WO2015071373A1

WO2015071373A1 - Formulations comprising s-adenosyl-methionine, quercetin and glycyrrhizin for liver health

Info

Publication number: WO2015071373A1
Application number: PCT/EP2014/074521
Authority: WO
Inventors: Caroline GREMILLET; Rosita GARCIA; Emmanuelle LEMAIRE
Original assignee: Ceva Sante Animale SA
Current assignee: Ceva Sante Animale SA
Priority date: 2013-11-13
Filing date: 2014-11-13
Publication date: 2015-05-21
Anticipated expiration: 2016-05-13

Abstract

The present invention relates to the field of therapeutic compositions for the treatment, protection, and repair of hepatic tissue in humans and other animals. More specifically, the compositions of the invention comprise S-adenosylmethionine, quercetin; and glycyrrhizin.

Description

FORMULATIONS COMPRISING S-ADENOSYL-METHIONINE, QUERCETIN AND

GLYCYRRHIZIN FOR LIVER HEALTH

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The mammalian liver is comprised of four lobes. It is the both the largest endocrine gland in the body and the largest internal organ. There are two major types of cells present in the liver: (1) hepatocytes (parenchymal); and (2) non-parenchymal cells. The l i v e r exhibits over five hundred essential physiological functions. Among the major known actions are the production of bile to remove wastes, production of plasma proteins, cholesterol synthesis, protein synthesis, carbohydrate and lipid metabolism, removal of toxins, and production of immunological agents. Hepatocytes perform the majority of these tasks, with non-parenchymal cells contributing ancillary functions.

In order to perform these myriad tasks, the liver has a dedicated vascular system, containing mainly of the hepatic artery and the portal vein. The hepatic artery carries oxygenated blood to the liver from the heart. The portal vein also supplies oxygenated blood, but additionally carries venous blood from the spleen and pancreas. Both the hepatic artery and the portal vein form a large capillary matrix throughout the liver. This prodigious network ensures the effective oxygenation of cells and greater surface area for the exchange of various metabolites, nutrients, and hormones between the liver and the circulatory system.

Specifically, the liver utilizes the portal vein system to process and distribute its various metabolic products, hormones, and digested nutrients from the gastrointestinal tract. The portal vein and its circulatory elements are thus one of the primary points of entry into the general circulatory system of the body.

As the primary gateway for many ingested chemicals, the liver is particularly susceptible to any ingested drugs or toxins. These drugs and toxins arrive in a completely undiluted form from the gastrointestinal tract, such that liver cells are maximally exposed. Ethyl alcohol, a toxin, is metabolized by hepatocytes, and can cause significant damage over time by inducing steatosis, alcoholic hepatitis, and cirrhosis. Even initially nontoxic compounds can cause devastating cytotoxicity in hepatocytes once they are activated by metabolic processes in the liver. One such drug, acetaminophen, is highly therapeutic as an analgesic and antipyretic. However, enzymatic oxidation of the drug in hepatocytes can form a highly reactive metabolite, resulting in acute cytotoxicity and liver failure. Other risks to liver tissue originate from blood-borne pathogens like bacteria or viruses, such as hepatitis A through E.

Types of liver damage include necrosis, degeneration of functional activity, fibrosis, and inflammation. Because the l ive r is at significant risk of damage from its unshielded exposure to various cytotoxic elements, it has a high level of regenerative capacity. While the complete loss of a lobe is permanent, other lobes in the liver can compensate through enlargement. As little as 25% of a complete liver can regenerate and become fully functional. Nevertheless, progressive damage to the liver tissue beyond a certain threshold will be irreversible and life-threatening, leaving liver transplantation as the sole remedy.

Treatment options for most forms of liver damage are limited to symptomatic relief because the only permanent cure for such conditions is the regeneration of healthy hepatocytes. For example, diuretics are used to combat tissue edema, white anti-encephalopathic drugs are used to clear the build-up of toxins. Such drugs can be used to mitigate the harmful effects of liver damage, though they generally produce unintended side effects and may become toxic to the liver itself if taken at high enough concentrations. Treatments by antiviral compounds for hepatitis treat the cause of the liver damage, but are also highly cytotoxic. Drugs like interferon and ribavarin can cause anemia, hemolysis, and clinical depression. Furthermore, the toxicity of these drugs and ether treatments is highly individualized, based on genetic factors, current liver functionality, and other unknowns. The dangers from present liver treatment options and the general dearth of understanding in disease pathways has created a need for treatment options that support liver function, structure, and regeneration, while minimizing side effects. There remains a need for compositions for maintaining liver health naturally with minimal side effects. SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide compositions for the maintenance, protection, treatment, and regeneration of liver tissue in humans and other animals while minimizing side effects. The composition contains S-adenosylmethionine, quercetin, and glycyrrhizin (and/or salts thereof). The preferred salt of glycyrrhizine is glycyrrhizinate ammonium. The S-adenosylmethionine is preferably present at about 0.5% to about 53% by weight of the total composition, more preferably about 4% to about 25%. Quercetin is preferably present at about 1% to 50% by weight of the total composition, more preferably about 1% to about 5%. Glycyrrhizinine is preferably present at about 0.05% to 10% by weight of the total composition, more preferably about 0.40% to about 1.60%.

It is a further object of the present invention to provide a method of using the novel compositions of the present invention for the maintenance, protection, treatment, and regeneration of liver tissue in humans and other animals.

It is a further object of the present invention to provide a method using the novel compositions of the present invention through which it is biochemically and physiochemically active in the treatment, protection, and repair of hepatic tissue in humans and other animals.

It is a further object of the present invention to provide a composition as defined above for use in the m ai nt e n an c e o f h e alth , mo r e s p e c i fi c al ly maintenance, protection, treatment, and regeneration of liver tissue, in humans and other animals (such as cows, dogs or cats).

These and other objects of the invention, as well as many of the intended advantages thereof, will become more readily apparent when reference is made to the following description, taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

FIG. 1 shows the molecular structure of S-adenosylmethionine.

FIG. 2 shows the S-adenosylmethionine cycle in the body.

FIG. 3 shows the major metabolic pathways of S-adenosylmethionine in the body. FIG. 4 shows the molecular structure of quercetin.

FIG. 5 shows the molecular structure of glycyrrhizinate ammonium. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In describing a preferred embodiment of the invention, specific terminology may be resorted to for the sake of clarity. However, the invention is not intended to be limited to the specific terms so selected, and it is to be understood that each specific term includes all technical equivalents that operate in similar manner to accomplish a similar purpose. Several preferred embodiments of the invention are described for illustrative purposes, it being understood that the invention may be embodied in other forms not specifically described.

The invention contemplated relates to novel compositions containing of S- adenosylmethionine, quercetin, and glycyrrhizin (and/or salts thereof), and to the use of that composition for the maintenance of health in a subject, more specifically maintenance, protection, treatment, and regeneration of liver tissue. In a particular aspect, the invention relates to a composition for use in the treatment of a liver disorder or condition. The foregoing composition described may be combined with any number of active and/or inactive compounds. Active compounds may include other biologically active substances, such as vitamins, minerals, amino acids, and combinations thereof. Inactive compounds may be, for example, excipients that are commonly used in pharmaceuticals and nutraceuticals, as well as physiologically inert additives that make the embodiment more palatable for oral administration, improving its taste or smell.

S-adenosylmethionine (hereinafter "SAMe") (FIG.l) is a physiologically active compound found naturally throughout the body. SAMe is synthesized in the body from the combination of ATP and methionine. SAMe functions as a reusable substrate in the body. Through the SAMe cycle (FIG.2), SAMe is formed, utilized, and regenerated as needed. SAMe is synthesized in vivo through an enzymatic reaction catalyzed by SAM- synthetase, which oversees the dephosphorylation of ATP and the formation of the sulfur bond. Akiva Gross et al., Applied Biochem. and Biotech. 8, 415-422 (1983). Upon donation of its methyl group in a number of enzymatic processes, SAMe is converted to S-adenosyl homocysteine ("SAH"). SAH is then hydrolyzed into homocysteine and adenosine by SAH hydrolase.

Subsequently, homocysteine is recycled into methionine by methionine synthase in the presence of folate in the rate-limiting step of the SAMe cycle. With adenosine and methionine again bioavailable, the cycle begins again, resulting in the regeneration of SAMe for future enzymatic reactions. Loenen et al., Biochem. Society Transactions 34(2):330-333, 331 (2006). The highest levels of SAMe are found in the l iver, where it participates in enzymatic reactions that DNA, amino acids, proteins, lipids, and secondary metabolites, like drugs (FIG. 3). SAMe also methylates mRNA and DNA, affecting transcription and translation. Its decarboxylation in polyamine synthesis further regulates cell growth. Additionally, SAMe participates as a cosubstrate in a number of biochemical reactions, functioning primarily in the transfer of methyl groups. The chemically reactive region of SAMe is found at the methyl group attached to the sulfur atom of methionine. This methyl group can be donated in a transmethylation reaction to a variety of substrates, as discussed above. SAMe is also responsible for the glutathione pathway, in that one of the products of the SAMe cycle, homocysteine may be co-opted by cystathionine β-synthase and cystathionine β-lyase to form cysteine. The cysteine is converted to glutathione through the actions of glutamate cysteine ligase and glutathionine synthetase. Glutathione acts as an endogenous antioxidant, quenching reactive oxygen species produced during enzymatic reactions, such as ethanol oxidation in the liver.

The invention disclosed is contemplated to treat a myriad of liver conditions. These treated liver conditions in animals and humans include, but are not limited to, cholestatic disorders, hepatic steatosis, viral hepatitis, chronic active hepatitis, primary or secondary cirrhosis, and chronic or acute liver toxicity linked to ingestion or medicinal side effects. While there are a myriad of agents and pathogens responsible for liver damage, ethanol is the best understood, and is therefore used as a representative example. Ethanol is able to affect the liver in a variety of ways. Other toxins and pathogens modulate similar oxidative damage pathways. Thus, an examination of the effects of ethanol on liver function is highly instructive in providing background on common pathogenic pathways for liver damage. Ethanol absorbed into the circulatory system is cleared from the body by the liver. Hepatocytes in the liver use a number of enzymatic reactions to convert ethanol into carbon dioxide and water. The primary system for ethanol clearance operates in the cytoplasm of hepatocytes. First, the enzyme, alcohol dehydrogenase, oxidizes ethanol to acetalaldehyde. Acetalaldehyde is a highly reactive oxygen species that must be quenched by an antioxidant, like glutathione. Prolonged exposure to reactive oxygen species can cause injury to DNA and mitochondria in surrounding tissue, leading to liver damage. Thus, sufficient quantities of glutathione and other antioxidants are essential in limiting damage prior to acetalaldehyde's oxidation. The enzyme, aldehyde dehydrogenase, is responsible for converting acetalaldehyde into acetic acid. The next step in ethanol oxidation is the conversion of acetic acid into acetyl-CoA, a core element of the citric acid cycle. The enzyme responsible is known as acetyl-coenzyme A synthetase. Once acetyl-CoA enters the citric acid cycle, it is facile ly converted into inert carbon dioxide and water. Ethanol can also be processed in the liver via the microsomal ethanol oxidizing system (hereinafter, the "MEO system"). This system utilizes the cytochrome P450 enzyme, CYP2E 1, and related enzymes. The MEO system generally functions as a back-up to the alcohol dehydrogenase, and is usually triggered by the chronic use of alcohol that overwhelms it. The MEO system is localized to microsomes formed from the smooth endoplasmic reticulum of hepatocytes. Similar to the alcohol dehydrogenase pathway, the MEO system produces acetalaldehyde, which is then introduced into the original metabolic pathway. Unlike the primary alcohol dehydrogenase system, the MEO system is a redox reaction that requires cofactor NADPH. As a redox reaction, the MEO system utilizes oxygen to oxidize ethanol, resulting in the production of free radicals and reactive oxygen species like superoxide, in addition to acetalaldehyde. Leiber et al., J. Biol. Chem. 245:2505-2512. Consequently, the MEO system exerts additional strain on hepatocytes and further drains stores of antioxidants like glutathione in the liver, exacerbating the risk for tissue damage. An additional downside of the MEO system operating in conjunction with the alcohol dehydrogenase pathway is that there is a surplus of acetal aldehyde, the precursor to acetyl-CoA. Excess acetyl-CoA in hepatocytes leads to an up-regulation of the citric acid cycle, which produces excess NADH, which has been shown to adversely affect gene regulation. Activation of the enzyme, CYP2E 1 , also has other unintended effects. The CYP2E1 enzyme is involved in the gluconeogenesis, fatty acid oxidation, and detoxification of xenobiotics. Activation of its expression in response to excess ethanol results in an up- regulation of all of these processes. Overproduction of glucose and fatty acid oxidation leads to fatty acid disease and steatohepatitis in the liver. Leiber, Drug Metab. Rev. 36(3-4):51 1-529, (Oct. 2004). Thus, it is clear that ethanol has numerous pathways by which to damage liver tissue.

SAMe deficiency has been shown to cause a wide variety of diseases, including colon cancer, breast cancer, coronary disease, pathologic brain function, and liver disease. Loenen et al., Biochem. Society Transactions 34(2):330-333, 332 (2006). In relation to ethanol oxidation, SAMe is necessary to ensure that there is sufficient glutathione to quench any free radicals present in the liver tissue. As discussed above, the primary reactive oxygen species formed during ethanol oxidation is acetalaldehyde. In alcoholics, the MEO system is activated as well, resulting in a proliferation of free radicals like superoxide, hydrogen peroxide, and hydroxyl, as well as overly active fatty acid synthesis. SAMe has a number of beneficial effects in hepatocytes that combat these acute negative effects of ethanol and other toxins. Studies in rats have shown that administration of SAMe has a restorative effect on hepatic levels of the antioxidant, glutathione. In the study, cirrhosis was artificially induced in the rats. Some were administered SAMe for an extended period of time, while others received no treatment. The study determined that administration of SAMe in the rats reduced liver injury, attenuated fibrosis and fibrogenesis in the liver. Further, rats that were administered SAMe concurrently with the cirrhosis-inducing agent were less likely to develop cirrhosis. Thus, SAMe produced both a preventative as well as a restorative effect on fibrosis and cirrhosis of the liver. The study concluded that SAMe, which was converted to glutathione in vivo, acted to prevent lipid peroxidation, one of leading causes of fibrosis and eventual cirrhosis in the liver. Thus, by halting the progression of fibrosis, SAMe prevents apoptosis and can provide time for hepatocytes to regenerate and prevent more serious liver problems. Gasso et al, J. Hepatology 25:200-205, 203-204 (1996). SAMe also has the additional benefit of preventing the build-up of reactive oxygen species and free radicals from bath alcohol dehydrogenase and the MEO system from damaging mitochondria in hepatocytes. In a recent study, SAMe was shown to attenuate damage from superoxides to the mitochondrial electron chain as well as to mitochondrial DNA. Bailey et al, Am. J. Physiol. Gastrointest. Liver Physiol. 291 :G857-G867, G865 (2006). Quercetin (FIG. 4) is a naturally occurring flavonoid (polyphenol) that is also biosynthesized in humans and other animals. It is found in a number of dietary sources, including tomatoes, red onions, kale, and cranberries. Quercetin is synthesized from the commonly ingested amino acid, phenylalanine, through a series of enzymatic reactions. As a polyphenol, quercetin operates as an antioxidant, neutralizing free radicals and reactive oxygen species that can harm cellular membranes and DNA. Quercetin has also been shown to inhibit the release of cytokines by cells in vitro, thereby mitigating inflammation. Other beneficial effects of quercetin include induction of apoptotic pathways in cancerous cells and the prevention of 1 ipid peroxidation in cell membranes. The antioxidant properties of quercetin are meditated through its ability to quench superoxide and nitric oxide reactive oxygen species in the liver. In addition to directly neutralizing reactive oxygen species in rats, quercetin is demonstrated to inhibit nuclear transcription factor NF-κΒ activation. By preventing this nuclear factor from initiating gene activation, quercertin is responsible for limiting the production of downstream nitric oxide gene products that form reactive oxygen species. Thus, quercetin operates by two independent pathways. It actively quenches reactive oxygen species and free radicals, while also preventing the expression of genes that would result in the production of additional free radicals. Dias et al, J. Nutr. 135:2299-2304, 2303 (2005). Quercetin has also been demonstrated to have protective and ameliorative effects on liver cells exposed to ethanol. In a recent mouse study, quercetin treatment prior to ethanol ingestion was found to increase the endogenous production of the antioxidant, glutathione, in liver tissue, as well as operating directly as an antioxidant on the lipid peroxides produced through the normal ethanol metabolism. The quercetin administered thus reduced cellular damage by removing reactive oxygen species via two separate mechanisms. Mouse hepatic cells treated with quercetin prior to the introduction of ethanol also maintained higher levels of intracellular glutathione, such that these hepatocytes could withstand greater oxidative stress from toxins. Molina et al., Biol. Pharm. Bull. 26(10), 1398-1402, 1399- 1401 (2003). Quercetin's anti-inflammatory properties are also well documented. As discussed above, the breakdown of toxins like ethanol results in the formation of reactive oxygen species like acetal aldehyde. In addition to direct intracellular damage, failure to quench these species can result in their binding to intracellular proteins, forming adducts. These adducts, when released into plasma, are not recognized by immune cells. The immune cells initiate the release of cytokines and other pro-inflammatory compounds in a misguided attempt to instigate an immune response to the area. This immune response results in pervasive damage to liver tissue and the eventual fibrosis. A major trigger of inflammation is nuclear factor NF-KB, which starts a downstream cascade that releases pro-inflammatory cytokines and reactive nitric oxide species. In particular, activation of the enzyme, nitric oxide synthase ( NOS), produces a significant portion of the nitric oxide species responsible for the inflammatory response. Quercetin is shown, in vitro, to attenuate the release of these cytokines and nitric oxide species through its inhibition of NF-κΒ, thereby lessening the immune response and ameliorating inflammation. Comalada et al, Eur. J. Immunol. 35:584-592, 590 (2005). The enzyme, cyclooxygenase-2 ("COX-2"), is another factor activated during an inflammatory response. Localized expression of COX-2 produces prostaglandins, which can attract a greater immune response to the site. In vivo quercetin administration is documented to reduce COX-2 expression in macrophages and human lymphocytes.

Gonzalez-Gallego et al, Nutr. Hosp. 22(3):287-293, 289 (2007). Thus, by preventing COX-2 expression, quercetin can reduce the inflammatory response in liver tissue, even after it has been initiated. Yet another documented pharmacologic effect of quercetin is the ability of its 3-O-galactoside, hyperoside, to inhibit key viral proteins, HBeAg and HBsAg, associated with hepatitis B. In this manner, quercetin's metabolites have the potential to act against viral infections in the liver. Acta Pharmacol. Sin. 28(3):404-409, 408 (Mar. 2007).

Glycyrrhizinate ammonium (FIG. 5) is the water-soluble salt of glycyrrhizin (glycyrrhizinic acid) which is primarily found in licorice root. As used herein, glycyrrhizinate ammonium, glycyrrhizin, and glycyrrhizinic acid are used interchangeably, except for dosage guidelines, which are specific to glycyrrhizinate ammonium. Glycyrrhizin or any other salt thereof can be provided in an amount equivalent to the amount glycyrrhizinate ammonium. Glycyrrhizin can be found in licorice products worldwide. As such, glycyrrhizin is typically extracted from licorice root, for example by solvent extraction. Tian et al, Int. J. Mol. Sci. 9:571-577 (2008). A typical licorice root extract contains about 3% to about 5% glycyrrhizin by weight. Isbrucker et al, Regul Toxicol Pharmacol. 46(3): 167-92 (2006). Glycyrrhizin is also used as an artificial sweetener internationally, because it is many times sweeter than sucrose. The compound contemplated in this composition could also include artificially synthesized glycyrrhizin. From a medical perspective, glycyrrhizin is found to operate as an anti-inflammatory agent, an antioxidant, and an anti-viral agent. In mice hepatocytes in vivo, it has been shown to be more effective than the widely used silymarin in protecting hepatocytes from oxidative injury which generally occurs from exposure to toxins like ethanol. In the study, glycyrrhizin attenuated the release of alanine aminotransferase and aspartate aminotransferase, two clinically significant enzymes released by hepatocytes in response to acute injury. This reduction in the release of these enzymes is indicative of the protective effects of glycyrrhizin on hepatocytes. The study identified the anti- inflammatory and antioxidant properties of glycyrrhizin as the reasons for this protective effect. It found that glycyrrhizin exhibited an anti-inflammatory effect on liver tissue by mediating the levels of key pro-inflammatory transcriptional factors. Glycyrrhizin was responsible for attenuating the production of the mR A of the pro-inflammatory COX-2 at the transcriptional level. This attenuation in COX-2 headed off an escalated immune response. The study also found that glycyrrhizin had some effect on two other proinflammatory factors, TNF-a and z^'NOS. Moreover, the study identified a mechanism by which glycyrrhizin acted as an antioxidant in hepatocytes. Cells treated with glycyrrhizin maintained a higher level of glutathione in response to oxidative stress and showed lower levels of lipid peroxidation. As a consequence of their glutathione stores, these cells were able to withstand greater stress from free radicals and reactive species. Lee et al., Biol. Pharm. Bull. 30(10):1898-1904, 1902-03 (2007). Another study confirmed these findings, showing that glycyrrhizin increased glutathione stores, and increasing the production of a number of enzymes that detoxify and aid in the excretion of various toxins. Further, the glycyrrhizin treatment increased IFN-γ and decreased the IL-4 cytokine, which is responsible for the production of naive CD4 T-cells that are responsible for inflammation. Rather, by inducing production of IFN-γ, glycyrrhizin increased the T-cell differentiation and maturation, stimulating the adaptive immune system while heading off its innate inflammatory response. Li et al., Int. J. Mol. Sci. 12:905-916, 912 (201 1). Glycyrrhizin's anti-inflammatory effects are mediated through a number of pathways. One of these pathways is directed through the selectin receptors found on lymphocytes and endothelial cells. Selectin receptors are translocated to the cell membrane in response to inflammatory cytokines. Glycyrrhizin is shown to block binding to selectin receptors, resulting in anti-inflammatory activity. Narasinga et al., J. Biol. Chem. 269(31): 19663- 19666, 19665 (1994). Glycyrrhizin has also been demonstrated to act as an antiinflammatory agent via other pro-inflammatory surface receptors. By blocking the NF-KB pathway as well as the pro-inflammatory Toll-Like Receptors TLR3 and TLR4 on the cell surface, glycyrrhizin has broad anti-inflammatory activity through its interaction with the membranes of affected cells. Schrofelbauer et al., Biochem. J. 421 :473-482, 480 (2009).

A recent study has also demonstrated that glycyrrhizin can act as an antiviral agent against hepatitis C. The study was based on past findings of glycyrrhizin's antiviral abilities. In previous studies, it had been shown to stimulate production of antiviral interferon in vivo, as well as inhibiting NF-κΒ and inducing IL-8 secretion. This study demonstrated the glycyrrhizin's ability to directly inhibit expression of the viral core protein, leading to a viral inhibition rates as high as 85%. Ashfaq et al, J. Trans. Med. 9: 112 (2011).

It is expected that elements of the combinations of the present invention will work synergistically because they have a myriad of complementary mechanisms by which to treat, protect, and repair hepatic tissue in humans and other animals. The complexities inherent in the diagnosis and treatment of liver conditions require the invention of a combination that can positively impact numerous liver damage pathways synergistically, thereby maximizing the efficacy of the combination. Those who diagnose liver conditions understand that it is favorable to treat such conditions with a broad spectrum approach, such that as many potential mechanisms for treatment are addressed as possible. Because it is generally not possible to immediately diagnose the precise cause of liver damage, it is highly beneficial to treat such a condition by attempting to mitigate future damage and maintain liver function while further examination is conducted. Symptomatic treatment is therefore one of the best methods in addressing liver conditions. The present invention strives to do just that, incorporating compounds that act across a number of pathways.

As discussed above, the compounds in the presently claimed invention demonstrate antioxidant properties, anti-inflammatory properties, anti-fatty acid properties, and antiviral properties. Studies have shown that S-adenosylmethionine acts an antioxidant, quenching free radicals and reactive species in hepatocytes. It has also been shown to prevent lipid peroxidation, which can lead to excessive build-ups of fatty acids in liver cells, resulting in the fatty acid disease that accompanies many liver conditions. Moreover, S-adenosylmethionine acts indirectly to increase intracellular glutathione, providing an additional antioxidant supply to combat future oxidative damage. Quercetin is demonstrated ta increase the endogenous production of the antioxidant, glutathione, in liver tissue, as well as operating directly as an antioxidant on the lipid peroxides produced through the normal ethanol metabolism. Further, quercetin is shown to act as an anti-inflammatory agent, attenuating the release of pro-inflammatory cytokines and nitric oxide species through its inhibition of transcriptional factor, NF-κΒ. Quercetin is also shown to act as an antiviral agent, inhibiting key proteins associated with hepatitis B. In combination, S-adenosylmethionine and quercetin thus produce beneficial effects for a number of liver conditions. Adding glycyrrhizin to the combination treats an even broader range of liver diseases. Like the other two compounds in the composition, glycyrrhizin has a number of beneficial properties. It is an antioxidant, an anti-inflammatory, a stimulant of adaptive immunity, and has been shown to exhibit antiviral properties against key viral proteins in hepatitis C. Similarly to the other compounds in the composition, glycyrrhizin mediates these actions through a variety of pathways. Thus, the combination of all three compounds in the composition acts synergistically to treat, protect, and repair the liver. This synergistic effect is further enhanced because the complementary actions of the compounds allow for a lower effective dose of the composition. Administration of a lower effective dose is economically favorable while also minimizing any possible side effects. It is important to note that S-adenosylmethionine, quercetin, and glycyrrhizinate ammonium are all very well tolerated in vivo and do not generally exhibit side effects. Nevertheless, it is known in the art that minimizing dosage of any drug is a sound medical strategy.

Additional antioxidants and otherwise beneficial compounds known in the art can be added to the composition claimed to increase its general protective effects on liver tissue. One embodiment of the invention claimed contemplates a composition containing of S- adenosylmethionine, quercetin, and glycyrrhizinate ammonium, with additional compounds including (1) n-acetyl cysteine; (2) 1-taurine; (3) zinc ascorbate; (4) vitamin E; (5) vitamin B6; and (6) any other B vitamins. Other embodiments of the invention can include any combination of the foregoing compounds in a composition containing of S- adenosylmethionine, quercetin, and glycyrrhizinate ammonium. The compositions of the present invention can be administered orally, parenterally, transdermally, sublingually, intravenously, intramuscularly, rectally, and subcutaneously. The foregoing methods are by no means the only vehicles for administration of the invention. Other conventionally accepted methods of administration will occur to those skilled in the art, and these may be used as well. The preferred route of administration is oral. Preferred daily doses of the compounds in the composition are as follows:

S-adenosylmethionine:

Daily per kilogram dosage range for all species: 1 mg/kg- 100 mg/kg

More preferable daily per kilogram dosage range for all species: 2mg/kg- 30mg/kg Preferred small domestic animal dose range: 1 mg/kg- 60mg/kg

Preferred human dose range: 3mg/kg- 30mg/kg

Preferred livestock dose range: 1 mg/kg- 30mglkg

Quercetin:

Daily per kilogram dosage range for all species: 0.5 mg/kg- 100 mg/kg

More preferable daily per kilogram dosage range for all species: 0.5 mg/kg- 50 mg/kg Preferred small animal dose range: 2 mg/kg- 25 mg/kg

Preferred human dose range: 2 mg/kg- 25 mg/kg

Preferred livestock dose range: 2 mg/kg- 25 mg/kg Glycyrrhizinate ammonium:

Daily per kilogram dosage range for all species: 0.1 mg/kg- 5.0 mg/kg

More preferable daily per kilogram dosage range for all species: 0.1 mg/kg- 4.0 mg/kg

Preferred small animal dose range: 0.5 mg/kg- 4.0 mg/kg

Preferred human dose range: 0.2 mg/kg-2.0 mg/kg

Preferred livestock dose range: 0.1 mg/kg- 4.0 mg/kg

The dosages for glycyrrhizin or its other salts can be provided in amounts equivalent to the dosages for glycyrrhizinate ammonium given above.

The composition of the present invention can be formulated into a dosage form convenient for administration. Dosage forms appropriate for the present invention include, but are not limited to: tablets, capsules (swallowed and chewable), powders, granulate, solutions, and suspensions. The composition may also be provided as a kit or blister pack, in which the various components are separately provided for coadministration. According to an embodiment, the invention provides a kit comprising a composition as defined in the present invention and a package leaflet or user instructions including the information that said composition is to be used for maintaining liver health in a mammal, preferably in a dog or a cat. Here, the components are mixed together prior to administration or are co-administered at the time of use. Moreover, it is contemplated that the dosage of the composition may be administered in various combinations in which the components may be present in a single dosage form or in more than one dosage form. Accordingly, the doses may be administered in combinations of more than one dosage form, such that each dosage form contains at least one component or in which two or more components are combined into a single dosage form. Accordingly, the kit or composition of the invention can more specifically allow a single dose or more of S- adenosylmethionine that provides from 1 mg/kg to about 1 00 mg/kg, a single dose or more of quercetin provides from about 0.5 mg/kg to about 100 mg/kg, and/or a single dose or more of glycyrrhizin and/or salts thereof provides an amount equivalent to about 0.1 mg/kg to about 5 mg/kg of glycyrrhizinate ammonium. Oral dosage forms are preferred. By weight, each dose preferably contains about 0.5 to about 53%, more preferably about 4 to about 24% S-adenosylmethionine; about 1% to about 50%, more preferably about 1% to about 5% mg quercetin; and about 0.05% to about 10%, more preferably about 0.40% to about 1.60% glycyrrhizinate ammonium. The daily dose of the composition can vary in a large extent depending on the condition of the subject to be treated. According to specific embodiments, the daily dose of the composition is about 1.6 mg/kg to about 205 mg/kg.

It will be appreciated that the compositions and treatment methods of the invention are useful in fields of veterinary medicine and human medicine. Thus, the subject for administration is a mammal, such as a human or other mammalian animal. For veterinary purposes, subjects include for example, livestock (farm animals) including cows, sheep, pigs, horses and goats; companion animals such as dogs and cats, exotic and/or zoo animals; laboratory animals including mice rats, rabbits, guinea pigs and hamsters; and poultry such as chickens, turkeys, ducks and geese, which may also be considered livestock.

Although certain presently preferred embodiments of the invention have been specifically described herein, it will be apparent to those skilled in the art to which the invention pertains that variations and modifications of the various embodiments shown and described herein may be made without departing from the spirit and scope of the invention. Accordingly, it is intended that the invention be limited only to the extent required by the appended claims and the applicable rules of law.

Claims

1. A composition comprising

(A) S-adenosylmethionine;

(B) quercetin; and

(C) glycyrrhizin and/or salts thereof.

2. The composition of claim 1, further comprising one or more of the following ingredients: (1) n-acetyl cysteine; (2) 1-taurine; (3) zinc ascorbate; (4) vitamin E; (5) vitamin B6; and (6) any other B vitamins.

3. The composition of claim 1 or 2, wherein the salt of glycyrrhizin is glycyrrhizinate ammonium.

4. The composition of claim 2, wherein the vitamin E is any of the four forms of tocopherol or any of the four forms of tocotrienol.

5. The composition of claim 2, wherein the vitamin B6 is biologically active pyridoxal phosphate.

6. The composition of anyone of claims 1-5, comprising about 0.5 to about 53 percent S-adenosylmethionine by weight.

7. The composition of anyone of claims 1-6, comprising about 1 to about 50 percent quercetin by weight.

8. The composition of anyone of claims 1-7, comprising about 0.05 to about 10 percent glycyrrhizinate ammonium by weight, or its equivalents in the form of glycyrrhizin, and/ or salts thereof.

9. The composition of anyone of claims 1-8, wherein the glycyrrhizin and/ or salts thereof are obtained from licorice extract.

10. The composition of anyone of claims 1-9, in a dosage form of a tablet, capsule, powder, granulate, solution, or a suspension.

1 1. A composition of anyone of claims 1-10 for use in maintaining health in a subject.

12. The composition for use of claim 11 , wherein the subject is an animal or a human.

13. The composition for use of claim 11 or 12, wherein the daily dose of the composition is about 1.6 mg/kg to about 205 mg/kg.

14. The composition for use of anyone of claims 11-13, wherein the dosage provides about 1 mg/kg to about 100 mg/kg of S-adenosylmethionine.

15. The composition for use of anyone of claims 11-14, wherein the dosage provides about 0.5 mg/kg to about 100 mg/kg of quercetin.

16. The composition for use of anyone of claims 11-15, wherein the dosage provides an amount equivalent to about 0.1 mg/kg to about 5 mg/kg of glycyrrhizinate ammonium.

17. A kit comprising a composition according any one of claims 1 to 16 and a package leaflet or user instruction including the information that said composition is to be used for maintaining liver health in a mammal, preferably in a dog or a cat.