Title:  Methods and compositions for the protection of mitochondria

United States Patent:  6,479,533

Issued:  November 12, 2002

Inventors:  Yarosh; Daniel B. (Merrick, NY)

Assignee:  Applied Genetics Incorporated Dermatics (Freeport, NY)

Appl. No.:  452585

Filed:  December 1, 1999

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 cosmetics and pharmaceuticals, radiation therapy, among others, is provided by a composition comprising L-ergothioneine, L-ergothioneine may be prepared in a cosmetically or pharmaceutically-acceptable base to form an agent for topical application to the skin, and for oral or parenteral administration. Effective application and delivery, of L-ergothioneine is enhanced by encapsulation in a liposomes, a preferred embodiment. Diagnostic methods for determining exposure and susceptibility to radiation, radical and reactive oxygen species in mammals is also provided.

DETAILED DESCRIPTION OF THE INVENTION

Inhibition of oxidative damage to mitochondria in various tissues of the mammalian body is of therapeutic benefit for the prophylaxis and treatment of many pathological conditions ranging from those responsible for significant morbidity and mortality, such atherosclerosis and cancer, to those of a less pathological but significant adverse psychological component, such as unsightly changes to the skin as a result of long-term photoaging. In diverse diseases such as cancer, diabetes, atherosclerosis, cataract, and certain neurological diseases, among others, ROS are implicated in the pathophysiology of the disease. Cancer chemotherapeutic agents such as adriamycin and bleomycin induce oxidant damage, as does anti-cancer radiation (e.g., X-ray) therapy. Therapies that use light combined with endogenous and exogenous chromophores and photosensitizers that generate ROS are also used to produce cosmetic and therapeutic results by damaging mitochondria. As critical subcellular organelles involved in aerobic energy metabolism and the oxidative reactions therein, mitochondria are sensitive to endogenous and exogenous influences and may be easily damaged or destroyed. Dysfunctional energy metabolism and, more severely, damaged mitochondria, may lead to cell senescence, apoptosis and death, and downstream tissue and organ dysfunction and damage. In the skin, increased oxidative damage as a consequence of UV light exposure can damage the cellular structure of the skin leading to premature, psychologically-debilitating changes related to aging, such as thinning of the skin, wrinkling, and abnormal pigmentation. Exposure of environmental oxidants to the lungs can induce mitochondrial and attendant cellular damage leading to chronic airway obstructive disorders.

In accordance with the present invention, protection is afforded to mitochondria by the application or administration of a composition comprising L-ergothioneine. Administration to the target cells, tissue, or organ may be parenterally; transmucosally, e.g., orally, nasally, rectally; or transdermally or intradermally. Parenteral administration is via intravenous injection, and also including, but is not limited to, intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, intrathecal and intracranial administration. It may be prepared in a tablet or capsule formulation for oral administration. For topical delivery, a solution of L-ergothioneine in water, buffered aqueous solution or other cosmetically or pharmaceutically acceptable carrier, or in a hydrogel lotion or cream, comprising an emulsion of au aqueous and hydrophobic phase. at a concentration of between 10 .mu.M and 5 mM, is used. In light of the current manufacturing costs and constraints of L-ergothioneine, a preferred concentration is about 20 .mu.M. To this may be added ascorbic acid or its salts, or other ingredients, or a combination of these, to make a cosmetically-acceptable formulation. Metals should be kept to a minimum. It may be preferably formulated by encapsulation into a liposome for oral, parenteral, or, preferably, topical administration. As will be seen below, a composition of L-ergothioneine within a liposome improves the efficacy of protection of mitochondria from oxidative damage resulting from radiation damage.

It was found unexpectedly that the use of a liposome formulation for L-ergothioneine enhances the effectiveness of the compound for the protection of mitochondria. While liposome delivery has been utilized as a pharmaceutical delivery system for many other compounds for a variety of applications [see Langer, Science, 1990, 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss: New York, pp- 353-365 (1989); Lopez-Berenstein, ibid., pp. 317-327; see generally ibid.], subcellular delivery of L-ergothioneine in an efficacious form was discovered by the inventor herein and is a preferred embodiment of the compositions and methods of the present invention. The function of the liposome is to increase the delivery of the L-ergothioneine to the mitochondria, and distinctly or additionally, to protect the L-ergothioneine until it reaches the target cell or tissue. A non-limiting example of a liposome formulation is that formed from phosphatidyl choline, phosphatidyl ethanolamine, oleic acid and cholesteryl hemisuccinate in a ratio of 2:2:1:5, encapsulating 10 mM L-ergothioneine. A final concentration of 1 .mu.M to 10 .mu.M L-ergothioneine is used, preferably about 12 .mu.M. This final concentration can be achieved by dilution of the purified liposomes in a cosmetically or pharmaceutically-acceptable base. Many other suitable liposome formulations are known to the skilled artisan, and may be employed for the purposes of the present invention. For example, see: U.S. Pat. No. 5, 190,762; "Method of Administering Proteins to Living Skin Cells" to Yarosh which is incorporated herein by reference. A general discussion of liposomes and liposome technology can be found in a three volume work entitled "Liposome Technology" edited by G. Gregoriadis, 1993, published by CRC Press, Boca Raton, Fla. The pertinent portions of this reference are incorporated herein by reference.

Transdermal delivery of L-ergothioneine, either as a liposome formulation or free L-ergothioneine, is also contemplated. Various and numerous methods are known in the art for transdermal administration of a drug, e.g., via a transdermal patch. It can be readily appreciated that a Transdermal route of administration may be enhanced by use of a dermal penetration enhancer.

In yet another aspect of the present invention, provided is cosmetic and pharmaceutical compositions of L-ergothioneine. Such compositions may be for administration for injection, or for oral, pulmonary, nasal, topical, intradermal or transdermal or other forms of administration. In general, comprehended by the invention are cosmetic and pharmaceutical compositions comprising effective amounts of L-ergothioneine together with cosmetic or pharmaceutically acceptable diluents, preservatives, solubilizers,. emulsifiers, adjuvants and/or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; additives such as detergents and solubilizing agents (e.g. Tween 80, Polysorbate 80), moisturizing agents (e.g. ceramides, alpha-hydroxy acids) anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., phenoxyethanol, phenonip, thimerosal, benzyl alcohol) and bulking substances (e.g., lactose. manitol); incorporation of the material into particulate preparations of polymeric compounds such as polylactic acid, polyglycolic acid, etc. or into liposomes (infra). Hylauronic acid may also be used. Such compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of L-ergothioneine. The compositions may be prepared in liquid or lotion form, or may be in dried powder, such as lyophilized form, or may be prepared as an aerosol.

Controlled release oral formulation may be desirable. The drug may be incorporated into an inert matrix which permits release by either diffusion or leaching mechanisms, e.g., gums. Slowly regenerating matrices may also be incorporated into the formulation. Some enteric coatings also have a delayed release effect. Another form of a controlled release of this composition is by a method based on the Ores therapeutic system (Alza Corp), i.e. the drug is enclosed in a semipermeable membrane which allows water to enter and push drug out through a single small opening due to osmotic effects.

Also contemplated herein is pulmonary delivery of the pharmaceutical compositions of the present invention, for the treatment or promotion of mitochondria from oxidative damage. Pulmonary delivery may be used to treat the lung tissue itself, or serve as a delivery route to the blood stream and thus other locations within the body. A pharmaceutical composition of the present invention is delivered to the lungs of an animal while inhaling and traverses across the lung epithelial lining to the blood stream. Contemplated for use in the practice of this invention are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, measured dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art. With regard to construction of the delivery device, any form of aerosolization known in the art, including but not limited to spray bottles, nebulization, aromization or pump aerosolization of a liquid formulation, and aerosolization of a dry powder formulation, can be used in the practice of the 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, in which oxidative damage is believed to account for a high incidence of cataracts. Other ophthalmic uses include treatment or prophylaxis of macular degeneration and degenerative retinal damage.

Nasal delivery of a pharmaceutical composition of the present invention is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present invention to the blood stream directly after, administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran. For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present invention into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present invention. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

In a further aspect, the L-ergothioneine liposomes can cross the blood-brain barrier, which would allow for intravenous or oral administration. Many strategies are available for crossing the blood-brain barrier, including but not limited to, increasing the hydrophobic nature of a molecule; introducing the molecule as a conjugate to a carrier, such as transferring, targeted to a receptor in the blood-brain barrier; and the like. In another embodiment, the molecule can be administered intracranally or, more preferably, intraventricularly. In yet another embodiment, L-ergothioneine can be administered in a liposome targeted to the blood-brain barrier. These methods of delivery are particularly important since many debilitating brain diseases, such as Parkinson's, Alzheimer's, amyotrophic lateral sclerosis and possibly schizophrenia, have been linked to elevated ROS in the brain through a deficit in the mitochondrial enzyme complex 1.

A subject in whom administration of L-ergothioneine is an effective therapeutic regiment for mitochondrial protection is preferably a human, but can be any animal. Thus, as can be readily appreciated by one of ordinary skill in the art, the methods and pharmaceutical compositions of the present invention are particularly suited to administration to any animal, particularly a mammal, and including, but by no means limited to, domestic animals, such as feline or canine subjects, farm animals. such as but not limited to bovine, equine, caprine, ovine, and porcine subjects, wild animals (whether in the wild or in a zoological garden), research animals, such as mice, rats, rabbits, goals, sheep, pigs, dogs, cars, etc., avian species, such as chickens, turkeys, songbirds, etc., i.e., for veterinary medical use.

The protection of mitochondria a from oxidative damage may be used for the prevention and treatment of a number of disorders, including effects of radiation to the body, disease processes, exposure to pollutants including tobacco combustion products, and protection against the damaging effects of certain pharmaceuticals whose mechanisms of action involve generation of ROS and other radicals. For example, certain anti-neoplastic agents induce oxidative radicals as their mechanism of action, but a significant and limiting side effect in patients is cardiotoxicity; higher doses and thus increased anti-cancer efficacy is achievable by protecting the mitochondria of the heart and other tissues with the compositions and methods of the present invention. In addition, various types of radiation used for anti-cancer therapy, as an alternative or adjunct to surgery, induces significant damage to tissues; prior administration of L-ergothioneine may be used to reduce or prevent the toxicity of radiation therapy to the body.

Furthermore, numerous disease processes involve ROS. In the eye, cataract, macular degeneration and degenerative retinal damage are attributed to ROS and may be treated with topical, oral or parentally-administered L-ergothioneine. A liposome formulation is preferred. ROS-related diseases of the lungs such as emphysema and bronchopulmonary dysphasia and including pathology induced by inhalation of tobacco combustion products and asbestos may be treated by an aerosolized form of L-ergothioneine as described above. Various diseases of the nervous system such as Parkinson's disease, Alzheimer's disease, muscular dystrophy, and multiple sclerosis may be treatable by oral or parenteral formulations or direct delivery to the central nervous system via intrathecal, intraventricular and intracranial administration. Iron overload diseases such as hemochromatosis and thalassemia may also be treated by the compositions and methods of the present invention. Other diseases include pancreatitis; diabetes; renal diseases including autoimmune nephrotic syndrome and heavy metal-induced nephrotoxicity; and radiation injuries.

In addition, destruction of mitochondria in non-target cells may be an unwanted side effect of cosmetic and sterilization treatment using light with endogenous or exogenous photosensitizers to generate ROS that destroy target tissue. For example, lasers for hair removal rely on generation of ROS in melanin-containing hair follicles. This may be augmented by delivery of a photosensitizing dye to the hair follicle. An unwanted side effect is the destruction of mitochondria in skin exposed to the light. Similarly, light is used in conjunction with photosensitizing dyes to inactivate viruses that contaminate blood products. An unwanted side effect is the destruction of mitochondria in the targeted blood cells.

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 mitochondrial damage and the level of L-ergothioneine necessary to afford protection may be assessed in vitro, exposing aliquots of a cellular sample from said mammal to the damaging agent or condition, said aliquots containing various concentrations of L-ergothioneine. The damage to mitochondria of the various aliquots is determined, as well as the lowest concentration, if any, of L-ergothioneine providing sufficient protection from damage. To determine the degree of therapeutic benefit of L-ergothioneine to a mammal after exposure to a mitochondrial damaging agent, a similar diagnostic test as described above may be employed, with a variation in that the various concentrations of L-ergothioneine, are applied to the cellular sample aliquots after exposure to the mitochondrial damaging agent. In another embodiment the extent of exposure of a mammal to ROS may be assessed by determining the effect of L-ergothioneine on a sample of cells taken from the mammal. These diagnostic utilities further offer assistance in selecting an effective therapeutic dose of L-ergothioneine.

In a further embodiment, the ability of L-ergothioneine to protect a cellular sample from the damaging effects of a therapeutic regimen that causes oxidative damage, such as an anti-neoplastic agent or radiation therapy to be administered to a mammal with cancer, can be performed in vitro by combining the anti-neoplastic agent with various concentrations of L-ergothioneine, applying the combination to identical aliquots of a cellular sample from a mammal, and determining the extent of mitochondrial damage in said series of samples. These data may be used to determine an effective dose of L-ergothioneine to prevent mitochondrial damage in the non-diseased cells of said mammal. In a parallel manner using a sample of diseased or cancerous cells from said mammal, it may be determined whether L-ergothioneine will effect any diminution of the anti-cancer activity of said anti-cancer agent; based on these two tests, a level of L-ergothioneine for co-administration with the anti-cancer agent may be selected to provide optimal protection of the non-diseased cells of the mammal from the anti-cancer agent while providing maximum anti-cancer therapy. These are non-limiting examples of useful diagnostic tests assessing the prophylactic and therapeutic benefits of the compositions and methods of the present invention. They may be used, for example to determine the optimal concentration of L-ergothioneine to be used in conjunction with PDT or laser treatments.

Claim 1 of 5 Claims

I claim:

1. A method to selectively inactivate prokaryotic organisms intermingled with eukaryotic cells by treating the mixture with an effective concentration of L-ergothioneine to protect the mitochondria of the eukaryotic organisms and then exposing the mixture to radiation, radicals, or reactive oxygen species sufficient to inactivate the prokaryotic organisms.
 

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