated aqueous solution or other pharmaceutically acceptable vehicle, or a hydrogel cream or lotion, comprising an emulsion of an aqueous and hydrophobic phase, at a concentration of 50 μM and 5mM. A preferred concentration is around 1 mM. To this may be added ascorbic acid or its salts, or other ingredients, or a combination thereof, to make a cosmetically acceptable formulation. Metals should be kept to a minimum. It is preferable that it be formulated by encapsulation in a liposome for oral administration, parenteral or preferably topical. As will be seen later, a composition of L-ET within a liposome improves the protection efficiency of mitochondria from oxidative damage resulting from radiation damage. Unexpectedly, it was found that the use of the liposome formulation for L-ET improves the effectiveness of the compound for the protection of mitochondria. Although liposome delivery has been used as a pharmaceutical delivery system for many other compounds in a variety of applications [see Langer, Science 249: 1527-1533 (1990); Treat and others, in Liposomes in the Therapy of Infectious Disease and Cancer, López, Berestein and Fidler (eds.), Liss; New York, pp. 353-365 (1989); López-Berestein, ibid., Pp. 317-327; see generally ibid.], the subcellular delivery of L-ET in an effective form was discovered by the inventor even and is a preferred embodiment of the compositions and methods of the present invention. The function of the liposome is to increase the supply of L-ET to the mitochondria and clearly or additionally, protect the L-ET until it reaches the target cell or tissue. A non-limiting example of a liposome formulation is that which is formed of phosphatidylcholine, phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate in a ratio of 2: 2: 1: 5, encapsulating 10 mM of L-ET. A final concentration of 1 μM to 10 mM L-ET is used, preferably around 12 μM. This final concentration can be achieved by diluting the purified liposomes in a pharmaceutically acceptable vehicle. Many other suitable liposome formulations are known to those skilled in the art, and may be employed for the purposes of the present invention. For example, see: the US patent. No. 5,190,762; "Method of Administering Proteins to Living Skin Cells" by Yarosh that is incorporated herein by reference. It is possible to find a general description of liposomes and liposome technology in a three volume work entitled "Liposome Technology" edited by G. Gregoriadis, 1993, published by CRC Press, Boca Raton, Florida. Relevant portions of this reference are incorporated herein by reference. Transdermal delivery of L-ET is also contemplated, either as a liposome formulation or as free L-ET. Several and numerous Methods are known in the art for transdermal administration of a drug, for example, through a transdermal patch. It can be appreciated that a transdermal route of administration can be improved through the use of a dermal penetration enhancer. In another aspect of the present invention, pharmaceutical compositions of L-ET are provided. Said pharmaceutical compositions can be administered by injection, orally, pulmonary, nasal or other forms of administration. In general, pharmaceutical compositions comprising effective amounts of L-ET together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, auxiliaries and / or vehicles are found in the invention. Such compositions include diluents of various regulator contents (eg, Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabilsulfite), preservatives (e.g., thimerosal, benzyl alcohol) and binding substances (e.g., lactose, mannitol ); the incorporation of the material into preparations of particles of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or in liposomes (infra). Hyaluronic acid can also be used. Such compositions can influence the physical state, stability, release rate in vivo, and the rate of in vivo elimination of L-ET. The compositions can be prepared in liquid form, or they can be in dry powder form, as in lyophilized form.

The controlled release oral formulation is also convenient. The drug can be incorporated in an inert matrix that allows the release either by diffusion or leaching mechanisms, for example, gums. It is also possible to incorporate slow degeneration matrices into the formulation. Some enteric coatings also have a delayed release effect. Another way of a controlled release of this therapeutic is through a method based on the therapeutic system of Oros (Alza Corp.), that is, the drug is enclosed in a semipermeable membrane that allows water to enter and push out the drug through a small opening due to osmotic effects. The pulmonary delivery of the pharmaceutical compositions of the present invention is also contemplated, for the treatment or protection of mitochondria from oxidative damage. The pulmonary supply can be used for the treatment of the lung tissue itself, or serve as a supply route for blood flow and other places in the body. A pharmaceutical composition of the present invention is sent to the lungs of a mammal upon inhalation, and traverses the epithelial lining of the lung into the bloodstream. The use of various mechanical devices in the practice of this invention designed for pulmonary delivery of therapeutic products is contemplated, including but not limited to nebulizers, metered dose inhalers, powder inhalers, all known to those skilled in the art. With respect to the structure of the delivery apparatus, any form of aerosolization known in the art, including but not limited to sprinkler bottles, mist, atomization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used in the practice of this invention. Ophthalmic delivery of the compositions of the present invention is also contemplated for the protection and treatment of mitochondria, for example, in the lens of the eye, where it is believed that oxidative damage is responsible for a high incidence of cataracts. Other ophthalmic uses include the treatment or prophylaxis of macular degeneration and degenerative damage to the retina. Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. The nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after administering the therapeutic product in the nose, without the need for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. For nasal administration, an effective device is a small, hard bottle to which a dose sprinkler fits. In one embodiment, the metered dose is delivered by extracting the pharmaceutical composition from the solution of the present invention to a chamber of defined volume, whose chamber has an aperture sized for aerosolization and aerosol formulation forming a sprinkler when a liquid is compressed in the chamber . The camera compresses to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. These devices are commercially available. In another aspect, the L-ET liposomes can cross the blood-brain barrier, allowing intravenous or oral administration. Many strategies are available to cross the blood-brain barrier, including but not limited to, increasing the hydrophobic nature of a molecule; introducing the molecule as a conjugate to a vehicle, such as transferrin, directed to a receptor in the blood-brain barrier; and similar. In another embodiment, the molecule can be administered intracranially or, most preferably, intraventricularly. In a further embodiment, the L-ET can be administered in a liposome directed to the blood-brain barrier. A subject in whose administration of L-ET is an effective therapeutic regimen for the protection of mitochondria is preferably a human, however it can be any animal. Therefore, as one skilled in the art can appreciate, the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but not limited to, domestic animals, such as subjects felines or canines, farm animals, such as but not limited to bovine, equine, caprine, ovine and porcine, wild animals (whether in their natural environment or in a zoo), laboratory animals, such as mice, rats, rabbits, goats, sheep, pigs, dogs, cats, etc., species of birds, such like chickens, turkeys, songbirds, etc., that is, for veterinary medical use. The protection of mitochondria from oxidative damage can be used for the prevention and treatment of a number of disorders, including effects of solar, electromagnetic and nuclear radiation on the body, disease processes, exposure to pollutants including tobacco combustion products, and protection against the harmful effects of certain pharmacists whose mechanisms of action involve the generation of reactive oxygen species and other radicals. For example, certain antineoplastic agents induce oxidant radicals as their mechanism of action, however an important and limiting side effect that occurs in patients is cardiotoxicity; higher doses and therefore, increased efficiency against cancer can be achieved by protecting the mitochondria of the heart and other tissues with the compositions and methods of the present invention. In addition, several types of radiation used for cancer therapy, as an alternative or auxiliary in surgery, induce significant tissue damage; The previous administration of L-ET can be used to reduce or prevent the toxicity of radiation therapy in the body. It can also be used in combination with fibrinolytic therapy, such as tissue plasminogen activator or streptokinase, where the clots of clogged blood vessels that cause embolism and heart attack dissolve. The tissue protection of ROS arising as a result of reperfusion is an object of the present invention.

In addition, numerous disease processes involve reactive oxygen species. In the eye, cataracts, macular degeneration and degenerative damage to the retina are attributed to ROS and can be treated with L-ET administered topically, orally or parenterally. A liposome formulation is preferred. Diseases related to ROS of the lungs such as emphysema and bronchopulmonary dysphasia and including pathology induced by inhalation of tobacco and asbestos combustion products can be treated through an aerosolized form of L-ET as described above. Various diseases of the nervous system such as Parkinson's disease, Alzheimer's disease, muscular dystrophy, and multiple sclerosis can be treated through oral or parenteral formulations or direct delivery to the central nervous system via intrathecal, intraventricular and intracranial administration. Excess iron diseases such as hemochromatosis and thalassemia can also be treated through the compositions and methods of the present invention. Other diseases include pancreatitis; diabetes; kidney diseases including autoimmune nephrotic syndrome and heavy metal-induced nephrotoxicity; and radiation damage. Local and system damage as a result of burns involves ROS damage. In addition to the aforementioned therapeutic and prophylactic uses of the compositions of the present invention, various diagnostic utilities are also contemplated. The potential of L-ergothioneine to protect a mammal from damage to motocondrias and the level of L-ET necessary to provide protection can be determined in vitro by exposing aliquots of a cellular sample of said mammal to the harmful agent or condition, said aliquots containing various concentrations of L-ET. The damage to the mitochondria of the different aliquots is determined, as well as the lowest concentration, if any, of L-ET providing sufficient damage protection. To determine the degree of therapeutic benefit of L-ET in a mammal after exposure to a harmful agent of the mitochondria, a diagnostic sample similar to that described above can be used, with a variation where several concentrations of L- ET are applied to aliquots of cellular sample after exposure to the harmful agent of mitochondria. In another embodiment, the degree of exposure of a mammal to ROS can be assessed by determining the effect of L-ET on a sample of cells taken from the mammal. These diagnostic utilities also offer help in selecting an effective therapeutic dose of L-ET. In another embodiment, the ability of L-ET to protect a cellular sample from the damaging effects of a therapeutic regimen that causes oxidative damage, such as an antineoplastic agent or radiation therapy to be administered to a mammal with cancer, can be performed in vitro by combining the antineoplastic agent with various concentrations of L-ET, applying the combination to identical aliquots of a cellular sample of a mammal, and determining the degree of damage to the mitochondria in said series of samples. These data can be used to determine an effective dose of L-ET to prevent damage to mitochondria in the non-diseased cells of said mammal. In parallel, using a sample of diseased or cancerous cells of said mammal, it can be determined whether the L-ET will effect any decrease in cancer activity of said cancer agent; Based on these two tests, a level of L-ET can be selected for co-administration with the anticancer agent to provide optimal protection of the non-diseased cells of the mammal against the cancer agent, while providing maximum therapy against the cancer. Cancer. These are not limiting examples of useful diagnostic tests that determine the prophylactic and therapeutic benefits of compositions and methods of the present invention. The application of L-ET and its effect on damage to mitochondria is demonstrated by the following experimental examples where mouse keratinocytes are treated with L-ET in unencapsulated or encapsulated liposome. Then the mitochondria undergo the potentially damaging effects of UV-B light and aloxan (which is known to induce oxygen radicals) and the measured results. Damage to mitochondria was detected by two methods: 1) the MTT test and 2) the JC-1 test.

EXAMPLE 1 UV-B light: Mouse keratinocytes were pretreated with different concentrations of L-ergothioneine (not encapsulated) and then exposed to ultraviolet B radiation (UV-B), the shortest wavelength scale of UV light present in sunlight and which is responsible for important photodamage on the skin. The light was generated by a FS20 solar lamp filtered with 2 Kodicell sheets to eliminate light having a wavelength less than 280 nm. MTT test: This test measures the specific activity of mitochondria to cut the tetrazolium ring of the soluble MTT dye [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] to form the form insoluble blue formazan. Living mitochondria metabolize MTT and form blue formazan; the dead mitochondria immediately stop forming blue formazán. In the MTT test, the mammalian cells receive previous treatment with L-ergothioneine and are then treated with the harmful agent of mitochondria, in this example UV-B. MTT is added and the blue dye formation is measured spectrophotometrically. Results: Table 1 provides the percentage optical density of the blue formazan present related to unexposed mitochondria (control) in cells for various levels of UV-B, expressed in joules per square meter, and concentrations of L-ergothioneine.

TABLE I These data show that in the absence of L-ET, the increased intensity of UV-B irradiation results in a decreasing number of living mitochondria in keratinocytes, as shown in the level of decreasing conversion responsive to MTT to formazan doses. Ultraviolet-irradiated keratinocytes are protected by L-ergothioneine: at 100 and 200 J / m2, the three levels of L-ET remained greater than 95% of viability of mitochondria; in the higher dose of UV-B, the L-ET continued to protect the mitochondria.

EXAMPLE 2 Aloxan: Mouse keratinocytes were previously treated with various concentrations of L-ergothioneine (not encapsulated) and then exposed to aloxan at various concentrations. MTT test: The effect of alloxan on pretreated mitochondria was determined by the MTT test as in example 1. Results: Table 2 provides the percentage optical density of the blue formazan present related to the unexposed mitochondria (control) in cells for several concentrations of aloxan and concentrations of L-ergothioneine. TABLE 2 Aloxan induces oxidative damage to the mitochondria; a dose-responsive reduction in the viability of mitochondria can be seen up to the level of 5 mM, where after the damage has formed a plateau. In the portion responsive to the dose of the curve, L-ergothioneine gave protection from oxidative damage. EXAMPLE 3 The mouse keratinocytes were pretreated with L-ergothioneine, both unencapsulated and encapsulated in liposomes (prepared as described below), at a final concentration of 12.5 μM. A control group was not treated with L-ergothioneine. Mouse keratinocytes were then exposed to UV-B as in Example 1. Liposome encapsulation: L-ergothioneine was encapsulated in liposomes composed of phosphatidylcholine, phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate in a ratio of 2: 2: 1 :5. To calculate the concentration of trapped L-ergothionein, the liposomes are extracted with chloroform, and the OD258 is measured in the aqueous layer. The concentration of L-ergothioneine is calculated using e25s of L-ergothioneine of 14,500. The final concentration of L-ET in the purified liposome was about 1.1 mM. This concentration was reduced by diluting the liposomes by cell culture media to a final concentration of 12.5 μM in the media. Unencapsulated L-ergothioneine was adjusted to the same concentration by dilution.

The effect of UV-B on the pretreated and untreated mitochondria was determined by the MTT test as in example 1. Results: Table 3 provides the percentage optical density of the blue formazan present in each case related to unexposed mitochondria (control) in cells for various levels of UV-B.

TABLE 3 In the absence of L-ergothioneine, the keratinocytes showed significant damage to the mitochondria in both doses of UV-B. L-ET not encapsulated would provide important but not complete protection under these conditions, however complete protection was achieved by the encapsulated formulation of L-ET, at the same concentration. Therefore, the liposome formulation of L-ET provides superior protection.

EXAMPLE 4 Aloxan: Mouse keratinocytes were pretreated with L-ergothioneine (not encapsulated) at 1 mM and then treated with 8 mM aloxan One day after treatment with L-ergothioneine, the cells were treated for 10 minutes with JC-1 and the cells were examined through a fluorescence microscope. Test JC-1: The JC-1 test makes use of the fluorescent dye JC-1 iodide of (d.d '.?.?' - tetrachloro-l .l '^. S'-tetramethyl-benzimidazoylcarbocyanine) (Molecular Probes, Inc., Eugene, OR). This dye immediately and specifically is intercalated in the membranes of the mitochondria. In charged live membranes the dye JC-1 is maintained in the membrane as a monomer, and fluoresces green. When the membrane of the mitochondria is damaged, aggregation of the JC-1 dye occurs in J-aggregates and the fluorescence changes to orange. The orange coloration is characteristic of the mitochondria membrane damage. Results: The untreated control cells were predominantly green. Cells treated with aloxan alone showed significant patches of orange. The cells treated with aloxan and L-ergothioneine showed much less orange than the cells treated with simple aloxan. The cells treated with L-ergothioneine showed green fluorescence. Therefore, using different means to determine the viability of the mitochondria, the protection obtained by the mitochondria is confirmed by L-ET. This invention may have other modalities or be carried out in other ways without departing from the spirit or essential characteristics of the invention. same The present description should be considered illustrative and not restrictive, the scope of the invention is indicated by the appended claims, and it is intended that all changes that occur within the meaning and scale of equivalence are covered therein. The different citations of the present literature are incorporated herein by reference.

Claims (33)

NOVELTY OF THE INVENTION CLAIMS

1. - The use of a composition containing L-ergothioneine in combination with a pharmaceutically acceptable vehicle for the preparation of a medicament that protects the mitochondria from damage caused by radiation, radicals and reactive oxygen species.

2. The use according to claim 1, wherein said composition comprises L-ergothioneine at a concentration in the range of 50 μm to 5 mM.

3. The use according to claim 1, wherein the L-ergothioneine is at a concentration of about 1 mM.

4. The use according to claim 1, wherein said composition is dispersed in a liposome.

5. The use according to claim 4, wherein said liposome is prepared from a composition comprising phosphatidylcholine, phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate in a ratio of about 2: 2: 1: 5.

6. The use according to claim 5, wherein the L-ergothioneine is present at a concentration in the scale of 1 μM to 10 mM.

7. The use according to claim 6, wherein L-ergothioneine is present at a concentration of about 12 μM.

8. The use according to claim 1, wherein said medicament is made available to said mitochondria through the parenteral, topical, transmucosal, pulmonary or transdermal administration of said medicament.

9. The use according to claim 8, wherein said transdermal administration comprises topical application to a surface of the skin.

10. The use according to claim 9, wherein said vehicle is a hydrogel lotion.

11. The use according to claim 8, wherein said transmucosal administration is selected from the group consisting of oral, rectal and nasal administration.

12. The use according to claim 8, wherein said parenteral administration is selected from the group consisting of intravenous, subcutaneous, intraarterial, intramuscular, intraperitoneal, intrathecal, intracranial and intraventricular administration.

13. The use according to claim 1, wherein said damage is the result of exposure to toxins in the air selected from the group consisting of tobacco combustion products, contaminants industrial, petroleum combustion products, ozone, nitric oxide, radioactive particles and combinations thereof.

14. The use according to claim 1, wherein said damage is the result of exposure to the group consisting of ultraviolet radiation, solar radiation, tanning radiation, thermal radiation, solar erythema radiation, gamma radiation, radiation of microwave, electromagnetic radiation, nuclear radiation and combinations thereof.

15. The use according to claim 1, wherein said damage is pathologically causative in a disease or condition selected from the group consisting of cataracts, macular degeneration, degenerative damage to the retina, lung cancer, skin cancer, melanoma, solar erythema, radiation poisoning, asbestosis, atherosclerosis, Parkinson's disease, Alzheimer's disease, muscular dystrophy, multiple sclerosis, burns, emphysema, bronchopulmonary dysphasia, iron excess diseases, hemochromatosis, thalassemia, pancreatitis, diabetes, nephrotic syndrome autoimmune, nephrotoxicity induced by heavy metals, and radiation damage.

16. The use according to claim 1, wherein said damage is induced by the exposure or consumption of nuclear waste, radioactive fallout, industrial chemicals or ethanol.

17. The use according to claim 1, wherein said damage is caused by a factor selected from the group consisting of reactive oxygen species, radicals, free radicals, oxidant stress, oxidant damage and combinations thereof.

18. The use of a composition comprising L-ergothioneine in combination with a pharmaceutically acceptable vehicle for the preparation of a medicament that protects the mitochondria from damage caused by the therapeutic administration of pharmaceutical agents or radiation.

19. The use according to claim 18, wherein said therapeutic agent is selected from the group consisting of anti-cancer agents, cancer radiation therapy, fibrinolytic therapy and combinations thereof.

20. The use according to claim 18, wherein said composition is arranged in a liposome.

21. A composition that provides protection to mitochondria against radiation, radicals and harmful reactive oxygen species, comprising L-ergothioneine in a pharmaceutically acceptable vehicle.

22. The composition according to claim 21, further characterized in that said vehicle is a hydrogel lotion.

23. The composition according to claim 21, further characterized in that L-ergothioneine is present at a final concentration in the range of 50 μM to 5 mM.

24. The composition according to claim 22, further characterized in that L-ergothioneine is present at a final concentration of about 1 mM.

25. - The composition according to claim 21, further characterized in that said composition is arranged in a liposome.

26. A composition that provides protection to mitochondria against radiation, radicals and harmful reactive oxygen species, which comprises L-ergothioneine at least partially encapsulated in a liposome.

27. The composition according to claim 26, further characterized in that said liposome is prepared from a liposome-forming composition comprising phosphatidylcholine, phosphatidylethanolamine, oleic acid and cholesteryl hemisuccinate in a ratio of about 2: 2: 1. :5.

28. The composition according to claim 26, further characterized in that said L-ergothioneine is present at a concentration in the scale of 1 μM to 5 mM.

29. The composition according to claim 28, further characterized in that said L-ergothioneine is present at a concentration of about 12 μM.

30. A method for determining the level of damage that radiation, radicals or reactive oxygen species cause to a mammal, comprising the sequential steps of: a) isolating a cellular sample from said mammal; b) dividing said cellular sample into a series of identical aliquots; c) expose each of said aliquots to a series of concentrations of L-ergothioneine in a pharmaceutically acceptable vehicle; d) determine the degree of damage to the mitochondria in said aliquots and e) use a predetermined correlation between the L-ergothioneine level and the degree of inhibition of various degrees of damage to the mitochondria in a cellular sample of a mammalian test exposed to radiation , radicals or reactive oxygen species, deriving the degree of damage to said mammal.

31.- A method for determining the susceptibility of a mammal to radiation, radicals or reactive oxygen species, comprising the sequential steps of: a) isolating a cellular sample from said mammal; b) exposing said cell sample to radiation, radicals or reactive oxygen species; c) dividing said cellular sample into a series of identical aliquots; d) exposing each of the aliquots to a series of compositions comprising various concentrations of L-ergothionein in a pharmaceutically acceptable carrier; e) determine the degree of damage to the mitochondria in said aliquots and identify the concentration or concentrations of L-ergothioneine that inhibit said damage to the mitochondria; f) comparing said concentrations with a predetermined ratio between the concentration of L-ergothioneine and the degree of protection against damage in the mitochondria of mammalian cells pre-exposed to radiation, radicals or reactive oxygen species, and in this way derive the susceptibility of said mammal to said damage.

32. A method for determining the degree of protection against damage by radiation, radicals or reactive oxygen species in a mammal provided by L-ergothionein, comprising the sequential steps of: a) isolating a cellular sample from said mammal; b) dividing said cellular sample into a series of aliquots; c) exposing each of said aliquots to one of the series of compositions comprising various concentrations of L-ergothionein in a pharmaceutically acceptable carrier; d) exposing said aliquots to radiation, radicals or reactive oxygen species; e) determine the degree of damage to the mitochondria in said aliquots and identify the concentration or concentrations of L-ergothioneine that inhibit said damage to the mitochondria; f) compare the level of such damage with the concentrations of L-ergothioneine at a predetermined ratio between the concentration of L-ergothioneine and the degree of protection against damage in the mitochondria of mammalian cells pre-exposed to radiation, radicals or oxygen species reactive, and in this way derive the susceptibility of said mammal to said damage.

33. The method according to claim 32, used to determine an effective dose of L-ergothioneine for the treatment of a mammal exposed to radiation, radicals or reactive oxygen species.