KR20070030815A - Use of mitochondrially targeted antioxidant in the treatment of liver diseases and epithelial cancers - Google Patents

Abstract

The present invention relates to the use of mitochondrially targeted antioxidants such as derivatives of vitamin E, coenzyme Q ₁₀ or glutamine peroxidase mimetic in the treatment and prevention of liver disease and / or epithelial cancer. The invention also relates to a pharmaceutical composition containing an antioxidant for use for purpose. The invention also relates to the manufacture of a medicament containing an antioxidant useful for such prophylaxis and treatment.

Mitochondria, antioxidants, vitamin E, coenzyme Q10, glutathione peroxidase, liver disease, epithelial cancer

Description

Use of mitochondrially targeted antioxidant in the treatment of liver diseases and epithelial cancers}

The present invention relates to the use of mitochondrially targeted antioxidants, such as derivatives of vitamin E, coenzyme Q ₁₀ or glutathione peroxidase mimetic, in the treatment and prevention of liver disease and / or epithelial cancer.

The categories of liver disease range from mild reversible fatty liver to progressive chronic liver disease and can lead to the occurrence of life-threatening conditions such as liver cirrhosis, liver failure and liver cancer.

The main causes of chronic liver disease are hepatitis B virus or hepatitis C virus, excessive alcohol intake and non-alcoholic fatty liver disease (NAFLD).

Hepatitis B virus (HBV) infection is a worldwide health problem. This is a major cause of cirrhosis and hepatocellular tumors (HCC) worldwide (Conjeevaram H.S. et. Al., 2003, Journal of Hepatology, 38: 90-103). Hepatitis C virus (HCV), the leading cause of liver-related morbidity and mortality worldwide, represents one of the major health and health problems (Alberti A. and Benvegnu L., Journal of Hepatology 2003, 38: 104-118). HCV infection often results in chronic hepatitis, which is associated with cirrhosis and the development of HCC (Cyong J. C. et al., 2002, Am J Chin Med, 28: 351-360).

Alcoholic liver disease (ALD) is the most common cause of cirrhosis in the West and is currently one of the ten most common causes of death. In the United States, ALD infects more than about 2 million people or nearly 1% of the population. The incidence of milder forms of ALD may be substantially greater because many patients do not seek any medical attention because they do not feel symptoms. The range of ALD ranges from fatty liver (fatosis), which is common in non-traditional drinkers, to fatty liver disease, bile tablets (characterized by bile secretion from the liver), fibrosis and ultimate cirrhosis (Stewart SF and Day CP, 2003, Journal of Hepatology) , 38: 2-13). Fatty liver can return to its original state by resection, but is a risk factor for progression to fibrosis and cirrhosis in patients who are constantly drinking, especially those with fatty liver disease.

Non-alcoholic fatty liver disease (NAFLD) refers to a wide variety of liver damage, ranging from simple fattyosis to fatty liver disease, cholestasis, antifungal fibrosis and cirrhosis. Fatty liver disease (non-alcoholic fatty liver disease) is expressed only in stages within the range of NAFLD (Anguilo P., 2002, N Engl. J. Med., 346: 1221-1231). Pathological characteristics are similar to those of alcohol-induced liver injury, but this occurs only in patients who do not abuse alcohol. NAFLD should be distinguished from hepatitis due to secondary causes or from steatosis without hepatitis, since these conditions clearly have different pathogens and outcomes. Secondary causes of fatty liver disease (fatosis) include nutrition (eg protein-calorie malnutrition, starvation, total parenteral nutrition, rapid weight loss, gastrointestinal surgery due to obesity), drugs (eg glucocorticoids, synthetic estrogens, aspirin, calcium-channels) Blockers, tetracyclines, valproic acid, cocaine, antiviral agents, pialuridine, interferon α, methotrexate, zidobudine), metabolic or genetic agents (such as dyslipidemia, beta-lipoprotein disorders, Weber-Christian disease, galactosemia, glycogen storage Disorders, gestational acute fatty liver) and other causes such as diabetes, obesity or hyperlipidemia (Anguilo P., 2002, N. Engl. J. Med., 346: 1221-1231; MacSween RNM et al., 2002, Pathology of the Liver.Fourth Edition.Churchill Livingstone, Elsevier Science).

Despite the prevalence of chronic liver disease, no effective therapy exists for most diseases of this class.

Many congenital and acquired liver diseases are associated with modifications of the hepatocyte intermediate filament (IF) cytoskeleton. One of the most common IF-related modifications is Mallory bodies (MB), which is used in some hepatocellular tumors (HCC) as well as alcoholic fatty liver disease and non-alcoholic (AH and NASH), chronic cholestasis, copper poisoning and other metabolic liver diseases. It is formed in hepatocytes. MB consists of several proteins (HSP27, HSP70, p62 and ubiquitin) involved in the unfolded protein reaction as well as the misfolded aggregated keratin as the main component. Misfolding of proteins typically occurs as a result of protein modification in the context of cellular stress, particularly oxidative stress. The chemical composition of MB indicates that keratin is a preferred target for misfolding in stress situations and that MB can be considered as a result of a cellular defense response to misfolded keratin (Denk et al., 2000, J. Hepatol. , 32: 689-702).

The most severe non-viral chronic liver disease, alcoholic fatty liver disease, and non-alcoholic fatty liver disease (ASH and NASH) cause high frequency cirrhosis, liver failure and liver cancer (such as HCC). ASH and NASH are indistinguishable by morphology evaluation in diagnostic pathology laboratories. However, increased fat placement due to fibrosis, inflammation and hepatocellular morphology presents a more serious condition. Cellular changes in ASH and NASH include increased size (expansion) and the presence of intracellular aggregates (such as MB), and it is believed that this range of hepatocellular pathology is capable of diagnosing these conditions.

Overall, definitive diagnosis via biopsy is unlikely to affect the treatment of this disease in patients as there are no special treatments for ASH and NASH.

Liver cancer is relatively common in the industrialized West, but is a major cause of cancer worldwide. Unlike many other types of cancer, the number of people who die from liver cancer is increasing.

Worldwide, primary liver cancer, such as HCC, is one of the most common malignant cancers and kills about one million people each year (Bruix, J. et al., 2004, Cancer Cell (5): 215-219). .

The major risk factors for liver cancer are viruses, alcohol intake, food affected by aflatoxin fungi and metabolic disorders. Alcoholism and chronic hepatitis B and C rates are on the rise. Thus, the rate of liver cancer is also expected to increase gradually, highlighting the need for new therapies in this area.

Primary liver cancer is difficult to treat. Surgical removal and liver transplantation of cancer is limited to small cancers and is not an applicable option for most patients because cancer is usually an advanced stage at diagnosis. Tumors are sometimes used for tumors that are not suitable for surgery, but no benefit lasts long. Therefore, the survival rate of primary liver cancer is extremely low. Conventional therapies have generally been found to be ineffective in treating liver cancer.

For example, in the case of HCC, there are no effective therapies except for resection and transplantation, but these therapies are only applicable to early stage HCC and are limited by the availability between donations and are associated with serious risk to the patient. These therapies are also very expensive. These cancers have extremely poor reactions with chemotherapeutic agents, most likely due to normal liver action to transport detoxification and harmful compounds. Several other therapies such as chemoembolization, cryotherapy and ethanol infusion are on the test and their efficacy is not yet established.

Thus, satisfactory therapies for treating liver disease and other epithelial cancers have not been developed until now.

It is already known that various antioxidants can target mitochondria by covalently binding to lipophilic cations through alkylene chains (Smith RAJ et al., 1999, Eur J. Biochem., 263: 709-716). And Kelso GF et al., 2001, J. Biol. Chem., 276: 4588-4596; James AM et al., 2005, J. Biol. Chem., 280: 21295-21312. This method directs the antioxidant to the primary production site of free radicals and reactive oxygen species in the cell rather than dispersing inconsistently.

In particular, vitamin E and coenzyme Q ₁₀ derivatives (US Pat. Nos. 6,331,532; WO 99/26954, WO2005 / 016322 and WO2005 / 016323) or glutathione peroxidase analogs (WO 2004/014927) are bound to triphenyl phosphonium ions. Targeting to mitochondria has been disclosed. In vitro experiments showed [2- (3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) ethyl] -triphenylphosphonium Bromide (Mito Vit E) and mitoquinol [10- (3,6-dihydroxy-4,6-dimethoxy-2-methylphenyl) decyl] triphenylphosphonium bromide and mitoquinone [10- (4,5- Mixture of dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl) decyl] tridecylphosphonium bromide (MitoQ) (Kelso GF et al., See citation and Smith RAJ et al., See citation) or MitoQ compounds (an James AM et al., 2005, J. Biol. Chem., 280: 21295-21312; WO 2005/016322 and WO2005 / 016323) wherein the anion is methanesulfonate It rapidly and selectively accumulates by mitochondria in isolated cells.

Mitochondrial-targeted derivatives of spin traps of phenyl-t-butylnitron (MitoPBN) have also been developed (Smith R.A.J., Bioenergetics Group Colloquim, 2003, 679th Meeting of the Biochemical Society: 1295-1299).

Importantly, the accumulation of these antioxidants by the mitochondria protected themselves from oxidative damage more effectively than untargeted antioxidants, while the harmful side effects of biomolecule accumulation in the mitochondria increased their efficacy. Indicates reduction (Murphy MP and Smith RAJ, 2000, Adv. Drug. Delivery Rev., 41: 235-250).

It has also been found that simple alkyltriphenylphosphonium cations TPMP, Mito Vit E and MitoQ can be safely supplied to mice over long periods of time to produce therapeutically effective concentrations in the brain, heart, liver and muscle (Smith RA et al. , 2003, PNAS, 100 (9): 5407-5412).

Industrial applications of these compounds (US Pat. Nos. 6,331,532, WO 99/26954 or WO 2004/014927, WO2005 / 016322 and WO 2005/016323) have shown Parkinson's disease, Friedrich's dyskinesia, Wilson's disease, diseases associated with mitochondrial DNA mutations, diabetes Can be used to prevent increased mitochondrial oxidative stress associated with neurodegenerative diseases such as motor neuron disease, stroke, heart attack, inflammatory and ischemic reperfusion tissue damage, and non-specific energetic damage associated with aging in organ transplantation and surgery It is claimed to exist. It is also recommended that these compounds be used as a preventive agent to protect organs during transplantation, to mitigate ischemic reperfusion injury during surgery, to reduce cellular damage following stroke and heart attack or as a preventive agent administered to premature infants with cerebral ischemia. It has been claimed in the patent document mentioned.

Interest in the potential value of antioxidant therapies in the treatment of alcoholic hepatitis (AH) has been augmented by the growing evidence that oxidative stress is a major mechanism in alcohol-mediated hepatotoxicity (Stewart SF and Day CP, 2003, Journal of Hepatology, 38: 2-13). These considerations have recently led to several trials investigating the effects of antioxidant supplementation in patients with severe AH (eg Philips M. et al., 2001, Journal of Hepatology, 34: 250A). In most recent studies (Stewart SF et al., 2002, Journal of Hepatolology, 36:16), the active group took N-acetylcysteine at a dose of 150 mg / kg over a week followed by 100 mg / kg / day Vitamin AE, biotin, selenium, zinc, manganese, copper, magnesium, folic acid and coenzyme Q were taken daily for 6 months. Such antioxidant therapy shows no benefit either alone or in combination with steroids. In sum, based on the data available to date, high doses of antioxidant therapy do not confer survival benefits in patients with severe AH (see Stewart S.F. and Day C.P., citations).

Oxidative stress is known to be involved in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). In studies using rats fed the choline deficiency diet, vitamin E, known to react with reactive oxygen species (ROS) by blocking the proliferation of radical reactions in a wide range of oxidative situations, prevents the development of fatty liver Neither did it reduce oxidative stress (Oliveira CP et al., 2003, Nutr. J., 2 (1): 9).

In studies of patients with cirrhosis and patients infected with hepatitis C virus (HCV) treated with alpha-tocopherol (VitE group), there was no improvement in liver function or inhibition of liver tumorigenesis and no improvement in cumulative survival (Tagaki). H. et al., 2003, Int. J. Vitam Nutr. Res., 73 (6): 411-5).

Any multicenter of 120 patients with chronic hepatitis C confirmed biopsies that did not respond to the previous alpha-interferon pathway, oral administration of N-acetyl cysteine (1200 mg / day) and vitamin E (600 mg / day) The study did not improve the poor efficacy of retreatment with alpha-interferon alone (Ideo, G., et al., 1999, Eur. J. Gastroenterol. Hepatol., 11 (11): 1203-7).

Summary of the Invention

The present invention relates to the use of mitochondrially targeted antioxidant compounds comprising a lipophilic cation covalently bound to an antioxidant moiety for the treatment or prevention of liver disease and / or epithelial cancer.

details

Surprisingly, the use of mitochondrially targeted antioxidants such as vitamin E derivatives, coenzyme Q ₁₀ or glutamine peroxide analogs have been found to be useful in the treatment and prevention of liver disease and / or epithelial cancer.

In its broadest aspect, the present invention provides mitochondrial treatment and prevention for liver disease and / or epithelial cancer, comprising lipophilic cations covalently bound to antioxidant residues that can be transported through the mitochondrial membrane and accumulate in complete mitochondrial cells. Provide targeted antioxidants. In particular, the compounds according to the invention prevent cell damage due to oxidative stress (or free radicals) in the mitochondria.

The term “liver disease” according to the invention affects the anatomy, physiology, metabolism and / or genetic activity of the liver and in the short term, temporarily, chronically or permanently, in whole or in part in a pathological manner of new liver cells. It refers to and includes all kinds of disorders affecting production and / or regeneration of the liver.

In particular alcohol-induced diseases (such as ASH), non-alcoholic fatty liver changes (such as NAFLD including NASH), nutrition-mediated liver damage (such as starvation), and other toxic liver damage (such as but not limited to acetaminophen (paracetamol) Chlorinated hydrocarbons (such as CCl ₄ ), amiodarone (cordarone), valproate, tetracycline (venous only), isoniaside (Drug-induced liver disease 2004. Lazerow SK, Abdi MS, Lewis JH.Curr Opin Gastroenterol., 2005, 21 (3): 283-292) or ingesting mushrooms containing aflatoxins (preferably B1 aflatoxins) or taking certain metals (such as copper or cadmium) or natural drugs (milk thistles, oak vines) Food poisoning, which can lead to acute or chronic liver disease, such as ingestion of herbal products used in homeopathic therapy, such as Kawa-Kawa; interference with bilirubin metabolism, hepatitis-like symptoms, cholestasis, and granulation Includes mediated liver injury (such as that caused by radiation therapy) - a non-specific hepatitis), trauma and surgery (e.g., Pringle operation), radiation induced by drugs, such as lesions, intrahepatic vascular lesions and curing.

Liver diseases are understood to include infectious liver diseases (such as caused by hepatitis B virus (HBV) and hepatitis C virus (HCV) infection) and autoimmune-mediated liver disease (such as autoimmune hepatitis). It also includes liver damage caused by sepsis.

Liver diseases are also understood to include genetic liver diseases (such as hemochromatosis and alpha antitrypsin deficiency) and other inherited metabolic liver diseases (eg fatty liver disease (MSH)).

Preferred examples of liver disease to be treated include alcoholic liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), steatosis, cholestasis, sclerosis, acute and chronic hepatitis, hemochromatosis and alpha 1 antitrypsin deficiency.

The term “liver disease” according to the invention in the sense of the present invention also encompasses tumors (primary liver neoplasia) and tumor-like lesions of the liver (such as focal nodular hyperproliferation, FNH).

Liver diseases are also understood to include benign liver neoplasms (such as hepatocellular adenomas) as well as liver neoplastic diseases such as liver cancer such as hepatocellular tumors (HCC). HCC also includes subtypes of the above-mentioned diseases, including hepatic cancer characterized by intracellular proteinaceous inclusion bodies, HCC characterized by hepatocellular steatosis, and fibrous lamellar HCC. For example, macroplastic regeneration (hyperproliferative) nodules as well as lesions characterized by increased hepatocyte cell size (large cell change) and lesions characterized by reduced hepatocyte cell size (small cell change) are included in precancerous lesions ( Anthony P. in MacSween et al., Eds. 2001, Pathology of the Liver, Churchill Livingstone, Edinburgh, UK).

The term "epithelial cancer" within the meaning of the present invention includes tumors of organs other than the liver, such as the lungs, kidneys, pancreas, prostate, skin and breast, and tumors of the gastrointestinal system such as the stomach, kidneys and colon. The term "epithelial cancer" according to the present invention refers to a disease of organs in which the epithelial cell component of the tissue is modified resulting in malignant tumors identified according to standard diagnostic procedures generally known to those skilled in the art.

Preferred embodiments include alcoholic liver disease, non-alcoholic fatty liver disease, steatosis, cholestasis, cirrhosis, nutrition-mediated liver injury, toxin liver injury, infectious liver disease, liver damage in sepsis, autoimmune-mediated liver disease, hemochromatosis, Mitochondria comprising lipophilic cations covalently bound to antioxidant residues in the treatment and prevention of liver diseases selected from the group consisting of alpha1 antitrypsin deficiency, radiation-mediated liver injury, liver cancer, benign liver neoplasia and focal nodular hyperplasia By using the targeted antioxidant compound.

Other embodiments include alcoholic liver disease, non-alcoholic fatty liver disease, steatosis, cholestasis, cirrhosis, nutrition-mediated liver injury, toxin liver injury, infectious liver disease, liver damage in sepsis, autoimmune-mediated liver disease, hemochromatosis, Indicates the use of mitochondrially targeted antioxidant compounds comprising lipophilic cations covalently bound to antioxidants in the treatment and prevention of liver diseases selected from the group consisting of alpha1 antitrypsin deficiency, radiation-mediated liver injury.

The present invention relates to the use of mitochondrially targeted antioxidant compounds comprising a lipophilic cation covalently bound to an antioxidant moiety in the manufacture of a medicament for treating or preventing liver disease and epithelial cancer.

Preferred embodiments include alcoholic liver disease, non-alcoholic fatty liver disease, steatosis, cholestasis, cirrhosis, nutrition-mediated liver injury, toxin liver injury, infectious liver disease, liver damage in sepsis, autoimmune-mediated liver disease, hemochromatosis, The use of mitochondrially targeted antioxidants according to the invention in the treatment and prevention of liver diseases selected from the group consisting of alpha1 antitrypsin deficiency, radiation-mediated liver injury, liver cancer, benign liver neoplasia and focal nodular hyperplasia Indicates.

Other preferred embodiments include alcoholic liver disease, non-alcoholic fatty liver disease, steatosis, cholestasis, liver cirrhosis, nutrition-mediated liver injury, toxin liver injury, infectious liver disease, liver damage in sepsis, autoimmune-mediated liver disease, hemochromatosis The use of mitochondrially targeted antioxidants according to the invention in the manufacture of a medicament for the treatment or prevention of a liver disease selected from the group consisting of alpha 1 antitrypsin deficiency, radiation-mediated liver injury, liver cancer.

Another preferred embodiment is the use of a mitochondrially targeted antioxidant according to the invention wherein the liver disease is an alcoholic liver disease or a non-alcoholic fatty liver disease.

A more preferred embodiment is the use of a mitochondrially targeted antioxidant according to the invention wherein the liver disease is an alcoholic fatty liver disease or a non-alcoholic fatty liver disease.

Another preferred embodiment is the use of a mitochondrially targeted antioxidant according to the invention wherein the liver disease is an alcoholic fatty liver disease.

Another preferred embodiment is the use of a mitochondrially targeted antioxidant according to the invention wherein the liver disease is a non-alcoholic fatty liver disease.

The term "disease according to the invention" within the meaning of the present invention includes liver disease and epithelial cancer as defined above.

Preferred embodiments indicate the use of mitochondrially targeted antioxidant compounds for the treatment or prevention of diseases according to the invention wherein the lipophilic cation is a triphenylphosphonium cation.

Other lipophilic cations that may be covalently bound to antioxidants in accordance with the present invention include tribenzyl or triphenyl ammonium cations or tribenzyl or substituted triphenyl phosphonium cations.

A compound wherein the mitochondrial enemy targeting in accordance with the invention In another preferred embodiment of the formula ^{_{P (Ph) 3 + XR ·}} Z - a ^- (the anion is again R is an antioxidant moiety wherein, X is a linking group, Z) And said lipophilic cation represents a triphenylphosphonium cation as represented by the formula:

As the linking group, X may be a carbon chain, one or more carbon rings, or a combination thereof and one or more carbon atoms are substituted by oxygen (ether or ester formation) and / or by nitrogen (amine or amide formation) It may be such a chain or a ring.

Carbon chains are generally alkylene groups, but carbon chains comprising one or more double or triple bonds are also within the scope of the present invention. Also included are carbon chains comprising one or more substituents (such as oxo, hydroxyl, carboxylic acid or carboxamide groups) and / or one or more side chains or branches selected from unsubstituted or substituted alkyl, alkenyl or alkynyl groups.

Preferably, X is C ₁ -C ₃₀ , more preferably C ₁ -C ₂₀ , most preferably C ₁ -C ₁₅ carbon chain. Preferably, X is (CH ₂ ) _n , where n is an integer from 1 to 20, more preferably from about 1 to about 15.

In some particularly preferred embodiments, the linking group X is an ethylene, propylene, butylene, pentylene or decylene group.

In one particularly preferred embodiment the antioxidant residue R is quinone. In another preferred embodiment the antioxidant R residue is quinol. Quinones and corresponding quinols are equivalents because they are converted to each other by reduction and oxidation, respectively.

In another embodiment the antioxidant residue R is a general radical destroyer including vitamin E and vitamin E derivatives, chain breakdown antioxidants, butylated hydroxyanisole, butylated hydroxytoluene, induced fullerenes, 5,5- Spin traps comprising derivatives of dimethylpyrroline N-oxide, t-butylnitrosobenzene, α-phenyl-tbutylnitron and related compounds.

In another preferred embodiment, the antioxidant residue R is a vitamin E or vitamin E derivative.

In another preferred embodiment the antioxidant residue R is butylated hydroxyanisole or butylated hydroxytoluene.

In a more preferred embodiment the antioxidant residue R is derivatized fullerene.

In some particularly preferred embodiments the antioxidant residue R is 5,5-dimethylpyrroline N-oxide, t-butylnitrosobenzene, α-phenyl-tbutylnitron and derivatives thereof.

Preferably, Z ⁻ is a pharmaceutically acceptable anion. Such pharmaceutically acceptable anions are formed from organic or inorganic acids. Suitable inorganic acids are, for example, hydrochloric acid, halogen acids such as hydrobromic acid, sulfuric acid or phosphoric acid. Suitable organic acids are for example carboxylic acids, phosphonic acids, sulfonic acids or sulfamic acids such as acetic acid, propionic acid, octanoic acid, decanoic acid, dodecanoic acid, glycolic acid, lactic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid , Malic acid, tartaric acid, citric acid, amino acids such as glutamic acid or aspartamic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, cyclohexanecarboxylic acid, adamantanecarboxylic acid, benzoic acid, salicylic acid, 4-aminosalicylic acid, phthalic acid, phenylacetic acid, Mandelic acid, cinnamic acid, alkanesulfonic acid such as methane- or ethane-sulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, arylsulfonic acid such as benzenesulfonic acid, 2-naphthalenesulfonic acid, 1,5- Naphthalene-disulfonic acid or 2-, 3- or 4-methylbenzenesulfonic acid, methyl sulfuric acid, ethyl sulfuric acid, dodecyl sulfuric acid, N-cyclohexyl sulfamic acid, N-methyl-, N-ethyl- or N-propyl-sulfamic acid Or other organic amount Sex assets such as ascorbic acid.

In one preferred embodiment Z ⁻ is a halide. In another preferred embodiment Z ⁻ is bromide.

In another preferred embodiment, Z ⁻ is an anion of alkane- or arylsulfonic acid. In one particularly preferred embodiment Z ⁻ is methanesulfonate.

In another particularly preferred embodiment, the mitochondrially targeted antioxidants and all stereoisomers thereof useful for the treatment and prevention of liver disease and / or epithelial cancer have the formula:

Wherein Z ⁻ is a pharmaceutically acceptable anion, preferably Br ⁻ . This compound is called "Mito Vit E".

In another preferred embodiment the mitochondrially targeted antioxidants useful for the treatment and prevention of the diseases according to the invention have the formula:

Wherein Z ⁻ is a pharmaceutically acceptable anion, preferably halogen, m is an integer from 0 to 3, and each Y is independently selected from groups, chains and aliphatic and aromatic rings having electron donating and accepting properties (C) _n is a carbon chain which optionally has at least one double bond or triple bond and optionally has at least one substituent and / or unsubstituted or substituted alkyl, alkenyl or alkynyl side chain and n is An integer from 1 to 20.

Preferably, each Y is independently selected from the group consisting of alkoxy, alkylthio, alkyl, haloalkyl, halo, amino, nitro, optionally substituted aryl, or if m is 2 or 3, two Y groups They combine with the attached carbon atoms to form an aliphatic or aromatic carbocyclic or heterocyclic ring fused to an aryl ring. More preferably each Y is independently selected from methoxy and methyl.

Preferably, (C) _n is an alkyl chain of formula (CH ₂ ) _n .

In a particularly preferred embodiment, the mitochondrially targeted antioxidants according to the invention have the formula:

In the food,

Z ⁻ is a pharmaceutically acceptable anion, preferably Br ⁻ . The compound is called "mitoquinol" or "mitoquinone" as the oxidized form of the compound (hydroquinone of the formula is quinone). Mixtures of various amounts of mitoquinol and mitoquinone are referred to as "MitoQ".

More preferably, mitochondrially targeted antioxidants according to the invention have the formula:

In the formula, the pharmaceutically acceptable anion Z ⁻ is methanesulfonate. In this embodiment, a mixture of various amounts of mitoquinol and mitoquinone is referred to as "MitoS".

Further preferred embodiments according to the invention represent mitochondrially targeted derivatives of spin trap phenyl-t-butylnitrone of the formula: hereinafter referred to as "MitoPBN":

In another embodiment according to the invention, the mitochondrially targeted antioxidant is a glutathione peroxidase analog, such as a selenium organic compound, ie an organic compound comprising at least one selenium atom. Preferred classes of selenium organic glutathione peroxidase analogs include benzisoselenazolones, diaryl diselenides and diaryl selenides.

In particular, the glutathione peroxide mimetic moiety is of the formula hereinafter referred to as "Ebelsen" (2-phenyl-benzo [d] isoselenazol-3-one):

Preferred compounds of the present invention have the formula:

Wherein Z ⁻ is a pharmaceutically acceptable anion, preferably Br ⁻ and L is a monosaccharide.

Particularly preferred embodiments according to the invention have the formula:

Wherein Z ⁻ and (C) _n are as defined above, W is O, S or NH, preferably O or S, and n is 1 to 20, more preferably 3 to 6.

In another embodiment, the present invention provides an effective amount of a mitochondrial targeted antioxidant according to the present invention to a physiological saline solution, demineralized water, stabilizer (β-cyclodextrin, preferably 1: 2 ratio) and / or proteolysis. Provided is a pharmaceutical composition suitable for treating and / or preventing a patient suffering from liver disease and / or epithelial cancer, including with one or more pharmaceutically acceptable carriers or diluents, such as enzymease inhibitors.

As used herein, the term “pharmaceutically acceptable” is suitable for use in contact with the tissues of a subject (preferably human) of interest within the scope of healthy medicinal judgments, with excessive toxicity, irritation , Compound, ingredient, substance, composition, dosage, form, etc., having a reasonable benefit / risk ratio balanced without causing an allergic reaction or other problem or complication. Each carrier, diluent, excipient, etc. must be "acceptable" in the sense of being compatible with the other ingredients of the formulation.

In another embodiment, the invention is a liver disease and / or epithelial cancer that will be assisted by oxidative stress reduction comprising administering to a liver disease and / or epithelial cancer patient a mitochondrially targeted antioxidant as defined above. Provided are methods for treating or preventing a patient.

The term "treatment" within the meaning of the present invention is intended to cure a patient with one or more diseases according to the present invention and / or for a temporary, short term (a few hours to several days), long term (weeks, months or years). Means a treatment that improves the physiological condition of a patient with one or more symptoms associated with the disease, wherein the improvement in the physiological condition is as constant or increased as long as the overall effect indicates a marked improvement in symptoms as compared to the control patient. Or decrease, or may be continuous fluctuations or vibrations.

In addition, the term “treatment” as used herein in connection with the treatment of liver disease and / or epithelial cancer generally relates to the treatment and treatment of humans or animals (such as veterinary applications), with some desired therapeutic effect being the progression of the condition. Can be obtained by suppressing the rate of progression, and the rate of progression, the rate of progression, the relaxation of the state and the healing of the state.

The term "treatment" according to the present invention includes combination treatments and therapies, for example, combining two or more treatments or therapies sequentially or simultaneously. Treatment also includes prophylactic measures (ie prophylaxis).

Treatment according to the invention can be carried out in a manner generally known to those skilled in the art, such as by oral administration or intravenous injection of the pharmaceutical composition according to the invention.

Therapeutic efficacy and toxicity, such as ED ₅₀ and LD ₅₀ , are measured by standard pharmaceutical procedures in cell culture or experimental animals. The dose ratio between therapeutic and toxic effects is the therapeutic index and can be expressed as LD ₅₀ / ED ₅₀ . Pharmaceutical compositions that exhibit large therapeutic indices are preferred. Dosing should be selected based on the age, weight and condition of the individual to be treated, as well as the route of administration, dosage form and medication plan and the desired outcome, and the exact dosage should be determined by the physician.

The actual dosage depends on the nature and severity of the disease to be treated and is within the discretion of the physician and may be varied by titration of the dosage to produce the desired therapeutic effect for the particular situation of the invention. However, pharmaceutical compositions comprising from about 0.1 to 500 mg / kg, preferably from about 0.1 to 100 mg / kg, most preferably from about 0.1 to 10 mg / kg of active ingredient per individual dose are suitable for therapeutic treatment. .

In general, suitable dosages according to the invention range from about 0.1 mg to about 250 mg per kilogram of body weight of the patient to be treated.

The active ingredient may be administered once or several times a day. In certain cases satisfactory results can be obtained with very low doses of 0.1 mg / kg intravenously (i.v.) and 1 mg / kg orally (p.o.). Preferred ranges are from 0.1 mg / kg / day to about 10 mg / kg / day i.v. And 1 mg / kg / day to about 100 mg / kg / day p.o.

The invention also provides an antioxidant according to the invention useful for the treatment and / or prevention of liver disease and / or epithelial cancer comprising mixing or dissolving the active compound with a suitable pharmaceutical carrier using standard methods known in the art. It relates to the manufacture of a medicament containing a compound. Such methods include mixing the active compound with a carrier comprising at least one accessory component. In general, the preparations according to the invention are prepared by uniformly and intimately mixing the active compounds with a carrier (such as a liquid carrier, a finely divided solid carrier) and then molding the product if necessary. Suitable carriers, diluents and excipients for use in the present invention can be found in standard pharmaceutical literature (eg Handbook for Pharmaceutical Additives, 2001, 2nd edition, eds. M. Ash and I. Ash).

Antioxidant compounds according to the invention, such as derivatives of vitamin E, coenzyme Q ₁₀ or glutamine peroxidase analogues, can be prepared by the compounds described in US Pat. No. 6,331,532, WO 99/26954, WO 2004/014927 or WO 2003/016323. It can synthesize | combine according to the well-known method for manufacturing.

It will be appreciated by those skilled in the art that various modifications can be made to the compositions, methods and processes of the present invention. Accordingly, it is to be understood that the invention includes such modifications and variations as long as they come within the scope of the appended claims and their equivalents. All references cited herein are hereby incorporated by reference.

In order to evaluate the effect of mitochondrially targeted antioxidants such as derivatives of vitamin E, coenzyme Q ₁₀ or glutamine peroxidase analogs in the treatment and / or prevention of liver disease according to the invention, the context (Gliason Trias) Presence of morphological modifications such as inflammatory cells and hepatocellular damage (necrosis, destruction of the cytoskeleton (Example 3, FIG. 1) and swelling of hepatocytes, formation of dense keratin intermediate filament (IF) networks, reduction in the density of keratin IF and The presence of Mallory bodies (MB), which represents one of the most frequent IF-related cytoskeletal modifications in various congenital and acquired liver diseases, was evaluated by treatment with or without treatment according to the present invention (Examples 2 and 3).

Morphological modifications, including MB, are reproduced in mice by chronic intoxication with the fungal drug griseofulvin (GF) or porphyrogenic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). (Denk H. et al., 1975, Lab. Invest: 773-776; Tsunoo C. et al., 1987, J. Hepatol., 5: 85-97). MB formation can be induced in mouse liver by ingesting a diet containing DDC- or GF (see Example 1). Oxidative damage induced by methyl radicals is a common pathological principle in the liver of DCC or GF-ingested animals and humans with ASH or NASH, and not only by cytochrome P450-mediated oxidation of ethanol, but also by acetaldehyde and free fatty acid high loads. Free radicals produced by induced mitochondrial damage are central features (Lieber CS, 2000, J. Hepatol., 32: 113-128; Anguilo P., 2002, N Engl. J. Med., 346: 1221- 1231).

In addition, changes in the chemical composition of MB as well as the IF keratin cytoskeleton in DDC- or GF-fed mice are very similar to those found in human ASH and NASH, if not identical (Denk H. et al., 2000, J. Hepatol., 32: 689-702). In this regard, other mouse models for alcoholic liver disease based on alcohol-containing diet ingestion show obstruction of fat metabolism and to some extent inflammation of human ASH, but no modification of keratin IF cytoskeleton and MB. It does not result in formation.

In one type of experiment (Example 2), the appearance of large MBs, typically located in the periplasmic cytoplasmic region, is characterized by conventional tissue immunochemistry (hemoxillin and eosin staining) or immunization using antibody SMI 31 against p62 protein. Detection was performed in mice tested at 6-10 weeks of intoxication using fluorescence microscopy standard methods (Zatloukal K. et al., 2002, Am J Pathol. 160 (1): 255-63). p62 was originally identified as a phosphotyrosine-independent ligand of the SH2 domain of p56 ^lck and as a cytoplasmic non-proteasome ubiquitin-binding protein (Vadlamudi RK et al., 1996, J. Biol. Chem., 271: 20235- 20237). The general role of p62 in cellular stress response was implied because p62 expression was increased by various stress stimuli, in particular oxidative stress (Ishii T. et al., 1996, Biochem Biophys. Res Comm., 226: 456-460 ).

 At 4 weeks of recovery from intoxication there is a group of hepatocytes that do not have cytoplasmic keratin filaments but still contain a small amount of MB around the cell in combination with the desmosome. When the mouse is reexposed to GF or DDC, multiple MBs reappear within 24 to 72 hours (Stumptner C. et al., 2001, J. Hepatol., 34: 665-675). This, when re-addiction ceased, enhanced MB formation as a toxic memory effect, similar to an allergic reaction.

In order to assess the effect of the antioxidant according to the invention on the progression of morphological changes in early stage DDC or GF addicted mice livers, a positive control animal group (3-7 days exposure only to GF or DDC) was subjected to 3-7 days. Mito Q (mitoquinol [10- (3,6-dihydroxy-4,5-dimethoxy-2-methylphenyl) decyl] triphenylphosphonium bromide and mitoquinone [10- (4,6-dimethoxy) -2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl) decyl] triphenylphosphonium bromide) or Mito Vit E [2- (3,4-dihydro-6-hydride Compared to DDC- or GF addicted mice treated with Roxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) ethyl] -triphenylphosphonium bromide). Mice tested were treated with intraperitoneal injection (ip) or intravenous (iv) (tail vein) injections comprising an antioxidant compound according to the invention, such as Mito Q or Mito Vit E, and these mice were excipient-injected. Control mice (PBS supplemented with sufficient DMSO to maintain the solubility of the antioxidant) and other suitable control mice (Example 3).

MitoS (mitoquinol [10- (3,6-dihydroxy-4,5-dimethoxy-2-methylphenyl) decyl] triphenylphosphonium methane sulfonate and mitoquinone [10- (4,6-dimeth) A mixture of methoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl) decyl] triphenylphosphonium methane sulfonate or a MitoQ or MitoQ derivative such as Mito Vit E are incorporated into the diet. Dosage is determined by measuring water or liquid diet consumption and mouse weight (Smith RAJ et al., 2003, PNAS, 100 (9): 5407-5412).

In another type of experiment, a control group treated with DDC- or GF addicted animals with an antioxidant according to the invention (such as Mito Q or Mito S) for 3 to 7 days and then exposed to DDC or GF only for 3 to 7 days, Comparison was made (Example 3).

In another experiment, 3-12 mg / kg of Mito Q was intraperitoneally administered to DDC-addicted mice for 3 days and compared with control animals. In order to evaluate the effect of mitochondrially targeted antioxidants according to the present invention in a short time experiment, the presence (absence) of inflammatory cells and portal expansion and / or Mallory body analysis (DDC or GF) around the context (trias of Gleason) Hepatocellular damage, such as necrosis, destruction of the cytoskeleton (see FIGS. 2 and 3), was compared with a suitable control (typical for long-term harvesting) (see FIGS. 1-3). Cell expansion and Mallory bodies are not suitable for these experiments because they do not form to the extent that statistical evaluation is possible when exposed to DDC for a short time.

In total, normal structures, represented by hepatocyte strands bounded by sinusoids under Mito Q treatment, are again observed. Hepatocyte morphology is normal in size and shape of the nucleus and in the structure of the cytoplasm. In addition, the number of inflammatory cells (eg, neutrophils, lymphocytes, phagocytes, macrophages) was significantly reduced upon the antioxidant treatment according to the present invention (FIGS. 1 to 3).

For long term experiments using mice intoxicated with DDC for 8 to 10 weeks, the presence of cell swelling and / or Mallory bodies (MB) in liver samples of treated animals was measured and compared with control animals (Example 3, 4-6).

To determine the effectiveness of these antioxidants in the treatment and / or prevention of chronic liver metabolic diseases and epithelial cancer upon 10 weeks of intoxication with DDC or GF, test mice were tested for antioxidant compounds such as Mito Q, Mito S or Injections containing Mito Vit E were intraperitoneally or intravenously (tail vein) for 7 days and compared with excipient-injected control mice and other suitable controls (see Example 3).

Alternatively, after 10 weeks of DDC poisoning, the test animals were intraperitoneally injected with Mito Q (1.25 mg / kg) twice for 7 days (on days 1 and 4 of 7) and analyzed by conventional histology ( Standard haematoxylin / eosin staining according to Luna LG, 1968, Manual of Histologic staining methods of the Armed Forces Institute of Pathology, 3rd edition, McGraw Hill, New York). The degree of cell expansion and the number of Mallory bodies were significantly reduced in Mito Q treated animals addicted to DDC when compared to a suitable control (see Example 3, FIGS. 4-6).

In another experiment, Mito Q, Mito S or Mito Vit E was fed for 7 to 14 days to mice poisoned 8 to 10 weeks with DDC or GF via drinking tap water (Example 3).

As another experiment, 6-week DDC- or GF addicted mice were treated simultaneously with Mito Q, Mito S or Mito Vit E for 4 weeks using Mito Q, Mito Q or Mito VitE at a maximum tolerated dose of 10-50%. The antioxidant according to the present invention was administered and then compared to a control animal group intoxicated with DDC or GF for 10 weeks (see Example 3).

As another experiment, mice poisoned for 10 weeks with DDC or GF were recovered for 4 weeks. In this experiment the toxic memory effect (resulting from 24 to 72 hours of re-exposure to DDC or GF poisoning) was reduced or eliminated by simultaneous treatment with the antioxidant according to the present invention.

To evaluate the prophylactic effect of the antioxidant according to the invention in liver disease, a group of mice ingested with DDC- or GF was co-treated with Mito Q, Mito S or Mito Vit E at a maximum allowable dose of 10-50%. Compared to the control animal group treated with DDC or GF only for 10 weeks (Example 3).

Alternatively, poisoned with DDC or GF for 24 to 74 hours and then administered Mito Q, Mito S or Mito Vit E within the initial recovery period for 4 weeks, wherein treated mice were exposed to 2.5 months of DDC- or GF exposure. It was then compared to control animals that were not treated with antioxidants.

Administration of antioxidants, such as derivatives of coenzyme Q, vitamin E or glutathione peroxidase analogs, may cause morphological abnormalities such as hepatocellular expansion, intracellular inclusions of misfolded proteins and MB in livers of DDC- or GF addicted animals. Provide a significant reduction. These results (Examples 3, FIGS. 1-6) show that such cellular damage is reduced by mitochondrially targeting of antioxidant compounds according to the present invention. DDC- or GF addicted mouse models are similar to those observed in patients with NASH or ASH and are intended to study the role of antioxidants such as coenzyme Q ₁₀ derivatives and vitamin E in the treatment or prevention of the diseases according to the invention. Provides robust in vivo and in vitro systems.

Treatment and / or prevention of human patients with liver disease according to the invention using mitochondrially targeted antioxidants significantly reduces the cause of liver pathology and thus provides a therapeutic and / or prophylactic effect as a therapeutic agent for the disease.

In order to assess oxidative stress in treating control and DDC-addicted mice with or without the antioxidant according to the invention, isolated liver mitochondria (all tested animal groups were prepared in preparation in Example 3). Thus prepared) tocopherol quinone (TQ) content (Gille L. et al., 2004, Biochemic. Pharmacology, 68: 373-381). Mouse liver mitochondria are described in Staniek K and Nohl H, 1999, Biochem. et Biophys. Acta, 1413: 70-80; Mela L. and Sietz S., 1979, Methods in Enzymology, Academic Press Inc .: Prepared according to the modification procedure of 39-46 and the TQ content is based on several variables and complex I (NADH dehydrogenase), including protein and cytochrome concentrations. , Complex II (succinate dehydrogenase), complex III (cytochrome bc1) and complex IV (cytochrome oxidase) are normalized by activity tests. Overall, these experiments show elevated TQ amounts in DDC addicted mice as compared to mice treated with Mito Q and control animals (Example 4).

As another experiment for evaluating proteins induced by oxidative stress, hemooxygenase (HO-1) was used using extracts derived from mice that were DDC addicted and concurrently exposed to Mito Q for short time (3 days, Example 5). ) Western blot analysis of the amount of expression was performed. Significant reduction in DDC-induced overexpression of HO-1 (known to be induced by reactive oxygen species (ROS), Suematsu M. and Ishimura Y., 2000. Hepatology, 31 (1): 3-6) is oxidative It is shown that the stress is significantly reduced in the liver by the antioxidant according to the invention (Example 5, Figure 7).

The amount of protein expression of fatty acid binding protein (FABP) (Monbaliu D. et al., 2005, Transplant Proc., 37 (1): 413-416), which represents a sensitive marker for hepatocellular damage, was also associated with DDC poisoning when compared to the control group. Mice exhibited a wise reduction of FABP protein. Under the Mito Q treatment of this animal group, FABP protein expression almost reached control mouse FABP expression values, which again demonstrates the effect of Mito Q in the treatment or prevention of the diseases according to the invention (Example 5).

As an experiment to confirm the effect of the antioxidant according to the invention in DDC- or GF addicted mice versus control mice, the serum levels of certain enzymes of the liver are characterized by the extent of liver damage and fibrosis characteristic of several diseases according to the invention in particular. It is monitored as Actitest (Biopredictive, Houilles, France) which provides (see Example 6). Serum amounts of a ₂ -macroglobulin, haptoglobin, γ-glutamyl transpeptidase, total bilirubin, apolipoprotein A1 and alanine aminotransferase are measured from controls treated with DDC- or GF and corresponding DDC- or GF treated animals are exposed to mitochondrially targeted antioxidants according to the general timeline according to Example 3 using the method described in Poynard, et al., 2003, Hepatology 38: 481-492.

Actitest performed using human serum as a measure of liver damage, especially fibrosis, can similarly be used to monitor the effectiveness of treating patients with the disease using the antioxidants according to the invention.

Alternatively, variables indicative of liver damage in serum from various test animal groups such as bilirubin, alanine-aminotransferase (ALT / GPT), aspartate aminotransferase (ASAT / GOT) and glutamate dehydrogenase (GLDH) was measured according to the standard procedure in a clinical diagnostic method using a commercially available kit (Example 6). Reduction of serum liver enzymes (such as alanine- and aspartate aminotransferases, see FIG. 8) in animals treated with the compounds according to the invention indicates a reduction in liver damage in the treated samples and these in diseases according to the invention. Provide support for the therapeutic efficacy of the compounds.

To assess the production of reactive oxygen species (ROS), for example, control and DDC- and GF addicted according to standard techniques (Brandes RP et al., Free Radic Biol Med. 2002; 32 (11): 1116-1122). Dihydroethidium (DHE) staining of liver secretions (such as frozen portions) prepared from animals can be used. This method is similar to the observation in patients with disease according to the invention as it allows induction of ROS production in vivo in the liver of DDC- or GF addicted animals (Example 7). Other methods of assessing ROS formation in DDC- or GF fed mice include Lucigenin chemiluminescence assay (Goerlach A. et al., 2000, Circ Res., 87 (1): 26-32).

This experiment involved treating DDC- or GF-fed animals with the targeted antioxidant according to the invention (Example 6). The general timing and dosage of test animals for their treatment with DDC- or GF-addiction and antioxidants is determined by the method used to determine morphological abnormalities, such as DDC- or GF-addiction according to Example 3. It is identical to the experimental method used for intracellular inclusions of proteins and MBs that were misfolded in the liver of the animal.

Application of antioxidants, such as derivatives of vitamin E, coenzyme Q ₁₀ or glutamine peroxidase analogs, using the general procedure according to Example 7 significantly reduces ROS formation, thus providing therapeutic effects in liver diseases according to the present invention. (Example 8).

If desired, liver cancer cell lines (such as HepG2 or Hep3B), SNU-398 cell lines derived from hepatocellular carcinoma (ATCC No. CRL-2233, manufactured by LGC Promochem, HUH-7 human carcinoma cells (Japanese collection of Research Biosources JCRB) 0403) or in vitro experiments with Tib-73 mouse embryonic cell line (ATCC TIB 73 = BNL CL2 derived from BAL / c mice, Maryland, USA) allow measurement of ROS production in hepatocytes upon DDC poisoning. (Example 9). Glutathione synthesis inhibitor L-butionine- (S, R) -sulfoxime (BSO) may be applied to increase endogenous oxidative stress (Kito M. et al., 2002, Biochem Biophys Res Commun., 291 ( 4): 861-867).

Since CoCl ₂ has recently been shown to affect mitochondria to measure ROS production in differentiated cell lines (Jung JY and Kim WJ., 2004, Neurosci Lett., 371: 85-90), HepG2 (ATT No. HB -8065, Maryland, USA) can be stimulated by 100 μΜ CoCl ₂ (Sigma) (Bel Aiba RS., Et al., 2004, Biol. Chem. 385: 249-57).

Other well established methods in cultured cells (as well as isolated organelles or whole tissues) are described in Chem Biol Interact. 2000 Jul 14; ROS production induced by antimycin A (FIG. 10) or by rotenone according to 127 (3): 201-217 by Lucigenin chemiluminescence assay (Goerlach A. et al., 2000, Circ Res., 87 ( 1): Allow measurement using 26-32).

Hepatocyte cultures intoxicated for less than 3 days by DDC (concentration = 50 μg / ml medium), BSO (up to 100 μM) or antimycin A (or rotenone) or CoCl ₂ (100 μM) induce ROS production in vitro Thus, another suitable model similar to the observation in a diseased patient according to the present invention is provided.

By applying the standard procedure according to Example 9, it is poisoned by DDC, BSO, antimycin (or rotenone) or CoCl ₂ (100 μM) and at the same time Mito Q or Mito Vit E (Jauslin ML et al., 2003, FASEB J., (13): according to 1972-4, a concentration corresponding to EC ₅₀ = 0.51 nM for Mito Q and EC ₅₀ = 461 nM for Mito Vit E) or later in a concentration range of 0.5 to 10 μM. Treated differentiated cell lines (such as liver cancer cells) significantly reduced ROS formation, thus confirming the therapeutic effect of mitochondrially targeted antioxidants in liver disease according to the present invention (see FIG. 9, Example 10).

MB is found in chronic cholestasis, such as primary cholangiocytosis and primary sclerotic cholangitis. In order to measure the effect of mitochondrially targeted antioxidants according to the invention in treating and / or preventing chronic cholestatic conditions, the treatment described above for DCC- or GF addicted mice (Example 3) Was carried out. The recovered drug-treated animals were treated with normal bile duct ligation (CBDL) or with bile acid (CA) -supplemented diet for 7 days with or without Mito Q and Mito Vit E (Fickert P. et. al. 2002, Am. J. of Pathology, 161 (6): 2019-2026) and compared with a suitable control.

A general technique for determining the effect of mitochondrially targeted antioxidants in the treatment and / or prevention of liver fibrosis and sclerosis is carbon tetrachloride (CCl ₄ ) -induced liver injury in an antioxidant treated mouse or rat model according to the present invention. (Arias IM et al., 1982, The Liver Biology and Pathobiology.Raven Press, New York).

In order to measure the effect of mitochondrially targeted antioxidants according to the invention on the treatment and / or prevention of epithelial cancer according to the treatment methods described above for the DDC- or GF addicted mice, a human epithelial cell carcinoma xenograft ( Immunocompensated mice with nude mouse tumor xenografts, such as CD1 nu / nu mice manufactured by Charles River Laboratories, USA, are used. Tumors xenografted subcutaneously according to standard methods known in the prior art (Li K. et al., 2003, Cancer Res., 63 (13): 3593-3597) are colon adenocarcinomas, invasive coronary breast cancers, small and non-small. Cell lung cancer, prostate cancer, pancreatic cancer and gastric cancer.

Treatment of these mice shows reduced tumor growth, improved tumor necrosis and reduced vascularization of tumor xenografts. Similarly, the amount of ROS in nude mouse tumor xenografts was also monitored and reduced in xenograft tumors treated with the antioxidant according to the invention (Example 11).

Compared with the treatment or prophylaxis techniques of liver disease and liver and other epithelial cancers, the treatment method according to the present invention is surprisingly improved and provides a continuous and more effective treatment.

The present invention will be described in detail with reference to the following drawings and examples, and preferred embodiments and features of the present invention are not limited thereto.

1 To  3: DDC Of hepatocellular damage in mouse liver when exposed to short time (3 days) Mito  Q effect

Figure 1: Normal livers are characterized by hepatocytes arranged mostly on strands, with central veins (triangles) and oyster-shaped blood vessels (C, originally white, located between these strands of hepatocytes, with several red spots representing red blood cells). Oriented). The nuclei of hepatocytes (originally blue in A, H & E staining) are large, non-condensing and mostly show one distinct nucleolus, cytoplasm (marked B, stained relatively uniform pink in H & E staining). No exudates with lymphocytes or granulocytes around the portal vein (Asterisk display) were detected (200 × magnification).

Figure 2: After 3 days of poisoning with DDC, the structure of the liver is severely impaired: the regular array of hepatocytes is impaired. In particular, effusion of lymphocytes and granulocytes was observed around the portal vein (marked with asterisk) (arrows). Hepatocytes show different degrees of cellular damage: the cells lose contact when in contact with other cells, the nucleus is concentrated and the cytoplasm is blue-pink as a marker of apoptosis. Cell size increase (expansion) and cytoplasm are heterogeneous, and clumps of cytokeratin may be seen. In addition, cells lose the plasma membrane as an indication of necrosis. A, B, and C are the same as in FIG. Inflammatory cells (marked with arrows) and damaged hepatocytes (clear cell boundaries, cell expansion) are detected around the portal vein (marked with asterisk). Deposition of protoporphyrins (small brown spots) shows DDC-specific effects on protoham perillase. A, B, and C are the same as in Fig. 1 (magnification: 400x).

Figure 3: Normal structure with hepatocyte strand bound by oyster blood vessels after co-treatment with Mito Q (Mito Q in PBS / 1% DMSO (225 nmol / animal / day corresponding to 6 mg / kg)) You can see it again. Hepatocyte morphology is normal in terms of nucleus size and morphology and cytoplasmic structure (Example 3). Absence of inflammatory cells around the portal vein (marked as asterisk); Hepatocytes appear normal except for a slight indication of cell swelling and protoporphyrin deposition. A, B, and C are the same as in Figs. 1 and 2 (magnification: 400 ×).

4 To  6: DDC Of hepatocellular injury in mouse liver after prolonged exposure (10 weeks) Mitoq Effect

Figure 4: In normal non-DDC-addicted mice (age: 4 months), liver structure is generally similar to that of non-DDC-addicted young mice shown in Figure 1A (A = nucleus, B = cytoplasm, C = Oyster vessel). There is no inflammation around the portal vein (asterisk mark) and the liver cells are arranged in strand form. The cytoplasm of these cells is regularly stained and of uniform size; No expansion of the cells or formation of malorisomes was seen (magnification: 400 ×).

Figure 5: The structure is the same as shown in Figures 1-4 (reference A = nucleus (original blue), B = cytoplasm (original pink), C = oyster-shaped vessel (white with original red dot)) and D original Brown represents pigment (primarily protoporphyrin) in the dulce conduit. After 10 weeks of poisoning by DDC and then recovery for 1 week without DDC, the arrangement of hepatocytes is still disturbed. Hepatocytes exhibit varying degrees of cellular damage, from disruption of cell structure to cell expansion and the formation of arrowhead bodies (arrows). The accumulation of protoporphyrin was especially seen in the bile duct (arrowhead), magnification: 400 ×.

Figure 6: After 10 weeks of DDC poisoning, recovery phase (1 week without DDC) treatment was performed with two MitoQ injections. The improvement is remarkable during recovery; Hepatocellular morphology changes were minimal. Many hepatocytes appear normal and there was no cell expansion and / or malorisome formation. The accumulation of protoporphyrins in the bile ducts around the portal vein (Asterisk mark) was significantly reduced. The structure was the same as in FIGS. 1-5 (reference A = nucleus (original blue), B = cytoplasm (original pink), C = cavernous vessels (original white with red spots) and D, originally brown, pigment (mainly Protoporphyrin). Magnification: 400x.

Figure 7: Mitoq Treated with DDC  In poisoned mouse Of hemoxigenase (HO-1)  Expression of the induced form

Western blot analysis of HO-1 (32 kDa, indicated by arrows) shows significant induction in DDC intoxication (lane numbers 4, 5, 6, for solvent control with DDC), while treatment with MitoQ (lane 7, 8 = 112 nmol / kg MitoQ without DDC and lanes 9, 10 = 112 nmol / kg MitoQ with DDC) resulted in a strong decrease in HO-1 protein expression. Lanes 1 to 3 represent solvent controls without DDC. Low molecular weight protein markers (22, 36, 55, 64, 98 and 148 kDa) are used.

DDC poisoning to normalize HO-1 protein expression in lanes 1-10 in contrast to homologous HO-2 (36 kDa) constitutively expressed using Chemimager 5500 software (Alpha Innotech) A 7-fold reduction in HO-1 was seen in DDC-addicted animals treated with MitoQ when compared to the control indicated by mice. In total, DDC poisoned mice (3 days) are analyzed by intraperitoneal injection with MitoQ in PBS / 1% DMSO daily and compared with the appropriate control.

Figure 8: DDC  Simultaneous Addication of Mice Mitoq  Serum Variables Under Treatment

Activity of serum liver enzymes showing hepatic damage in serum from various animal groups, ie bilirubin, alanine aminotransferase (ALT / GPT; diagram as white bar), aspartate aminotransferase (ASAT / GOT black) Bar diagrams) are determined according to the standard procedure for clinical diagnosis by using a commercially available kit (No: 11552414; 11876805216; 11876848216, all purchased from Loch Ague, Switzerland) on the Hitachi / Lactin 917 Analyzer. .

Lanes: 1 and 2 represent non-DDC addicted animal groups and DDC addicted mice. Lanes 3 to 5 represent DDC addicted (day 3) and MitoQ treated animals at concentrations of 3-, 6- and 12 mg / kg. The most significant decrease in enzyme activity is alanine aminotransferase (ALT / GPT, indicated by white bars), aspartate aminotransferase (AST / GOT, black bars), while bilirubin activity does not show any change ( Data not shown).

Figure 9: Mitoq Concurrently processed by HepG2  100 μM in cells CoCl ₂  By (0, 10, 20, 30 minutes) ROS   production

5 μM MitoQ can reduce basal ROS production in unstimulated cells. (See lane 2). CoCl ₂ -induced ROS production (100 μM CoCl ₂ ) was reduced by 5 μM MitoQ. These results show that 5 μM MitoQ significantly reduces basal and CoCl ₂ stimulated ROS levels in HepG2 cells (Example 10). "A" (lanes 4, 5, 6) means that HepG2 cells are stimulated by CoCl ₂ . The X axis shows the concentration range of MitoQ [μM] and the y axis shows the relative DCF fluorescence [%]. * P <0.05 vs unstimulated (0 μM MitoQ); #p <0.05 vs CoCl ₂ .

Figure 10: Lucigenin  1 μM using chemiluminescent assay Antimycin  by HUH Stimulation of -1 Cell

HUH-7 cells were cultured in 6 plates and concentrations from 0 to 25 μM (0, 1 and 5 with or without simultaneous use of MitoQ in the 0 to 1000 nmol concentration range dissolved in DMEM (manufactured by Gibco) for 3 hours at 37 ° C. stimulation with antimycin A of μM). The light reaction between superoxide and lucigenin was detected. The x-axis represents the concentration of MitoQ [nM] and the y-axis is the chemiluminescent signal expressed as the average number per minute [cpm] after normalizing with the number of cells measured by a cell counter. Overall, this diagram shows a marked decrease in ROS formation, which confirms the therapeutic benefit of mitochondrially targeted antioxidants in liver disease according to the present invention.

Example  1: Mallory body (MB) of  Experiment induction

MB can be induced in mouse liver by chronic poisoning of various mouse strains: for example male Swiss albino mice: 3,5-diethoxycarbonyl-1,4-dihydrocolidine (1,4-dihydro-2 , 4,6-trimethylpyridine-3,6-dicarbonate diethyl ester, DDC, Cat.no. 13703-0, manufactured by Sigma-Aldrich, Steinheim, Germany) or Griseofulvin (GF Strain Him OF1 SPF (Institut of Laboratories Animal Research, University of Vienna, Himburg, Austria) processed by Cat. No. 85,644-4, manufactured by Sigma-Aldrich.

A reference diet containing 2.5% GF or 0.1% DDC (manufactured by Sniff Spezialdiaten GmbH, Joest, Germany) was produced into pellets by Sniff.

Animals were placed in sterile separators or conventional cages with a 12 hour day-night cycle. These animals are manufactured by the National Academy of Sciences and are available from the National Institute of Health; It was painlessly administered according to the criteria outlined in "Guide for the Care and Use of Laboratory Aminals," published by NIH publication 86-23, revised 1985.

Mice (8 weeks old) received a standard diet containing 0.1% DDC or 2.5% GF for 2.5 months.

Mouse liver first responds to DDC- or GF poisoning by swelling hepatocytes and forming a dense keratin IF network. After 6 weeks of intoxication, expanded hepatocytes show reduced keratin IF density and early MB can be observed as fine granules associated with the keratin IF network. Continued intoxication results in the presence of large MBs located in the cytoplasmic cytoplasmic region. Most hepatocytes containing large MBs reduce the cytoplasmic IF keratin network to a significantly reduced or undetectable level. Upon intoxication, MB disappears within a few weeks. At 4 weeks of recovery from intoxication, there is a group of hepatocytes that do not have cytoplasmic keratin filaments but contain a residual amount of MB around the cells combined with the desmosome. When these mice are again exposed to DDC or GF, multiple MBs appear within 24 to 72 hours (Stumptner C. et al., 2001, J. Hepatol., 34: 665-675). This improves the formation of MB as a toxic memory effect, similar to an allergic reaction when re-addiction is blocked.

Mice are killed at different time points of intoxication by lumbar spine dislocation and the liver is immediately snap-frozen with methylbutane pre-cooled with liquid nitrogen for immunoglobulin or fixed in 4% buffered formaldehyde for conventional tissues and immunohistochemistry. .

Example  2: evaluation of liver deformation; Mallory body ( MB ) Detection

Liver samples prepared according to Example 1 were used for simple tissue staining by hematoxylin and eosin (Luna LG, 1968, Manual of Histologic staining methods of the Armed Forces Institute of Pathology, 3rd edition, McGraw Hill, New York). . Single-label immunohistochemistry or double-label immunofluorescence microscopy was also performed to detect MB in the animals tested.

A) Single-label immunohistochemistry on paraffin-embedded portions: Deparaffinize portions (4 μm thick) in xylene, grade ethanol (100%, 90%, 80%, 70%, 50% ethanol) and PBS ( 50 mM potassium phosphate, 150 mM NaCl, pH 8.0-8.5). For antigen recovery, the rehydrated portion was incubated with 0.1% protease type XXIV (Sigma Steinheim, Germany) for 10 minutes at room temperature (for ubiquitin daco primary antibody) or 10 minutes in 10 mM citrate buffer, pH 6.0. Incubate with 750 W microwave (energy controlled conventional home microwave oven) (for polyclonal K8 / 18 antibody 50K160, monoclonal K8 antibody K8.8 [Neomarkers], monoclonal K18 antibody DC- 10 [Neomarkers] and p62PC: polyclonal guinea pig antibodies against the C-terminal peptide of p62; Matroka K. et al., 2002, Am. J. Pathol., 160: 255-263). After washing with PBS, the endogenous peroxidase was blocked by incubating for 10 minutes in 1% H ₂ O ₂ (Merck) in methanol and then washed with PBS. The next step was incubated for 60 minutes at room temperature with the primary antibody in a humidity chamber (Nunc) and then washed three times with PBS. Subsequently, the portion was incubated for 30 minutes at room temperature with Multi Link Swine anti-goat, mouse, and rabbit immunoglobulin (Dako) diluted 1: 1 with PBS, washed three times with PBS, and further treated with Streptavidin biotin cauliflower pu. Incubate with oxidase complex ABC / HRP (Dako; in Sol A 1: 100 and Sol B 1: 100 PBS) for 30 minutes. Alternatively, the cells were incubated with peroxidase-bound rabbit anti guinea pig immunoglobulin secondary antibody (Dako) diluted 1: 100 in PBS for 30 minutes and then washed three times with PBS. Then, tyramide amplification was performed by applying biotinyl tyramide solution 1:50 in amplification diluent (TSA ^™ Biotin System, NEN, Boston, Mass.) For 5 minutes, washed three times with PBS, and then streptavidin-per Incubated with oxidase solution (1: 100 in PBS) for 30 minutes.

P62CT antibody binding was detected using the TSA ^™ Biotin System. Reactivity of ubiquitin and K8 / 18 antibody and detected using a ABComplex system (Dako), was applied to a sealed portion of the cover slip, and then rinsed with tap water the (mounting medium) Aquatex ^® (Merck Ltd.).

For color development, incubate with 3-amino-9-ethylcarbazole (AEC, manufactured by Dako) for 5 minutes, wash three times with PBS, counterstain with Mayr's haemalaun, rinse with tap water and cover slip. Aquatex ^R (manufactured by Merck) was enclosed in the flask.

B) Double-label immunofluorescence microscopy on the frozen portion: The frozen portion (3 μm thick) was cut and air dried using Cryocut (Freezer) (Leica CM3050, Leica, Nussloch, Germany) and dried at -20 ° C., acetone. Fixed for 10 minutes. Alternatively (particularly where preservation of the nuclear structure is needed) the part was fixed in PBS-buffered 4% formaldehyde for 15 minutes at room temperature followed by acetone for 5 minutes at -20 ° C. This part is fixed and then air dried or rinsed with PBS.

Then, the first primary antibody p62CT (polyclonal guinea pig antibody against the C-terminal peptide sequence of p62) (Zatlouka K. et al., Am. J. Pathol., 2002, 160: 255-263), K8 Antibodies (Ks 8.7, Progen, Heidelberg, Germany), K18 (Ks 18.04, Progen), K8 / 18 (50K 160) and ubiquitin (ID Labs Inc., Nuncrosilde, Canada) were wetted in Bioassay plate, Nunc, Denmark Rosilde), and applied at room temperature for 30 minutes. Alternatively, the antibody was applied overnight at 4 ° C. and washed three times with PBS for 5 minutes.

In the next step, the first secondary antibody was applied under light protection for 30 minutes at room temperature in a wet room and then washed three times with PBS for 5 minutes. Apply a second primary antibody for 30 minutes at room temperature in a light protected room, then wash three times with PBS for 5 minutes. Apply a second secondary antibody for 30 minutes at room temperature in a photoprotective chamber and then wash three times with PBS for 5 minutes. After the last antibody incubation, the slides are washed with distilled water followed by several seconds with ethanol and then air dried.

Secondary antibodies used are, for example, fluorescein isothiocyanate (FITC) -linked goat anti-mouse IgG (Zymed, San Francisco, CA) or Alexa 488 nm-linked goat anti-mouse IgG (Molecular Probes, Leiden, The Netherlands) And tetramethylenerodamine isothiocyanate (TRITC)-or FTTC-linked porcine anti-rabbit Ig (Dako, Danish Glosstrop) and TRITC-linked rabbit anti guinea pig Ig (Dako).

Finally, the specimen is enclosed with Mowiol (17% mowiol 4-88 [Calbiochem Nr. 475904], 34% glycerol in PBS) or other commercially available encapsulants.

All antibodies are diluted in PBS and incubated separately sequentially. The fluorochrome-bound antibody is centrifuged at 16,000 x g for 5 minutes to remove aggregates before applying to the slides. For negative control, the first antibody is replaced with PBS, pre-immune serum or isotope-matched immunoglobulins.

Immunofluorescence specimens were analyzed by a laser scanning microscope (LSM510 laser-scanning microscope, Obercoken Zeiss, Germany). Awards are obtained using a multitrack form for co-localization analysis (dual labeling). The combined picture appears green / red pseudo-color and yellow at the co-localization site. Store slides at + 4 ° C. while protecting them from light.

Example  3: effect of antioxidant according to the invention on liver pathology

To assess the effect of the antioxidant according to the invention on the regression of morphological modifications in DDC- or GF addicted mouse livers, a positive control animal group (3-7 days exposure only to DDC or GF) was subjected to DDC- or GF poisoning. Mitoquinol [10- (3,6-dihydroxy-4,5-dimethoxy-2-methylphenyl) decyl] triphenylphosphonium bromide and mitoquinone [10- (4,5-dimethoxy-2-methyl- 3,6-dioxo-1,4-cyclohexadien-1-yl) decyl] -tridecylphosphonium bromide (provided by Key Organics Ltd, London, UK) or Mito Vit E [2- (3,4 -Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl) ethyl] -triphenylphosphonium bromide from Key Organics Ltd, London, UK Compared to mice treated for 3-7 more days.

For injection, MitoQ or Mito Vit E is dissolved in PBS supplemented with sufficient DMSO (preferably 1%) to maintain the solubility of the antioxidant. Mice were intraperitoneally or intravenously injected (tail vein) and compared with excipient-injected controls. These are Smith R.A. J et al., 2003, PNAS, 100 (9): at a maximum allowable dose of 20 mg MitoQ / kg / day (750 nmol) and 6 mg Mito Vit E / kg / day (300 nmol) according to 5407-5412. Corresponds.

MitoS (mitoquinol [10- (3,6-dihydroxy-4,5-dimethoxy-2-methylphenyl) decyl] triphenylphosphonium methane sulfonate and mitoquinone [10- (4,5-dimethoxy- MitoQ or MitoQ derivatives such as 2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl) decyl] -tridecylphosphonium methane sulfonate or a mixture of Mito Vit E) Supplemented. Dosage is determined by measuring water or liquid dietary consumption and mouse weight. Mice have no indication of toxicity, 500 μM or 1 mM MitoQ or MitoS (maximum permissible dose of 232 μmol / kg / day or 346 μmol according to Smith RA, J. et al., 2003, PNAS, 100 (9): 5407-5412 / kg / day, corresponding to 154 and 230 mg / kg / day for 500 μM and 1 mM diets respectively or 500 μM Mito Vit E (maximum permissible dose 105 μmol / kg / day, 60 mg of Mito Vit E corresponding to kg / day) for 3 to 7 days of drinking water.

As another test, the DDC- or GF addicted animals were simultaneously added Mito Q (MitoS) or Mito Vit E for 3-7 days and compared with controls exposed to DDC or GF only for 3-7 days.

As another experiment, 3-12 mg / kg of Mito Q dissolved in 1% DMSO in PBS was intraperitoneally administered to DDC-addicted mice for 3 days and compared with control animals (positive control indicates negative while DDC-addicted mice) Control group is an excipient-injected animal without DDC poisoning).

In order to substantially evaluate the effects of mitochondrial targeted antioxidants according to the present invention in these short-term experiments, the presence (or absence) of inflammatory cells and the necrosis of the cytoskeleton instead of necrosis, cell swelling around the portal veins (trias of Gleason) Destruction (see FIGS. 2 and 3) and / or Mallory bodies analysis (typical when prolonged exposure to DCC or GF) was compared to a positive control. Cell expansion and Mallory bodies are not formed to the extent that statistical evaluation is possible due to the fact that they do not form upon short exposure to DDC.

In total, the normal structure, represented by strands of hepatocytes bounded by oyster blood vessels under Mito Q treatment, is seen again. Hepatocyte morphology is normal in size and shape of the nucleus and in the structure of the cytoplasm. In addition, the number of inflammatory cells (eg neutrophils, lymphocytes) is significantly reduced upon treatment with the antioxidant according to the invention.

In long-term experiments using mice addicted to DDC (8-10 weeks), determine the presence (or absence) of cell swelling and / or malorisomes (MB) in liver samples of treated animals and compare with control animals do.

In one experiment, when poisoned by DDC or GF for 10 weeks, test animals were intraperitoneally or intravenously injected (tail vein) with a pair of mice for 7 days with Mito Vit E or Mito Q (or MitoS) and excipient-injected controls. Compare with

As another experiment, MitoQ (1.25 mg / kg) is intraperitoneally injected (Days 1 and 4 of the corresponding week) for 7 days after being intoxicated by DDC for 10 weeks and analyzed according to conventional histology. (Luna LG, 1968, Manual of Histologic staining methods of the Armed Forces Institute of Pathology, 3rd edition, McGraw Hill, New York). The degree of cell expansion and the number of Mallory bodies are markedly reduced in MitoQ treated animals intoxicated with DDC when compared to a suitable control (FIGS. 4-6).

As another experiment, mice poisoned for 8-10 weeks by DDC or GF are fed MitoQ (MitoS) or Mito Vit E through drinking water for 7-14 days.

As another experiment, mice that were poisoned for 6 weeks by DDC or GF were administered simultaneously with DDC or GF for 4 weeks using Mito Vit E or MitoQ (MitoS) at a maximum allowable dose of 10-50% and for 10 weeks with DDC or GF alone. Compare to toxic control animals.

As another experiment, mice intoxicated with DDC or GF for 10 weeks are recovered for 4 weeks. Subsequent re-addiction with DDC or GF and treatment with Mito Vit E or MitoQ / MitoS resulted in a toxic memory effect (resulting in 24-72 hours of re-exposure to DDC or GF poisoning) by treatment with mitochondrially targeted antioxidants. Reduced or eliminated.

To assess the prophylactic effect of antioxidants in liver disease, a group of mice fed DDC- or GF were simultaneously administered a maximum allowable dose of 10 to 50% of MitoQ, MitoS or Mito Vit E followed by DDC- or GF only. Compare to control animals poisoned for 10 weeks.

As one experiment, MitoQ, MitoS or Mito Vit E is administered during the initial recovery period (4 weeks) and then addicted to DDC or GF for 24 to 72 hours and compared with the untreated control animals.

Those skilled in the art will appreciate that various modifications of the general scheme are possible.

Overall, in both short and long term poisoning by DDC or GF, noticeable liver modifications are significantly alleviated or reduced by the application of the antioxidants of the invention used for the treatment or prevention of liver disease and / or epithelial cancer.

Example  4: with or without treatment of antioxidant according to the invention DDC  In mitochondria isolated from poisoned mice Oxidative  Determination of Stress (Measurement of Tocopherol Quinone Content)

4.1. Mouse liver mitochondria ( MLM ) Separation

Lett Heart Mitochondria (Staniek K. and Nohl H., 1999, Biochem. Et Biophys. Acta, 1413: 70-80; Mela L. and Sietz S., 1979, Methods in Enzymology, Academic Press Inc .: 39-46) The method for was applied to mouse liver (about 10% by weight relative to rat liver) isolated from the various animal groups according to Example 3. Separation of the liver was carried out at 4 ℃. Each liver was operated and stored by shock-freezing in liquid nitrogen (N ₂ ). Preparation buffer (0.3 M sucrose, 1 mM EDTA, 20 mM triethanolamine pH 7.4 in addition to 10 mg / L BHT (di-tert-butyl-hydroxytoluene) and 1 mM diethylenetriaminepentaacetic acid to prevent tocopherol oxidation After thawing in Fe chelator), the tissue is cut into small pieces, washed four times with prep buffer, homogenized five times with Potter pistil in 15 ml buffer, diluted to 30 ml Centrifuged at 570 g for 10 minutes. The supernatant was filtered through two layers of cheese cloth. Mitochondria were pelleted at 7400 g for 10 minutes, resuspended by hand in 30 ml buffer, repellet as above, washed and finally resuspended in about 200 ml buffer. Protein concentration was measured by the Burette method (using BSA as standard, at least 200 mg protein for double determination) to obtain the expected yield of 3-6 mg.

For normalization purposes, the cytochrome concentration is 0.2% (w / v) Triton X-100 (amico DW2000 photometer, requires about 0.5-1 mg mitochondrial protein for double crystals) (Williams JN, Jr., 1964, Archives of Biochemistry and Biophysics, 107: 537-543) and then calculated by subtracting the air oxidized difference spectrum from reduced dithionite; The expected concentrations of Cyt (a + a ₃ ), Cyt c, Cyt c _1, and Cyt b in healthy mitochondria are extrapolated from the rat liver mitochondria, 0.1-0.3 nmol / mg protein, respectively (Wakabayashi T. et al., 2000 , Pathology International, 50: 20-33).

As a control, crude homogenous equivalents (after first homogenization) containing all cell membranes should be maintained. The whole membrane containing the lightest fraction (microsome) is pelleted in 165 000 g for 40 minutes (ultracentrifugation) according to Murias M. et al., 2005, iochemical Pharmacology, 69: 903-912, and 5 mL preparative. Wash with buffer and resuspend and finally resuspend in about 200 mL buffer. Protein concentration is measured as follows.

4.2. Tocopherol Quinone ( TQ ) Analysis

The full MLM (see paragraph 4.1) may be used. The amount of 2-5 mg protein (mitochondria, whole membrane or various fractions) in 1 ml H ₂ O was mixed with 5 mM SDS and 2 nmol UQ ₆ (ubiquinone-6, internal reference) and 3 ml anaerobic ethanol / hexane ( 2: 5). The organic phase was evaporated under argon and the residue was dissolved in 120 ml ethanol. 40 ml was used for HPLC analysis (dual analysis per sample) on a Waters LC1 module with a C18 column. Quinones and tocopherols were eluted at 1 ml / min with 50 mM NaClO ₄ in ethanol / methanol / acetonitrile / HClO ₄ (400: 300: 300: 1) and optically (268 nm for TQ, 268 nm, UQ _6). And 275 nm for endogenous UQ ₉ and UQ ₁₀ ) or electrochemically (+0.6 V, for tocopherol and quinol) according to Gille L. et al., 2004, Biochemical Pharmacology, 68: 373-381; The expected TQ content in healthy mitochondria is 1-5% relative to cotoperol or ubiquinone (Gille L. et al., 2004, Biochemical Pharmacology, 68: 373-381).

Overall, these tests show elevated TQ levels in mitochondria of DDC addicted mice compared to control and DDC mice treated with antioxidants according to the present invention.

4.3. Mouse liver mitochondria ( MLM Additional enzyme) Complex  analysis

MLMs derived from various groups of animals (see the sequence of Example 3) were frozen and thawed 2-3 times to break the membranes, thereby reducing various membranes according to Fato R. et al., 1996, Biochemistry, 35: 2705-2716 (See reference). Photometric analysis can be performed at 25 ° C. (Aminco DW2000 dual wavelength photometer) and requires about 5-20 mg of mitochondrial protein per assay.

a) Aconitase (marker for superoxide damage) (James AM et al., 2005, JBC, March 23, 2005, Manuscript M501527200). The assay contains 0.6 mM MnCl ₂ , 5 mM Na citrate, 0.2 mM NADP ⁺ , 0.1% Triton X-100 0.4 U / mL isocitrate dehydrogenase and 50 mM Tris pH 7.4. NADPH production is carried out at 340-410 nm; The expected activity in healthy mitochondria is about 60 nmol / min for isolated mitochondria mg according to Senft AP et al., 2002, Toxicology and Applied Pharmacology 178: 15-21.

b) Complex I (NADH dehydrogenase) (denatured from Estornell E. et al., 1993, FEBS, 332, No. 1, 2: 127-131): this assay is 0.1 mM NADH, 0.05 mM decylubiquinone, 2 It contains mM KCN, 20 mM antimycin A and 20 mM Tris pH 7.5. NADH digestion is carried out at 340-410 nm. Inhibition with 2 mg / mL rotenone corrects for nonspecific quinone reduction; Expected activity in healthy mitochondria is about 100-300 nmol / (min. According to Stuart JA et al., 2005, Free Radical Biology & Medicine, 38: 737-745 and Barreto MC, 2003, Toxicology Letters, 146: 37-47. mg).

c) Complex II (Succinate Dehydrogenase) (modified by Gilille 2001): This assay contains 2 mM succinate, 0.05 mM decylubiquinone, 2 mM KCN, 20 mM antimycin A and 20 mM Tris pH 7.5 . Quinone decomposition is carried out at 275 -320 nm. Inhibition by 25 mM malonate corrects for nonspecific quinone reduction; The expected activity in healthy mitochondria is about 70-100 nmol / min per mg of minochondria isolated according to Barreto M.C., 2003, Toxicology Letters, 146: 37-47.

d) Complex III (cytochrome be ₁ ) (modified by Stuart JA et al., 2005, Feee Radical Biology & Medicine, 38: 737-745). This assay contains 0.05 mM decylubiquinol (prepared from decylubiquinone by dithionite reduction and hexane extraction), 15 mM Cyt c, mM KCN and 20 mM Tris pH 7.5. Cyt c reduction is carried out at 550-540 nm. Inhibition by 20 mM antimycin A corrects for nonspecific quinol oxidation; The expected activity in healthy mitochondria is about 80 nmol / min per mg of mitochondria isolated according to Stuart JA et al., 2005, Free Radical Biology & Medicine, 38: 737-745.

e) Complex IV (cytochrome oxidase) (modified by Stuart J.A. et al., 2005, Feee Radical Biology & Medicine, 38: 737-745). This assay contains 15 mM reduced Cyt c (prepared by dithionite reduction and air oxidation of excess dithionite) and 20 mM Tris pH 7.5. Cyt c oxidation is carried out at 550-540 nm. Activity in healthy mitochondria: about 1 nmol / min per mg of isolated mitochondria (Stuart J. A. et al., 2005, Free Radical Biology & Medicine, 38: 737-745).

Example  5: oxidative stress induced protein ( Hemoxigenase  I) evaluation

In order to assess the expression of hemoxigenase 1 (HO-1) protein known to be induced by oxidative stress (Suematsu M. and Ishimura Y., 2000, Hepatology, 31 (1): 3-6), DDC poisoning Standard Western blot analysis was performed using proteins extracted from mice treated with mice only or excipients (coated in Example 3 purely) or mice co-treated with MitoQ (diluted in 1% DMSO in PBS) for 3 days.

Liver tissue was ice cooled and RIPA supplemented with 2 μg / ml leupeptin, 2 μg / ml pepstatin, 2 μg / ml aprotinin, 1 mM phenylmethylsulfonylfluoride (PMSE) and 2 mM dithiothreitol Resuspend in buffer (50 mM Tris-HCL pH 7.4, 250 mM NaCl, 0.1% SDS, 1% deoxycholate, 1% NP-40) and then sonicate on ice (2 bursts for 5 seconds). Homogenized. After 20 minutes of incubation on ice, the lysate was made clear by centrifugation twice by microcentrifugation at 13 000 rpm at 4 ° C. for 15 minutes, and the supernatant was collected. Protein concentration was determined by the Bradford assay (Biorad) using bovine serum albumin as standard. Equal amounts of protein (typically 10-30 μg) were separated on a 12% SDS-PAGE gel and subjected to semidry-blotting (TE 70, made by Amersham) polyvinylidene difluoride (PVDF) membrane (Hybond-P, Electrophoresis). Blocking at room temperature for 1 h in blocking solution [5% milk in TBS-T (3 mM KCl with 25 mM Tris-HCl pH 7.4, 137 mM NaCl, 0.1% Tween-20)] and then the primary antibody solution (TBS-T / Incubated with stirring overnight at 4 ° C.). Antibodies specific for the following antigens were used: HO-1 (dilution: 1: 1000) that cross reacts with constitutively expressed isoforms HO-2 (36 kDa) and β-actin (1: 5000, manufactured by Sigma) ; Stress genes). After removing the primary antibody solution and washing several times with TBS-T, the membranes were incubated with HRP (Cabbali peroxidase) -bound secondary antibody (Rabbit anti-mouse, 1: 1000; manufactured by Dako) for 1 hour at room temperature. It was. After washing several times with TBS-T, detection is performed by exposure to chemiluminescence (ECL, manufactured by Amersham) and X-ray film (FIG. 7). The size of the bands can be analyzed by densitometry using ChemImager 5500 software (made by Alpha Innotech) and each signal can be analyzed by HO-1 when treated with Mito Q when compared to the corresponding HO-2 (DDC-addicted animal group). 7-fold reduction) or otherwise normalized to β-actin.

Significant reduction in DDC induced overexpression of hemoxigenase under MitoQ treatment (FIG. 7) means that oxidative stress is significantly reduced by the antioxidant according to the invention.

In long-term trials reported reduction in cytokeratin 8 and / or (N) ASH patients known to be increased in mice during DCC poisoning (Stumptner C. et al., 2001, Journal of Hepatology, 34: 665-675) The amount of protein expression of catalase can be assessed (Videla LA et al., 2004, Clinical Science, 106: 261-268).

Protein refraction of fatty acid binding protein (FABP) indicating a sensitive marker for hepatocyte damage (Monbaliu D. et al., 2005, Transplant Proc. 37 (1): 413-416) was measured. Western blot analysis shows a significant decrease in FABP protein in DDC addicted mice as compared to normal mice. Also, under MitoQ treatment of DDC-addicted animals, FABP reached almost control mouse FABP protein expression values (control represents the non-addicted animal group treated with excipients only, see Example 3), which is a disease according to the present invention. The effectiveness of MitoQ in the treatment or prevention of the disease

The amount of apoptotic cells in the frozen microcutter portion derived from DDC addicted mice treated with antioxidants according to the present invention was semi-quantified by anti-caspase 3 immunohistochemical reference methods known in the art (Brekken et al. , 2003, The Journal of Clinical Investigation, 111, 4: 487-495) and can be compared with a suitable control.

In addition, protoporphyrin levels in homogenates of DDC poisoned mice treated with antioxidants can be measured using fluorescence assays (Stumptner C. et al., 2001, Journal of Hepatology, 34: 665-675). Compared.

Example  6: evaluation of the effect of the antioxidant according to the invention on blood parameters

Serum levels of liver specific enzymes were monitored by Actitest (Biopredictive, Houilles, France) providing the extent of liver damage in accordance with the present invention. Serum levels of a ₂ -macroglobulin, haptoglobin, γ-glutamyl transpeptidase, total bilirubin, apolipoprotein A1, and alanine aminotransferase were determined to be DDC- or GF addicted, control and corresponding DDC- or GF. Animals treated with exposed and targeted antioxidants were measured following the method described in Poynard, et al., 2003, Hepatology 38: 481-492 and the general timeline according to Example 3.

Actitest conducted using human serum to the extent of liver damage, in particular to the degree of fibrosis, can similarly be applied to monitor the therapeutic effect of patients with these diseases using the antioxidants according to the invention.

In serum from various test animal groups according to variables indicative of liver damage, bilirubin, alumina aminotransferase (ALT / GPT), aspartate aminotransferase (ASAT / GOT) and glutamate dehydrogenase (GLDH) were hitachi / Clinical diagnostics using a kit commercially available on the Roche 917 analyzer (No. 11552414; 11876805216; 11876848216; 119299992, all manufactured by Roche AG, Switzerland) was determined according to standard procedures.

In animals treated with a compound according to the invention (eg alanine- and aspartate aminotransferase, see FIG. 8), a decrease in serum liver enzymes indicates a reduction in liver damage in such treated samples and in a disease according to the invention. Provide support for the therapeutic effects of the compounds.

Example  7: reactive oxygen species in the tissue fraction ( ROS ) Measurement

In order to detect the production of ROS in liver specimens and control tissues obtained from DDC- or GF intoxicated tissues, fluorescence microscopy using dihydroethidium (DHE, Molecular Probes) was performed according to standard methods (eg Brandes). RP et al., 2002, Free Radic Biol Med .; 32 (11); 1116-1122). DHE can be freely permeable to cells and oxidized to ethidium in the presence of O ₂ and entrained therein by intercalating with DNA in the nucleus. Ethidium is excited at 488 nm and has an emission spectrum at 610 nm.

Liver samples were embedded in OTC tissue tech (Sakura Finetek Europe, Zoeterwonde, The Netherlands) and frozen using liquid nitrogen-cooled isopentane. The sample is cut into portions (5 μm-30 μm) and placed on a glass slide. Dihydroethidium (5-20 μmol / L) was applied to each tissue portion. Slides were incubated for 30 min at 37 ° C. in wet, photoprotected and washed (2-3 times) with buffered saline (PBS) at 37 ° C. Cover the part with a cover slip. Images of DHE are obtained using fluorescence microscopy or laser scanning confocal images using a 585 nm long time pass filter.

Another method established in the art is DDC- or GF for control liver tissue using Lucigenin chemiluminescence assay (Goerlach A. et al., 2000, Circ Res., 87 (1): 26-32). Enables measurement of ROS production in poisoned tissues. Specimens of liver tissue were prepared with 1 ml of 50 mmol / L HEPES (pH 7.4), 135 mmol / L NaCl, 1 mmol / L CaCl ₂ , 1 mmol / L MgCl ₂ , 5 mmol / L KCl, 5.5 mmol / L glucose and Homogenized in a vial containing 5 μmol / L lucigenin as the electron acceptor. The photoreaction between superoxide and lucigenin was detected using a chemiluminescent reader. Chemiluminescent signals are expressed as mean counts / minute / mg dry tissue measured over 15-30 minutes. Chemiluminescent signal data is displayed after subtracting background chemiluminescence values observed without specimens.

This method is similar to that observed in patients with the disease according to the invention, as it shows an increase in ROS in the liver derived from DDC- or GF addicted mice.

Example  8: DDC  or GF Reactive mice in mice exposed to Oxidative  Effect of the antioxidant according to the invention on the reduction of damage)

The timeline for dosing with DDC- or GF poisoning and antioxidants in test animals and the general method for dosing schedule are the same as the experimental procedure used to determine morphological abnormalities (see Example 3).

Administration of antioxidants according to the invention, such as derivatives of vitamin E, coenzyme Q ₁₀ or glutamine peroxidase analogues, provide a significant reduction in ROS levels in livers exposed to DDC or GF. This result also indicates the effect of ROS on liver damage and indicates that such damage can be reduced by targeting MitoQ / MitoS or Mito Vit E to the mitochondria, a major cell source of ROS. The decrease in ROS levels measured by the method according to Example 7 when treated with a targeted antioxidant illustrates the therapeutic efficacy of the compounds according to the invention.

Example  9: reactive oxygen species in liver cell lines ROS ) Measurement

Liver cancer cell line (eg HepG2 or Hep3B), SNU-398 hepatocellular carcinoma-induced cell line (ATCC No. CRL-2233, LGC promochem, Germany), HUH-7 human carcinoma-induced cell line (Japanese coleection of Research Biosources JCRB 0403) or Tib A simple experiment applying the -73 mouse embryonic liver cell line (ATCC TIB 73 = BNL CL2 derived from BAL / c mice, Maryland, USA) allows measurement of ROS production in these cells upon DDC poisoning. Glutathione synthesis inhibitor L-butionine- (S, R) -sulfoxymin (BSO) is applied to increase endogenous oxidative stress (Kito M. et al., 2002, Biochem Biophys Res Commun., 291 (4) ): 861-867).

CoCl ₂ is to measure the mitochondrial ROS production in differentiated cell lines: Since known to affect the (Jung JY and Kim WJ, 2004 , Neurosci Lett, 371.. 85-90) recently, HepG2 is 100 μM CoCl ₂ (Manufactured by Sigma) (Bel Aiba RS, et al., 2004, Biol Chem. 385: 249-57).

Other well established methods on cultured cells (as well as isolated organelles or whole tissues) are described in Chem Biol Interact. 2000 Jul 14; Rotenone using antimycin A (FIG. 10) or lucigenin chemiluminescence assay (Goerlach A. et al., 2000, Circ Res., 87 (1): 26-32) according to 127 (3): 201-217 Enables measurement of ROS production induced by

For example, to measure ROS production in liver cancer cell lines, standard experimental methods according to Example 8 are used. Tested liver cancer cells were grown to 80% confluency in 96-well plates in culture medium (DMEM supplemented with 10% FCS (Gibco)) and then washed with HBSS and with DHE (10-50 μM) at 37 ° C. Incubate for 10 minutes in the dark. Cells are washed twice with Hank balance salt solution (HBSS, Gibco) to remove excess dye. Fluorescence was monitored by fluorescence microscopy (Olympus, Hamburg, Germany).

Alternatively, the production of ROS is fluoroprobe 5- (and -6) -chloro converted to fluorescent dichlorofluorosane (DCF; Djordjevic T. et al., 2004, Antioxidants & Redox Signaling, 6: 713-720). It was measured using methyl-2 ', 7'-dichlorodihydrofluorescein diacetate ester (CM-H ₂ DCFDA, Molecular Probes, Goettingen, Germnay). To measure DCF fluorescence using a microplate reader (Tecan, Crailsheim, Germany), cells (eg HepG2, Hep3B or SNU-398) were grown to 80% confluency in 96-well plates. The cells were washed twice with Hank balanced salt solution (HBSS, Gibco) and CM dissolved in N-ω-nitro-L-arginine methyl ester (L-NAME, 10 μM) at 37 ° C to prevent NO formation. Incubated for 10 min in the dark with -H ₂ DCFDA (8.5 μM). After washing several times with HBSS to remove excess dye, fluorescence was monitored by using 480 nm excitation and 540 nm emission wavelengths. DCF fluorescence was normalized to the number of viable cells using the Alamar Blue test according to the manufacturer's instructions (Biosource, Nivelles, Belgium). Briefly, cells are incubated with Almar Blue in phosphate-buffered saline (PBS), pH 7.4 at 37 ° C. to show a change from fully oxidized (blue) to reduced (red) form. Absorbance was measured at 580 nm wavelength.

If desired, ROS production was assessed by flow cytometry analysis of CM-H ₂ DCFDA stained cells. Cells were separated and collected by trypsinization, harvested by centrifugation and resuspended in HBSS at a concentration of 1 × 10 ⁶ cells / ml. 8.5 μM of CM-H ₂ DCFDA was added to the cells in the dark at 37 ° C. prior to stimulation. DCF fluorescence was monitored by analyzing 10,000 cells using 480 nm excitation and 540 nm emission wavelengths in a flow cytometer (Partec, Muenster, Germany).

Liver cancer cell lines cultured for up to 72 hours in culture medium supplemented with DDC (EC ₅₀ = 50 μg / ml medium) or 100 μM of BSO (DMEM and 10% FCS, Gibco) patients with disease according to the invention Another in vitro model similar to the observation in.

Example  10: DDC -, BSO -, Antimycin  A- (or Rotenon -) Addicted or CoCl ₂  Induced In culture medium Oxidative  Effect of the antioxidant according to the invention on the reduction of damage

Using the general strategy and standard techniques of timetables according to Example 9, DDC or BSO addicted and simultaneously MitoQ / MitoS or Mito Vit E (Jauslin ML et al., 2003, FASEB J., 2003, (13): 1972- According to 1974, human cell lines simultaneously treated with EC ₅₀ = 0.51 nM for MitoQ and EC ₅₀ = 416 nM for Mito Vit E) provide a significant reduction in ROS formation.

As another experiment, HepG2 stimulated with 100 μM CoCl ₂ (manufactured by Sigma) was used (Bel Aiba RS et al., 2004, Biol Chem. 385: 249-57). Following the experimental procedure described in Example 9, HepG2 cells were plated in 96-well plates and serum was not provided for 16 hours prior to testing. HepG2 was washed once with HBSS (Hank's equilibrium salt solution, Gibco) and incubated with MitoQ or corresponding amount of DMSO (manufactured by Sigma) at a concentration of 0.5-10 μM. After 15 minutes DCF was added to the cells (final concentration 8 μM) and the cells were incubated with the dye for 10 minutes. After loading the medium is removed and fresh HBSS containing MitoQ and CoCl ₂ (100 μM) is added. Fluorescence was measured on a plate-reader (Tecan Safire) after 0, 10, 20 and 30 minutes (Djordjevic T, et al., 2005, Free Radic Biol Med. 38: 616-30).

Unstimulated cells treated with 5 μM MitoQ showed a decrease in basal ROS production. CoCl ₂ -stimulated ROS production (100 μM CoCl ₂ ) was significantly reduced by 5 μM MitoQ, meaning that the antioxidant according to the present invention significantly reduced basal and CoCl ₂ -stimulated ROS levels in these cells (FIG. 9).

Another method enables the measurement of ROS production induced by antimycin A or rotenone, such as in HUH-7 or Tib-73 using lucigenin chemiluminescence (experimental equipment as in Example 9). HUH-7 cells were cultured in 6 well plates and stimulated with antimycin A at 0-25 μM concentrations (preferably 0, 1 and 5 μM) and dissolved in DMEM (Gibco) for 3 hours at 37 ° C. It is not stimulated or stimulated simultaneously by Mito Q (or MitoS) in the range of from 1000 nmol. After three successive washes, the cells were washed with 1 ml of 50 mmol / L HEPES (pH 7.4), 135 mmol / L NaCl, 1 mmol / L CaCl ₂ , 1 mmol / L MgCl ₂ , 5 mmol / L KCl, 5.5 mmol / Balance the plates containing L glucose and 5 μmol / L lucigenin as electrode receptor. The photoreaction between superoxide and lucigenin was detected using a chemiluminescent reader (Lumistar, BMG Laboratories, Germany). Chemiluminescent signals are expressed in average counts / minute and normalized to cell number as measured by a cell counter (Casy Technology Instrument, Scharfe-System, Germany).

Overall, these experiments show a significant decrease in ROS formation (FIG. 10), thus confirming the therapeutic effect of mitochondrially targeted antioxidants in liver disease according to the present invention.

Example  11: Evaluation of the Effect of Antioxidant Compounds in Nude Mice

A general strategy for measuring the effect of mitochondrial targeted antioxidants according to the invention in the treatment and / or prevention of epithelial cancer is that human epithelial cancer xenografts (nude mouse tumor xenografts are supplied by, for example, Charles River Laboratories, USA). Except for the application of immunocompensated mice with CD1 nu / nu mice), the treatment regime described above is followed for DDC- or GF addicted mice (according to Examples 2-7). Tumor cell lines or primary tumors subcutaneously xenografted according to standard methods (Li K. et al., 2003, Cancer Res., 63 (13): 3593-3597) are rectal adenocarcinomas, invasive coronary breast cancers, small and non-small. Cell lung cancer, prostate tumor, pancreatic tumor and gastric tumor.

Tumor-induced cell lines (growing in DMEM / 10% FBS) are collected in log-phase growth, washed twice with PBS, resuspended in 1 ml PBS (2.5 × 10 ⁷ cells / ml) and nude mice (Hsd: Subcutaneous injection of 5 x 10 ⁶ cells / mouse (0.2 ml) to the right of athymic nu / nu, Harlan Winkelamnn; 6-6 weeks old). Tumor growth was monitored every other day for the indicated period (depending on cell type). Tumor size was determined by two vertical diameters and heights on the skin surface.

Mouse treatment with MitoQ (MitoS) reduces tumor growth, increases tumor necrosis and reduces vascularization of tumor xenografts. Similarly, the level of ROS in nude mouse tumor xenografts was monitored as described above and decreased in xenograft tumors treated with the antioxidant according to the invention.

It will be appreciated by those skilled in the art that various modifications are possible to the compositions and methods of the present invention. Accordingly, the invention is to be understood as including all such modifications and variations as long as they fall within the scope of the appended claims and their equivalents. All documents exemplified herein are incorporated herein by reference.

Claims (31)

Use of a mitochondrially targeted antioxidant compound comprising a lipophilic cation covalently bound to an antioxidant moiety for the treatment or prevention of liver disease and / or epithelial cancer. Use according to claim 1, wherein the lipophilic cation is a triphenylphosphonium cation. The use according to claim 1 or 2, wherein the compound has the formula

In the food, X is a linking group, Z ⁻ is an anion and R is an antioxidant moiety. 4. Use according to claim 3, wherein the antioxidant residue R is quinone or quinol. 5. Use according to claim 4, wherein the compound has the formula:

4. Use according to claim 3, wherein the antioxidant residue R is a glutathione peroxidase analog. Use according to claim 6, wherein the glutathione peroxidase analog drug moiety is of the formula:

4. The radical radical scavenger according to claim 3, wherein the antioxidant moiety R is a general radical destroyer including vitamin E and vitamin E derivatives, chain breakdown antioxidants, butylated hydroxyanisole, butylated hydroxytoluene, induced fullerene, Use selected from the group comprising spin traps comprising derivatives of 5-dimethylpyrroline N-oxide, t-butylnitrosobenzene, α-phenyl-tbutylnitron and related compounds. 4. Use according to claim 3, wherein the antioxidant residue R is a vitamin E or vitamin E derivative. Use according to claim 9, wherein the compound has the formula:

4. Use according to claim 3, wherein the antioxidant residue R is butylated hydroxyanisole or butylated hydroxytoluene. 4. Use according to claim 3, wherein the antioxidant residue R is derived fullerene. 4. Use according to claim 3, wherein the antioxidant residue R is 5,5-dimethylpyrroline N-oxide, t-butylnitrosobenzene, α-phenyl-t-butylnitron or derivatives thereof. The use according to claim 13, wherein the compound has the formula

The C ₁ -C ₃₀ carbon according to claim 3, wherein the linking group X optionally has one or more double or triple bonds and optionally comprises one or more unsubstituted or substituted alkyl, alkenyl or alkynyl side chains. Uses that are chains. Use according to claim 15, wherein the linking group X is (CH ₂ ) _n , wherein n is an integer from 1 to 20. 17. Use according to claim 16, wherein the linking group X is an ethylene, propylene, butylene, pentylene or decylene group. 18. Use according to any one of claims 3 to 17, wherein the anion Z ⁻ is a pharmaceutically acceptable anion. Use according to claim 18, wherein Z ⁻ is a halide. 20. The use of claim 19, wherein Z ⁻ is bromide. The use according to claim 18, wherein Z ⁻ is an anion of alkane- or arylsulfonic acid. Use according to claim 21, wherein Z ⁻ is methanesulfonate. The use of claim 22, wherein the compound has the formula:

The method of claim 1, wherein the liver disease is in alcoholic liver disease, non-alcoholic fatty liver disease, steatosis, cholestasis, cirrhosis, nutrition-mediated liver injury, toxin liver injury, infectious liver disease, sepsis. Use selected from the group consisting of liver damage, autoimmune-mediated liver disease, hemochromatosis, alpha1 antitrypsin deficiency, radiation-mediated liver injury, liver cancer, benign liver neoplasia and focal nodular hyperplasia. The method of claim 1, wherein the liver disease is in alcoholic liver disease, non-alcoholic fatty liver disease, steatosis, cholestasis, cirrhosis, nutrition-mediated liver injury, toxin liver injury, infectious liver disease, sepsis. Use selected from the group consisting of liver damage, autoimmune-mediated liver disease, hemochromatosis, alpha1 antitrypsin deficiency, radiation-mediated liver injury. The use according to any one of claims 1 to 23, wherein the liver disease is alcoholic liver disease or non-alcoholic fatty liver disease. Use according to any one of claims 1 to 23, wherein the liver disease is an alcoholic fatty liver disease or a non-alcoholic fatty liver disease. Use of a mitochondrially targeted antioxidant compound according to any one of claims 1 to 23, wherein the liver disease is an alcoholic fatty liver disease. Use according to any one of claims 1 to 23, wherein the liver disease is a non-alcoholic fatty liver disease. Use of a mitochondrially targeted antioxidant compound comprising a lipophilic cation covalently bound to an antioxidant moiety for treating or preventing liver disease and epithelial cancer according to any one of claims 1 to 29. 30. A method of treating patients with liver disease and epithelial cancer comprising administering to a patient in need thereof a therapeutically effective amount of a compound according to any one of claims 1 to 29.

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