HK1141711A - Methods for the treatment of liver diseases using specified matrix metalloproteinase (mmp) inhibitors - Google Patents

Abstract

Provided herein are methods for treatment of a liver disease by administering a matrix metalloproteinase inhibitor. Also provided are methods for reducing liver damage associated with a liver disease by administering the matrix metalloproteinase inhibitor described herein. Further provided are methods for lowering an elevated level of liver enzymes by administering the matrix metalloproteinase inhibitor.

Description

Methods of treating liver diseases using specific Matrix Metalloproteinase (MMP) inhibitors

1. Priority requirement

Spada claims priority from U.S. provisional application Ser. Nos. 60/904,322 and 60/937,301, filed on 8/2/2007 and 26/6/2007, respectively. The contents of the above-mentioned applications are incorporated by reference in their entirety.

2. Field of the invention

The present invention provides methods of treating liver disease by administering a matrix metalloproteinase inhibitor.

3. Background of the invention

Liver disease is acute or chronic damage to the liver, usually caused by infection, injury, exposure to drugs or toxic compounds, alcohol, impurities in food, and abnormal elevation of normal substances in the blood, autoimmune processes, or by genetic defects (e.g., hemochromatosis). Sometimes the exact cause of the damage may be unknown. Liver disease can be classified as either acute or chronic liver disease based on the duration of the disease. In acute liver diseases such as acute hepatitis and Acute Liver Failure (ALF), the history of the disease does not exceed six months. Liver diseases of longer duration are classified as chronic liver diseases.

Common liver diseases include cirrhosis, liver fibrosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic ischemia-reperfusion injury, Primary Biliary Cirrhosis (PBC), hepatitis (including viral and alcoholic hepatitis). The most common forms of viral hepatitis are hepatitis b and c (referred to as HBV and HCV, respectively). Chronic hepatitis may lead to cirrhosis. Cirrhosis caused by chronic hepatitis c infection causes 12,000 deaths each year in the united states at 8,000-.

In all types of liver disease, it is common for hepatocytes to die by a process called apoptosis. Apoptosis of hepatocytes is associated with liver fibrosis and other liver diseases. Prevention of excessive apoptosis of hepatocytes is an important component in the treatment of acute and chronic liver diseases (see Guicciardi et al, Gut, 2005: 54, 1024-once 1033, and Ghavami et al, Med. Sci. unit., 2005: 11 (11): RA 337-345).

The occurrence of active liver disease is often detected by the presence of elevated levels of enzymes in the blood. In particular, blood levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) that exceed the clinically acceptable normal range are known to be indicative of ongoing liver damage. The progression of liver disease during medical treatment is measured clinically by routine monitoring of ALT and AST blood levels in patients with liver disease. Lowering elevated ALT and AST within acceptable normal ranges is considered clinical evidence reflecting a reduction in the severity of ongoing liver damage in patients. (Kim W.R. et al, Hepatology, 2008, ready for preprinting online, accessible 2/20/2008 at the publisher's website).

Given that liver disease affects a vast patient population worldwide and has a tragic impact on infected patients, there remains a strong need to provide new effective agents for treating liver disease.

4. Overview

In one aspect, the invention provides methods of treating various liver diseases by administering a matrix metalloproteinase inhibitor. In some embodiments, the method comprises treatment of acute and/or chronic liver disease. In one embodiment, the liver disease is a condition caused by damage to the liver. In one embodiment, the damage to the liver is caused by toxins, including abnormal elevations of alcohol, certain drugs, impurities in food, and normal substances in the blood. In another embodiment, the damage to the liver is caused by an infection or an autoimmune disorder. In some embodiments, the exact cause of the damage is unknown. In some embodiments, liver diseases caused by damage to the liver include, but are not limited to, fatty liver, cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and alpha 1-antitrypsin deficiency.

In one embodiment, the liver disease includes, but is not limited to, cirrhosis, liver fibrosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic ischemia-reperfusion injury, hepatitis (including viral and alcoholic hepatitis), and Primary Biliary Cirrhosis (PBC).

In some embodiments, the liver disease is not secondary fatty liver disease. In some embodiments, the matrix metalloproteinase inhibitor is not marimastat.

In some embodiments, the present invention provides methods of treating liver disease in a patient who has undergone treatment for failure of liver disease. In some embodiments, the present invention provides methods of treating hepatitis c. In some embodiments, the present invention provides methods of treating hepatitis c in a patient undergoing treatment for hepatitis c failure. In one embodiment, the methods provided herein reduce liver damage associated with chronic and/or acute liver disease. In one embodiment, the methods provided herein reduce elevated liver enzyme levels, such as elevated ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels.

In some embodiments, the present invention provides methods of inhibiting Hepatitis C Virus (HCV) replication in a cell infected with HCV by administering a compound provided herein. In some embodiments, the present invention provides methods of inhibiting Hepatitis C Virus (HCV) replication in a patient infected with HCV by administering a compound provided herein.

In one embodiment, the matrix metalloproteinase inhibitor compound used in the methods provided herein is selected from:

and pharmaceutically acceptable derivatives thereof.

In one embodiment, the compound used in the methods provided herein is

Or a pharmaceutically acceptable derivative thereof.

In one embodiment, the compound used in the methods provided herein is

Or a pharmaceutically acceptable derivative thereof.

The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of one or more of the compounds provided herein and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is useful in preventing, treating or ameliorating one or more symptoms of a liver disease.

The invention further provides an article of manufacture comprising packaging material, a compound provided herein or a pharmaceutically acceptable derivative thereof for use in treating, preventing or ameliorating one or more symptoms associated with a liver disease, and a label indicating that the compound or pharmaceutically acceptable derivative thereof is for use in treating, preventing or ameliorating one or more symptoms of a liver disease.

5. Detailed description of the preferred embodiments

5.1 definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, application publications, and other publications are incorporated by reference in their entirety. If a term herein has multiple definitions, the definition in this section controls unless otherwise specified.

As used herein, an "individual" is an animal, e.g., a mammal, including a human, e.g., a patient.

Biological activity, as used herein, refers to the in vivo activity of a compound, or the physiological response that a compound, composition, or other mixture will elicit when administered in vivo. Thus, biological activity includes the therapeutic effects and pharmacokinetic behavior of the above compounds, compositions and mixtures. Such activity can be observed in vitro systems designed to test biological activity.

Pharmaceutically acceptable derivatives of the compounds used herein include salts, esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs thereof. Such derivatives can be readily prepared by those skilled in the art using known procedures for such derivatization. The prepared compounds can be administered to animals or humans without substantial toxic effects, and the prepared compounds are pharmaceutically active or prodrugs. Pharmaceutically acceptable salts include, but are not limited to, amine salts such as, but not limited to, N '-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-p-chlorobenzyl-2-pyrrolidin-1' -ylmethylbenzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxymethyl) aminomethane; alkali metal salts such as, but not limited to, lithium, potassium and sodium salts; alkaline earth metal salts such as, but not limited to, barium, calcium and magnesium salts; transition metal element salts such as, but not limited to, zinc salts; and inorganic salts such as, but not limited to, sodium hydrogen phosphate and disodium hydrogen phosphate; but also include, but are not limited to, salts of inorganic acids such as, but not limited to, hydrochlorides and sulfates; and salts of organic acids such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, mesylate, and fumarate. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, aralkyl, and cycloalkyl esters of acidic groups including, but not limited to, carboxylic, phosphoric, phosphonic, sulfonic, sulfinic, and boronic acids. Pharmaceutically acceptable solvates and hydrates are complexes of the compound with one or more solvent or water molecules, or 1 to about 100, or 1 to about 10, or 1 to about 2,3, or 4 solvent or water molecules.

Treatment as used herein refers to any means of ameliorating or beneficially altering one or more symptoms of a disease or disorder. Treatment also includes any pharmaceutical use of the compositions herein, for example to treat liver disease.

As used herein, ameliorating the symptoms of a particular disorder by administering a particular compound or pharmaceutical composition refers to any reduction, whether permanent or temporary, sustained or transient, that results from or is accompanied by administration of the composition.

The term "controlling", as used herein, unless otherwise indicated, includes preventing the recurrence of a particular disease or disorder in a patient who has already suffered from the disease or disorder, and/or prolonging the time a patient suffering from the disease or disorder remains in remission. The term encompasses modulating the threshold, progression, and/or duration of the disease or condition, or altering the patient's way of responding to the disease or condition.

It is to be understood that the compounds provided herein may contain chiral centers. Such chiral centers may be in the (R) or (S) configuration, or may be mixtures thereof. Thus, the compounds provided herein can be enantiomerically pure, or stereoisomeric or diastereomeric mixtures. Likewise, one skilled in the art will recognize that for a compound that undergoes epimerization in vivo, administration of the compound in the (R) form and administration of the compound in the (S) form are equivalent.

Substantially pure, as used herein, refers to sufficiently homogeneous to exhibit no readily detectable impurities as detected by standard analytical methods used by those skilled in the art to assess such purity, such as Thin Layer Chromatography (TLC), gel electrophoresis, High Performance Liquid Chromatography (HPLC), and Mass Spectrometry (MS); or sufficiently pure that further purification cannot detectably alter its physical and chemical properties, such as the enzymatic and biological activity of the substance. Methods of purifying compounds to prepare substantially chemically pure compounds are known to those skilled in the art. However, a substantially chemically pure compound may be a mixture of stereoisomers. In such cases, further purification may increase the specific activity of the compound. The disclosure herein will include all such possible isomers, as well as their racemic and optically pure forms. The optically active (+) and (-), (R) -and (S) -, or (D) -and (L) -isomers may be prepared using chiral synthons or chiral reagents, or separated using conventional techniques such as reverse phase HPLC. When a compound described herein contains an olefinic double bond or other center of geometric asymmetry, unless otherwise specified, it is meant that the compound contains both E and Z geometric isomers. Likewise, all tautomeric forms are also included.

In some embodiments, the compounds used in the methods provided herein are "stereochemically pure". A stereochemically pure compound has a stereochemically purity level which would be recognized as "pure" by those skilled in the art. In some embodiments, "stereochemically pure" refers to a compound that is substantially free of other isomers. In particular embodiments, the compound is 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% free of other isomers.

As used herein, "treating a liver disease" refers to treatment with any drug known and commercially available or under development for treating a liver disease. For example, treating hepatitis c refers to treating a patient with a drug for treating HCV that is available on the market. Several representative drugs are described below in the "combination therapy" section.

As used herein, "patients who have undergone failed therapy" refers to the patient population described in section 4.3 below, including patients who have previously been treated for liver disease with any of the drugs currently available on the market, and who have failed to respond to the treatment or who have temporarily alleviated the liver disease.

As used herein, "liver injury" refers to acute or chronic injury to the liver, typically caused by infection, injury, exposure to drugs or toxic compounds, alcohol, impurities in food, and abnormally elevated normal substances in the blood, autoimmune processes, transplant-related transplant rejection, or by genetic defects (e.g., hemochromatosis). Damage to the liver includes, but is not limited to, inflammation, scarring of liver tissue, and fibrosis.

The term "in combination" as used herein refers to the use of more than one treatment (e.g., MMP and interferon). The use of the term "in combination" does not limit the order in which treatments (e.g., MMPs and interferons) are administered to an individual with a disorder. A first treatment (e.g., an MMP inhibitor) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or after (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second treatment (e.g., an interferon) to a subject having a disorder.

The term "synergistic" as used herein refers to a combination of an MMP inhibitor and a second agent, e.g., an interferon, that is more effective than the additive effects of treatment with the two compounds administered alone. The synergistic effect of the therapeutic combination (e.g., a combination of MMPs and interferons) allows for lower doses of one or more treatments and/or less frequent administration of treatments to individuals with a disorder. The ability to use lower doses of treatment (e.g., MMPs and interferons) and/or to administer the treatment less frequently reduces the toxicity associated with administering the treatment to an individual without reducing the efficacy of the treatment in the prevention or treatment of the disorder. In addition, synergistic effects may lead to an improvement in the efficacy of the agent in the prevention or treatment of a disorder. Finally, the synergistic effect of therapeutic combinations (e.g., a combination of MMPs and interferons) can avoid or reduce the adverse or unwanted side effects associated with the use of either therapy alone.

The term "other agent" or "second agent" as used herein refers to any agent or combination of agents that can be used to treat liver disease in combination with the MMP inhibitors described herein. In some embodiments, the additional or second agent is an anti-hepatitis c virus interferon, an anti-hepatitis c virus polymerase inhibitor, an anti-hepatitis c virus protease inhibitor, or a combination thereof.

The term "elevated liver enzyme levels" or "elevated liver enzyme levels" as used herein means that liver enzyme levels in the blood are outside the normal clinically acceptable range for liver enzymes in the blood. The compounds provided herein reduce elevated liver enzyme levels to normal clinically acceptable levels of liver enzymes in the blood. Methods for measuring liver enzyme levels are well known in the art (see, e.g., Jeong S.Y. et al, Sandwich ELISA for a media of a cytological enzyme in a serum from a substrate with liquid disorders, Clin chem., 2003; 49 (5): 8269 and Burn des Roziers N. et al, A microbiological plate assay for a media of a serum amino transferase in a blood probes, Transfusion, 1995; 35 (4): 3314, each of which is incorporated herein by reference in its entirety).

5.2 Compounds used in the Process

The compounds used in the methods provided herein are matrix metalloproteinase inhibitors (MMP inhibitors). Several MMP inhibitors have been reported in the literature. Certain representative MMP inhibitors useful in the methods of the present invention are described by Fisher et al in Cancer Metastasis rev., (2006) 25: 115-136; rao in Current Pharmaceutical Design, 2005, 11, 295-; bender et al in U.S. Pat. No. 5,932,595; watanabe in U.S. patent nos. 6,207,698 and 6,831,178; levin et al in U.S. patent No. 6,225,311; purder et al in WO 2007/016390; and Alwayn et al in Am J Physiol Gastrointest Liver Physiol 291: G1011-G1019, 2006. The contents of these documents are hereby incorporated by reference in their entirety.

In one embodiment, the compound used in the methods provided herein is selected from

XL784 and pharmaceutically acceptable derivatives thereof.

In one embodiment, the compound used in the methods provided herein is selected from

Or a pharmaceutically acceptable derivative thereof.

In one embodiment, the compound used in the methods provided herein is

Or a pharmaceutically acceptable derivative thereof.

In one embodiment, the compound used in the methods provided herein is

Or a pharmaceutically acceptable derivative thereof.

In one embodiment, the compound is

Or a pharmaceutically acceptable derivative thereof.

In some embodiments, the compounds described herein have efficacy after oral administration of 0.001-1000mg/Kg in a model of acute liver disease. In some embodiments, the compounds described herein have efficacy after oral administration of 0.01-100mg/Kg in an acute liver disease model.

5.3 methods of treatment

In some embodiments, the methods provided herein include the treatment of acute and/or chronic liver disease. In one embodiment, the method is for treating acute liver disease. In one embodiment, the method is for treating chronic liver disease. In one embodiment, the method is for reducing liver damage associated with chronic and/or acute liver disease. Without being bound to any particular theory, it is believed that the MMP inhibitors used in the methods provided herein may function, in part, by inhibiting the TNF-a signaling cascade. Thus, in one embodiment, the invention provides a method of inhibiting the signaling cascade of TNF- α by administering a compound described herein.

In one embodiment, the liver disease is a condition caused by damage to the liver. In one embodiment, the damage to the liver is caused by toxins, including abnormal elevations of alcohol, some drugs, impurities in food, and normal substances in the blood. In another embodiment, the damage to the liver is caused by an infection or an autoimmune disorder. In some embodiments, the exact cause of the damage is unknown.

In one embodiment, the liver disease includes, but is not limited to, cirrhosis, liver fibrosis, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), hepatic ischemia-reperfusion injury, hepatitis (including viral and alcoholic hepatitis), and primary biliary cirrhosis. In one embodiment, the liver disease is manifested as elevated liver enzymes (e.g., ALT and AST), or pathological evidence of progressive liver injury that exists as a result of steatosis (fatty liver), fibrosis, and/or cirrhosis. In one embodiment, NASH manifests as elevated liver enzymes (e.g., ALT and AST), or pathological evidence of steatosis (fatty liver), fibrosis, and/or cirrhosis.

In some embodiments, the invention provides methods for treating fatty liver (also known as hepatic steatosis), including non-alcoholic fatty liver disease. Fatty liver is defined as the excessive accumulation of triglycerides in hepatocytes. In some embodiments, in a patient with non-alcoholic fatty liver disease, the liver comprises more than about 5% total weight of the liver fat or more than 30% of the hepatocytes in the lobules of the liver have fatty deposits. The most common causes of non-alcoholic fatty liver disease are obesity, diabetes, and elevated serum triglyceride levels. Other causes include malnutrition, inherited metabolic disorders (e.g., glycogen storage disorders), and medications (e.g., corticosteroids, tetracycline, and aspirin). In some embodiments, the fatty liver does not produce symptoms. In other embodiments, fatty liver causes jaundice (white and yellow skin and eyes), nausea, vomiting, pain, and abdominal tenderness. In one embodiment, the methods provided herein are useful for treating one or more symptoms of a non-alcoholic fatty liver disease.

Fatty liver with liver inflammation that is not caused by alcohol is called non-alcoholic steatohepatitis or NASH. In some embodiments, NASH may result from any of the causes described above as possible causes of nonalcoholic fatty liver disease. In one embodiment, the invention provides a method of treating NASH.

In one embodiment, the methods provided herein are for the treatment of hepatitis or liver inflammation, including viral and alcoholic hepatitis. The viral hepatitis may be acute or chronic. In some embodiments, the acute viral hepatitis is caused by hepatitis a, hepatitis b, hepatitis c, hepatitis d, or hepatitis e virus. In other embodiments, the acute viral hepatitis is caused by hepatitis b or c virus. In some embodiments, the methods provided are for treating chronic viral hepatitis. In one embodiment, chronic viral hepatitis is caused by hepatitis b or c virus. In some embodiments, provided are methods of treating a hepatitis c patient who has undergone a treatment for failure of hepatitis c. Representative methods for treating hepatitis C are described by Strader et al in Hepatology, 39(4), 2004.

In some embodiments, the patient has never received treatment or prevention of HCV infection. In a further embodiment, the patient has previously received treatment or prevention for HCV infection. For example, in some embodiments, the patient is non-responsive to HCV therapy. As is known in the art, under current interferon therapy, as many as 50% or more of HCV patients do not respond to treatment. In some embodiments, the patient may be one who has received treatment but still has HCV or one or more symptoms thereof. In some embodiments, the patient may be one who received treatment but failed to achieve a sustained response. In some embodiments, the patient has been treated for HCV infection but failed to show a 2log HCV RNA level after 12 weeks of treatment₁₀And (4) descending. It is believed that serum HCV RNA did not show more than 2log after 12 weeks of treatment₁₀Reduced patients, 97-100% have a chance of no response.

In some embodiments, the patient is a patient who has discontinued HCV treatment for one or more adverse events associated with treatment. In some embodiments, the patient is not eligible for current therapy. For example, certain treatments for HCV are associated with neuropsychiatric events. Interferon (IFN) - α + ribavirin is associated with a very high rate of depression. Depressive symptoms are associated with worse outcomes in many medical conditions. Life-threatening or fatal neuropsychiatric events include suicidal, suicidal and human-cidal ideations, depression, drug dependence/overdose recurrence, and aggressive behavior, occurring in patients who have and have not previously suffered from a psychiatric disorder during HCV treatment. Interferon-induced depression is a limitation of the treatment of chronic hepatitis c, particularly in patients with psychiatric disorders. Psychiatric side effects are common to interferon therapy and are responsible for about 10% to 20% of the current treatment discontinuations for HCV infection.

Accordingly, the present invention provides methods of treating or preventing hepatitis c in patients who are contraindicated to existing HCV treatment due to risk of neuropsychiatric events, such as risk of depression. The present invention also provides methods of treating or preventing hepatitis c in patients who require cessation of existing HCV therapy due to a neuropsychiatric event such as depression or such risk. The present invention further provides methods of treating or preventing hepatitis c in patients in need of a reduction in the dosage of existing HCV treatments due to neuropsychiatric events such as depression or such risk.

Existing treatments are also contraindicated in patients allergic to interferon, or ribavirin, or both, or any other component of a pharmaceutical product for which interferon or ribavirin is administered. Existing treatments are inappropriate in patients with hemoglobinopathies (e.g., thalassemia major, sickle cell anemia) and other patients at risk for haematological side effects to current treatments. Common haematological side effects include myelosuppression, neutropenia and thrombocytopenia. Additionally, ribavirin is toxic to red blood cells and is associated with hemolysis. Thus, the methods provided by the present invention are useful in patients allergic to interferon, or ribavirin, or both, patients with hemoglobinopathies (e.g., thalassemia major patients and sickle cell anemia patients), and other patients with risk of hematological side effects for existing treatments.

In some embodiments, the patient receives HCV treatment and the treatment is terminated prior to administration of the methods provided herein. In a further embodiment, the patient is treated and continues to receive treatment in addition to administering the methods provided herein. The methods of the invention may be administered in combination with other treatments for HCV, as will be appreciated by those skilled in the art. In some embodiments, the methods or compositions of the present invention can be administered in combination with other treatments for HCV that reduce the dosage.

In some embodiments, the invention provides methods of treating a patient refractory to interferon therapy. For example, in some embodiments, the patient may be one who has failed to respond to treatment with one or more agents selected from interferon, interferon alpha, peginterferon alpha, interferon + ribavirin, interferon alpha + ribavirin, and peginterferon alpha + ribavirin. In some embodiments, the patient may be one who has an adverse therapeutic response to one or more agents selected from the group consisting of interferon, interferon alpha, peginterferon alpha, interferon + ribavirin, interferon alpha + ribavirin, and peginterferon alpha + ribavirin.

In one embodiment, chronic HCV infection is manifested by elevated liver enzymes (e.g., ALT, AST), sustained HCV RNA levels (e.g., greater than six months), and/or histological evidence of liver damage, fibrosis, and/or cirrhosis. In one embodiment, the methods provided herein reduce the elevation of liver enzyme levels, such as ALT and AST levels. Methods for measuring liver enzyme levels are well known in the art (see, e.g., Jeong S.Y. et al, Sandwich ELISA for measurement of cellular enzyme aminotransferase from substrates with devices, Clin chem., 2003; 49 (5): 8269 and Burendes Roziers N. et al, A microbiological plate assay for measurement of serum enzyme in diodes, transfusion, 1995; 35 (4): 3314, each of which is incorporated herein by reference in its entirety). In one embodiment, an increase in the level of one or more liver enzymes, such as ALT or AST, or an increase in the level of total liver enzymes outside of the normal range, is reduced by more than about 90% or more than 95%. In one embodiment, one or more elevated levels of liver enzymes, such as elevated ALT or AST levels, or elevated total liver enzyme levels, are reduced by at least 95%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, at least 2%, or at least 1%.

In some embodiments, the invention provides methods of treating patients infected with hepatitis C virus and having normal serum transaminase levels. It has been reported that up to 60% of primary blood donors and drug-injected users infected with HCV have normal ALT levels (see Strader et al, Hepatology, 39(4), 2004). In one embodiment, a person who has been tested two or more times within six months or more is considered normal for ALT levels within the approved normal test range. It is known in the art that biopsies of those with normal transaminase values show bridge-like fibrosis or cirrhosis in 1% to 10% and at least portal fibrosis in the greater part of the population (Strader et al, Hepatology, 39(4), 2004). In one embodiment, the compounds provided herein are useful in treating such patients.

In some embodiments, the present invention provides methods of inhibiting Hepatitis C Virus (HCV) replication in a cell infected with HCV by administering a therapeutically effective amount of a compound provided herein. In some embodiments, a therapeutically effective amount of the compound is an amount sufficient to detectably reduce HCV replication. In one embodiment, the compound used in such a method is RO-113-0830. Methods for detecting HCV replication are known to those skilled in the art, including HCV replicon assays. A representative test is described by Pietschmann, T, et al, J.Virol.76, 2002, 4008-. In some embodiments, HCV replication is inhibited by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 90%, or more.

In another aspect, the present invention provides a method of inhibiting HCV replication in a patient infected with hepatitis c virus. The method comprises the step of administering to the patient an effective amount of a compound provided herein. In one embodiment, the method comprises the step of administering to the patient an effective amount of RO-113 and 0830.

In some embodiments, the invention provides methods of treating alcoholic hepatitis. Alcoholic hepatitis (steatohepatitis) is a combination of fatty liver, diffuse liver inflammation, and hepatic necrosis (focal necrosis in some embodiments), with varying degrees of severity on a scale.

In one embodiment, the invention provides a method of treating liver fibrosis, lobular hepatitis, and/or junction bridge necrosis in a patient. Liver fibrosis is an excessive accumulation of extracellular matrix proteins, including collagen, and is present in most types of chronic liver diseases. In some embodiments, severe liver fibrosis causes cirrhosis and liver failure. In one embodiment, the invention provides a method of reducing the level of fibrosis, lobular hepatitis, and/or junction bridge necrosis in a patient. Methods for measuring changes in the degree of hepatic histology, such as fibrosis, lobular hepatitis, and portal bridge necrosis, are well known in the art. For example, in Hepatology, 2006, 43 (2): several non-invasive tests of liver fibrosis are described in S113-S120. Hepatology, 2007, 45 (1): 242-249 describes the measurement and treatment of liver fibrosis. Wright M. et al, in hepatitis C virus infection: a cross sectional and longitudinal study, gut. 2003; 52(4): 5749 measures and determinants of the natural history of liver fibrosis are described. Each of these documents is incorporated by reference herein in its entirety. In some embodiments, liver fibrosis is caused by hepatitis, chemical exposure, bile duct obstruction, autoimmune disease, blood outflow disorders from the liver, cardiac and vascular disorders, alpha 1-antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage disorders, diabetes, malnutrition, Wilson's disease, or hemochromatosis.

In one embodiment, the level of fibrosis in the form of fibrous tissue, a fibroid, or fibrosis is reduced by more than about 90%. In one embodiment, the level of fibrosis is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least 2%.

In one embodiment, the compounds provided herein reduce the level of fibroplasia. Liver fibrosis is a process called fibrosis that causes excessive deposition of extracellular matrix components in the liver. Liver fibroplasia is observed in a number of conditions, such as chronic viral hepatitis b and c, alcoholic liver disease, drug-induced liver disease, hemochromatosis, autoimmune hepatitis, Wilson's disease, primary biliary cirrhosis, sclerosing cholangitis, hepatic hemorrhoidal disease, and others. In one embodiment, the level of fiber proliferation is reduced by more than about 90%. In one embodiment, the level of fiber proliferation is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, or at least 2%.

In one embodiment, the level of lobular hepatitis wherein a foci of inflammatory cells is also present in the lobular sinusoids is reduced by more than about 99% or 95%. In another embodiment, the level of lobular hepatitis is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, at least 2%, or at least 1%. In another embodiment, the level of header bridge necrosis is reduced by more than about 90%. In another embodiment, the level of header bridge necrosis is reduced by at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, at least 2%, or at least 1%.

In one embodiment, the present invention provides a method of treating cirrhosis of the liver. In some embodiments, symptoms of cirrhosis include, but are not limited to, portal hypertension, abnormal neurological function, ascites (fluid accumulation in the abdominal cavity), male breast hypertrophy, hemoptysis or hematemesis, finger deformity (contracture of the aponeurosis of the palm), gallstones, hair loss, itching, jaundice, renal failure, hepatic encephalopathy, muscle loss, anorexia, palmar redness, hypertrophy of salivary glands in the cheek, testicular atrophy, small spider veins in the skin, weakness, weight loss, spider nevi (central arteriole radiating many small branch vessels), encephalopathy, and flapping-wing tremor (tremor). The symptoms of cirrhosis vary depending on the severity and the individual. In some embodiments, mild cirrhosis may not show any symptoms at all.

In one embodiment, the cause of cirrhosis is hepatitis c. In other embodiments, causes of cirrhosis include the use of certain drugs, chemical exposure, biliary obstruction, autoimmune diseases, disorders of blood flow from the liver (i.e., hepatic vein occlusion syndrome), cardiac and vascular disorders, alpha 1-antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage disorders, diabetes, malnutrition, genetic accumulation of excessive copper (Wilson's disease) or iron (hemochromatosis). In one embodiment, the cause of cirrhosis is alcohol abuse.

In one embodiment, the present invention provides a method of reducing the level of cirrhosis of the liver. In one embodiment, the pathological feature of cirrhosis is loss of normal microscopic leaflet structure, with fibrosis and nodule regeneration. Methods for measuring the degree of cirrhosis are well known in the art. In one embodiment, the level of cirrhosis is reduced by about 5% to 100%. In one embodiment, the level of cirrhosis is reduced by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% in the patient.

In some embodiments, a method of treating liver cirrhosis comprises administering an MMP inhibitor provided herein, with the proviso that the MMP inhibitor is not TMI-005.

In some embodiments, the present invention provides methods of treating Primary Biliary Cirrhosis (PBC). Primary biliary cirrhosis begins with inflammation of the bile ducts inside the liver. This inflammation impedes bile flow from the liver; thus, bile stays in the hepatocytes or overflows into the blood stream. As inflammation spreads from the bile duct to the rest of the liver, a lattice of scar tissue develops throughout the liver. In one embodiment, the method is for treating PBC in a female between 35 and 60 years of age. In some embodiments, PBC is caused by an autoimmune disorder. In one embodiment, primary biliary cirrhosis occurs with rheumatoid arthritis, scleroderma, or autoimmune thyroiditis. The methods provided by the present invention are useful in the treatment of one or more symptoms of primary biliary cirrhosis.

In one embodiment, the invention provides a method of treating hepatic ischemia reperfusion injury. Ischemia can occur in the liver as a result of several pathological conditions, such as liver transplantation, cardiogenic shock or hemodynamic shock, and resection of the liver as a result of trauma or tumor. When blood circulation is reestablished (reperfusion), a rapid increase in oxygen concentration causes the formation of various reactive oxygen species, which in turn causes extensive damage (necrosis and apoptosis) to the hepatocytes, causing Ischemia Reperfusion (IR) damage in the liver. In some embodiments, a method of treating hepatic ischemia-reperfusion injury comprises administering a MMP inhibitor provided herein, with the proviso that the MMP inhibitor is not ONO-4817. In some embodiments, the method of treating a hepatic ischemia reperfusion injury comprises administering RO-113-0830.

As known to those skilled in the art, excessive apoptosis of hepatocytes is associated with liver fibrosis and other liver diseases. Therefore, prevention or inhibition of excessive apoptosis of hepatocytes is an important component in the treatment of acute and chronic liver diseases. Apoptosis occurs primarily through two signaling pathways: a death receptor-mediated extrinsic pathway or a mitochondria-mediated intrinsic pathway. After the cytokine receptor families known as death receptors, such as tumor necrosis factor receptor 1(TNF-R1), Fas/CD95, and tumor necrosis factor-related apoptosis-inducing ligand receptors 1 and 2(TRAIL-R1 and TRAIL-R2), bind to their cognate ligands (TNF-, Fas ligand (FasL)/CD95L, TRAIL), the extrinsic pathway starts at the plasma membrane. See Guicciardi et al Gut, 2005: 54, 1024-: 11(11): RA 337-345. In some embodiments, the MMP inhibitors provided herein block damage to hepatocytes by preventing or inhibiting apoptosis. In some embodiments, the compounds provided herein inhibit the signaling cascade of α -Fas. In some embodiments, the compounds provided herein inhibit the signaling cascade initiated by TNF- α. Without being bound by any particular theory, it is believed that in some embodiments, prevention or inhibition of excessive apoptosis of hepatocytes by the compounds provided herein helps reduce liver damage associated with acute and/or chronic liver disease.

5.4 preparation of the Compounds

The compounds used in the methods provided herein can be prepared using conventional synthetic procedures, including the methods described by Bender et al in U.S. patent No. 5,932,595 and Watanabe in U.S. patent nos. 6,207,698 and 6,831,178. A representative method for preparing RO-113-0830 is described in example 1.

5.5 pharmaceutical composition formulations

The pharmaceutical compositions provided herein comprise a therapeutically effective amount of one or more compounds provided herein that are useful for preventing, treating, or ameliorating one or more symptoms of a liver disease, and a pharmaceutically acceptable carrier.

The compounds are formulated in suitable pharmaceutical formulations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs for oral administration or for parenteral administration in sterile solutions or suspensions, as well as transdermal patch preparations and dry powder inhalers. In one embodiment, the compounds described above are formulated into Pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Remington's Pharmaceutical Sciences, 20 th edition, Mack Publishing, Easton PA (2000)).

In such compositions, an effective concentration of one or more compounds or pharmaceutically acceptable derivatives is admixed with a suitable pharmaceutical carrier or vehicle. Prior to formulation, the compounds may be derivatized as described above to the corresponding salts, esters, acids, bases, solvates, hydrates, or prodrugs. The concentration of the compound in the composition is such that it is administered in an amount effective to treat, prevent or ameliorate one or more symptoms of the liver disease.

In one embodiment, the composition is formulated for single dose administration. To formulate a composition to an effective concentration to alleviate or ameliorate the condition being treated, the weight percent of the compound is dissolved, suspended, dispersed, or mixed in a selected carrier. Pharmaceutical carriers or vehicles suitable for administration of the compounds provided herein include any suitable carrier known to those of skill in the art for a particular mode of administration.

In addition, the compounds may be formulated as the sole pharmaceutically active ingredient in the composition, or may be combined with other active ingredients. Liposomal suspensions, including tissue-targeting liposomes, such as tumor-targeting liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as known in the art. Briefly, liposomes, such as multilamellar vesicles (MLV's), can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7: 3 molar ratio) on the inner wall of the flask. A solution of the compound provided by the invention in Phosphate Buffered Saline (PBS) lacking divalent cations was added and the flask was shaken until the lipid membrane was dispersed. The resulting vesicles were washed to remove the unfilled compounds, pelleted by centrifugation, and then resuspended in PBS.

The active compound is included in a pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the treated patient without the presence of undesirable side effects. Therapeutically effective concentrations can be determined empirically by testing the compounds in vitro and in vivo systems known in the art and then deducing the dosage to humans therefrom.

The concentration of the active compound in the pharmaceutical composition will depend on the absorption, inactivation, and excretion rates of the active compound, the physicochemical properties of the compound, the dosage schedule, the amount administered, and other factors known to those skilled in the art. For example, the amount administered is sufficient to ameliorate one or more symptoms of a liver disease.

In one embodiment, a therapeutically effective dose should result in a serum concentration of the active ingredient of from about 0.1ng/ml to about 50-100. mu.g/ml. The pharmaceutical composition, in some embodiments, should provide a dose of about 0.001mg to about 2000mg of the compound per kilogram of body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 1mg to about 1000mg and from about 10 to about 500mg of the principal active ingredient or combination of principal ingredients per dosage unit form.

The active ingredient may be administered in a single dose or may be divided into a plurality of smaller doses to be administered at intervals. It will be understood that the precise dose and duration of treatment is a function of the disease being treated and may be determined empirically using known test protocols or inferred from in vivo or in vitro test data. It should be noted that concentrations and dosage values may also vary with the severity of the condition requiring relief. It will be further understood that for any particular patient, the particular dosage regimen will be adjusted over time in accordance with the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

Pharmaceutically acceptable derivatives include acid, base, salt, ester, hydrate, solvate and prodrug forms. The derivatives are selected such that their pharmacokinetic properties are superior to those of the corresponding neutral compounds.

Thus, an effective concentration or amount of one or more compounds described herein, or a pharmaceutically acceptable derivative thereof, is combined with a suitable pharmaceutical carrier or vehicle for systemic, local, or regional administration to form a pharmaceutical composition. Comprising an amount of the compound effective to treat or prevent a liver disease or ameliorate one or more symptoms thereof. The concentration of the active compound in the composition will depend on the absorption, inactivation, and excretion rates of the active compound, the dosage schedule, the amount administered, the particular formulation, and other factors known to those skilled in the art.

The compositions will be administered by any suitable route, including orally, parenterally, rectally, topically and regionally. For oral administration, capsules and tablets may be used. The compositions are in liquid, semi-fluid or solid form and are formulated in a manner suitable for each route of administration. In one embodiment, the mode of administration includes parenteral and oral modes of administration. In some embodiments, oral administration is contemplated.

Solutions or suspensions for parenteral, intradermal, subcutaneous or topical application may contain any of the following ingredients: sterile diluents such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, dimethylacetamide, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol and methyl paraben; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate and phosphate; and tonicity adjusting agents such as sodium chloride or dextrose. The parenteral formulation may be enclosed in ampoules, disposable syringes, or single-dose or multi-dose vials made of glass, plastic, or other suitable material.

In the case where the solubility of the compound is insufficient, a method of solubilizing the compound may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEENOr dissolved in an aqueous sodium bicarbonate solution.

After mixing or adding the compounds, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture will depend on a variety of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate symptoms of the disease, disorder or condition being treated and can be determined empirically.

For administration to humans and animals, the pharmaceutical compositions are provided in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, and oral solutions or suspensions, and oil-water emulsions containing suitable amounts of the compound or a pharmaceutically acceptable derivative thereof. Pharmaceutically therapeutically active compounds and derivatives thereof, are formulated and administered in unit dosage form or multiple dosage forms. As used herein, unit dosage forms refer to physically discrete units suitable for human and animal patients and packaged individually as is known in the art. Each unit dose contains a predetermined amount of the therapeutically active compound sufficient to produce the desired therapeutic effect, in combination with a pharmaceutical carrier, vehicle or diluent as required. Examples of unit dosage forms include ampoules and syringes, and individually packaged tablets or capsules. The unit dosage form may be administered in parts or in multiples. Multiple dosage forms are those in which a plurality of identical unit dosage forms are packaged in a single container for administration as separate unit dosage forms. Examples of multiple doses include vials, bottled tablets or capsules, or bottled pints or gallons. Thus, a multiple dosage form is a plurality of unit doses that are not divided in a package.

Sustained release formulations may also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing a compound provided herein, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can be used inMolecules are released within 100 days, while some hydrogels release proteins for shorter periods of time. When the charged compounds are left in the body for a long time, exposure to moisture at 37 ℃ results in their potential for denaturation or polymerization, resulting in loss of biological activity and possible alteration of their structure. Depending on the relevant mechanism of action, rational strategies can be devised to achieve stability. For example, if the polymerization mechanism is found to be intermolecular S — S bond formation through thio-disulfide exchange, stability can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

Dosage forms or compositions can be prepared containing from 0.005% to 100% of the active ingredient with the remainder being non-toxic carriers. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by the addition of any of the usual excipients, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, talcum, cellulose derivatives, croscarmellose sodium, glucose, sucrose, magnesium carbonate, or sodium saccharin. Such compositions include solutions, suspensions, tablets, capsules, powders, and sustained release formulations such as, but not limited to, implants and microencapsulated delivery systems, as well as biodegradable biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain from 0.001% to 100% active ingredient, and in one embodiment, from 0.1% to 85% or from 75% to 95% active ingredient.

The active compound or pharmaceutically acceptable derivative may be formulated with a carrier that protects the compound from rapid elimination from the body, for example, as a time-release formulation or coating.

The composition may contain other active compounds to achieve a desired combination of properties. As described herein, the compounds provided by the present invention, or pharmaceutically acceptable derivatives thereof, may also be advantageously administered for therapeutic or prophylactic purposes, together with another agent known in the general art to be of value in the treatment of liver diseases. It will be appreciated that such combination therapy forms a further aspect of the compositions and methods of treatment provided by the present invention.

5.5.1 compositions for oral administration

Oral pharmaceutical dosage forms are solid, gel or liquid. Solid dosage forms are tablets, capsules, granules and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar-coated, or film-coated. The capsules may be hard or soft gelatin capsules and the granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients known to those skilled in the art.

In some embodiments, the formulation is a solid dosage form, such as a capsule or tablet. Tablets, pills, capsules, lozenges, etc., may comprise any of the following ingredients or compounds of similar nature: a binder; a diluent; a disintegrant; a lubricant; a glidant; a sweetener; and a flavoring agent.

Examples of binders include microcrystalline cellulose, gum tragacanth, dextrose solution, acacia mucilage, gelatin solutions, sucrose and starch pastes. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dibasic calcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colorants include, for example, any approved certified water-soluble FD and C dyes, and mixtures thereof; and water insoluble FD and C dyes suspended in alumina hydrate. Sweetening agents include sucrose, lactose, mannitol, and artificial sweeteners such as saccharin, as well as any number of spray-dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits, and synthetic mixtures of compounds that produce a pleasant sensation, such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Enteric coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. The film coat comprises hydroxyethyl cellulose, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

If oral administration is desired, the compound may be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition may be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The compositions may also be formulated in combination with antacids or other such ingredients.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. In addition, the dosage unit forms may contain various other materials which modify the physical form of the dosage unit, such as coatings for sugar and other enteric agents. The compounds may also be administered as components of elixirs, suspensions, syrups, wafers, sprays, chewing gums and the like. Syrups may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavoring agents.

The active material may also be mixed with other active materials that do not impair the desired action or materials that supplement the desired action, such as antacids, H2 blockers and diuretics. The active ingredient is a compound as described herein or a pharmaceutically acceptable derivative thereof. Higher concentrations, up to about 98% by weight of the active ingredient may be included.

Pharmaceutically acceptable carriers included in tablets are binders, lubricants, diluents, disintegrating agents, coloring agents, flavoring agents and wetting agents. Enteric coated tablets have a gastric acid resistant effect due to the enteric coating and dissolve or disintegrate in the neutral or alkaline intestine. Sugar-coated tablets are compressed tablets, using different layers of pharmaceutically acceptable substances. Film coated tablets are compressed tablets coated with a polymer or other suitable coating. Multiple compressed tablets are compressed tablets made by more than one compression cycle using the above-described pharmaceutically acceptable substances. Colorants may also be used in the above formulations. Flavoring and sweetening agents are used in compressed tablets, sugar-coated tablets, multiple-compressed tablets, and chewable tablets. Flavoring and sweetening agents are particularly useful in the formation of chewable tablets and lozenges.

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions, and/or suspensions reconstituted from non-effervescent granules and effervescent formulations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. The emulsion is oil-in-water or water-in-oil.

Elixirs are clear, sweet, hydroalcoholic formulations. Pharmaceutically acceptable carriers for use in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. An emulsion is a two-phase system of one liquid dispersed as beads in another liquid. Pharmaceutically acceptable carriers for use in emulsions are non-aqueous liquids, emulsifiers and preservatives. Suspensions employ pharmaceutically acceptable suspending agents and preservatives. In non-effervescent granules reconstituted into liquid oral dosage forms, pharmaceutically acceptable materials used include diluents, sweeteners and wetting agents. In effervescent granules reconstituted into liquid oral dosage forms, the pharmaceutically acceptable materials used include organic acids and a source of carbon dioxide. Coloring agents and flavoring agents are used in all of the above dosage forms.

Solvents include glycerol, sorbitol, alcohol and syrup. Examples of preservatives include glycerin, methyl and propyl parabens, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids used in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, gum arabic, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, veegum, and acacia. Diluents include lactose and sucrose. Sweetening agents include sucrose, syrup, glycerin, and artificial sweeteners such as saccharin. Humectants include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate, and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved certified water soluble FD and C dyes, and mixtures thereof. Flavoring agents include natural flavors extracted from plants, such as fruits, and synthetic mixtures of compounds that produce a pleasant taste sensation.

For solid dosage forms, solutions or suspensions in, for example, propylene carbonate, vegetable oils, or triglycerides can be encapsulated in gelatin. Such solutions, as well as their formulation and encapsulation, are described in U.S. patent nos. 4,328,245; 4,409,239, respectively; and 4,410,545. For liquid dosage forms, solutions, such as those in polyethylene glycol, may be diluted with a sufficient amount of a pharmaceutically acceptable liquid carrier, such as water, to facilitate the administration of the measurement.

Alternatively, liquid or semi-solid oral formulations may be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations, including but not limited to those comprising a compound provided herein, dialkylated mono-or poly-alkylene glycols (including but not limited to 1, 2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether, where 350, 550, and 750 refer to the approximate average molecular weight of the polyethylene glycol), and one or more antioxidants (e.g., Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates).

Other formulations include, but are not limited to, aqueous alcoholic solutions containing pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol. Acetals include, but are not limited to di (lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal.

In all embodiments, tablet and capsule formulations may be coated as known to those skilled in the art in order to modify or maintain dissolution of the active ingredient. Thus, for example, they may be coated with coatings that are normally digestible by the intestine, such as phenyl salicylate, waxes and cellulose acetate phthalate.

5.5.2 injections, solutions and emulsions

Parenteral administration, typically subcutaneous, intramuscular, or intravenous injection, is also contemplated herein. Injectables can be prepared in conventional forms as liquid solutions or suspensions, solid forms suitable for conversion to solutions or suspensions in liquid prior to injection, or emulsions. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizing agents, solubility enhancing agents, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins. The invention also contemplates the implantation of a sustained release or sustained release system to maintain a constant level of dosage. Briefly, the present invention provides compounds dispersed in a solid internal matrix, such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of acrylates and methacrylates, collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate, surrounded by an external polymeric film, such as polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, neoprene, polyvinyl chloride, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene vinyl acetate copolymers, silicone rubber, crosslinked polyvinyl alcohol, crosslinked partially hydrolyzed polyvinyl acetate, and the like, Chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, polyethylene terephthalate ionomers, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymers, ethylene/vinyl acetate/vinyl alcohol terpolymers, and ethylene/ethyleneoxyethanol copolymers, which are not soluble in body fluids. In the release rate controlling step, the compound diffuses through the outer polymeric membrane. The percentage of active compound included in such parenteral compositions is highly dependent on its specific properties, as well as the activity of the compound and the needs of the patient.

Parenteral administration of the compositions includes intravenous, subcutaneous, and intramuscular administration. Formulations for parenteral administration include sterile solutions ready for injection, sterile dried soluble products such as lyophilized powders (which are combined with a solvent prior to use), including subcutaneous injection tablets, sterile suspensions ready for injection, sterile dried insoluble products (which are ready to be combined with a carrier prior to use), and sterile emulsions. The solution may be an aqueous solution or a non-aqueous solution.

Suitable carriers, if administered intravenously, include physiological saline or Phosphate Buffered Saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous carriers, non-aqueous carriers, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable materials.

Examples of aqueous carriers include sodium chloride injection, ringer's injection, isotonic dextrose injection, sterile water injection, dextrose and lactate ringer's injection. Non-aqueous parenteral deliveryThe carrier includes fixed oil from plants, cottonseed oil, corn oil, sesame oil and peanut oil. Parenteral formulations packaged in multi-dose containers must be supplemented with a bacteria-inhibiting concentration or a fungi-inhibiting concentration of an antimicrobial agent, including phenol or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride, and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. The buffer comprises phosphate and citrate. The antioxidant comprises sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropylmethylcellulose, and polyvinylpyrrolidone. The emulsifier comprises polysorbate 80 (TWEEN)80). Sequestering or chelating agents for metal ions include EDTA. Pharmaceutical carriers also include water-miscible vehicles such as alcohols, polyethylene glycols, and propylene glycol, and pH-adjusted sodium hydroxide, hydrochloric acid, citric acid, or lactic acid.

The concentration of the pharmaceutically active compound is adjusted so that the injectable formulation provides an effective amount to produce the desired pharmacological effect. As is known in the art, the precise dosage depends on the age, weight and condition of the patient or animal.

A unit dose of the parenteral formulation, packaged in an ampoule, vial or syringe with needle. As known and practiced in the art, all formulations for parenteral administration must be sterile.

Illustratively, intravenous or arterial infusion of sterile aqueous solutions containing the active compounds is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing the active material which is injected to produce the desired pharmacological effect.

Injections are designed for regional and systemic administration. In some embodiments, a therapeutically effective dose is formulated to comprise the active compound at a concentration of at least about 0.1% w/w up to about 90% w/w or more, or more than 1% w/w, relative to the tissue being treated. The active ingredient may be administered in a single dose or may be divided into a number of smaller doses to be administered at intervals. It will be appreciated that the precise dose and duration of treatment is a function of the tissue being treated and may be determined empirically using known test protocols or inferred from in vivo or in vitro test data. It should be noted that concentrations and dosage values may also vary with the age of the individual being treated. It will be further understood that for any particular patient, the particular dosage regimen will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the formulation, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed formulation.

The compounds may be suspended in micronized or other suitable form, or may be derivatized to produce a more soluble active product, or to produce a prodrug. The form of the resulting mixture will depend on a variety of factors, including the intended mode of administration, and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate symptoms of the condition and can be determined empirically.

5.5.3 Freeze-dried powder

Lyophilized powders are also of interest for the present invention, which can be reconstituted into solutions, emulsions and other mixtures to be administered. They may also be reconstituted and formulated as solids or gels.

Sterile lyophilized powders are prepared by dissolving a compound provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. The solvent may comprise excipients that improve the stability or other pharmacological components of the powder or reconstituted solution prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. The solvent may also comprise a buffer, such as citrate, sodium or potassium phosphate, or other such buffers known to those skilled in the art, at a pH of about neutral. The solution is then sterile filtered, followed by lyophilization under standard conditions known to those skilled in the art to provide the desired formulation. Typically, the resulting solution will be dispensed into vials for lyophilization. Each vial will contain a single dose (10-1000mg or 100-500mg) or multiple doses of the compound. The lyophilized powder may be stored under appropriate conditions, for example at about 4 ℃ to room temperature.

Reconstitution of the lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, about 1-50mg, 5-35mg, or about 9-30mg of lyophilized powder is added per ml of sterile water or other suitable vehicle. The exact amount depends on the compound selected. Such amounts may be determined empirically.

5.5.4 topical application

Topical mixtures were prepared as described for regional and systemic administration. The resulting mixture may be a solution, suspension, emulsion, etc., and formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical application.

The compounds, or pharmaceutically acceptable derivatives thereof, may be formulated as aerosols for topical application, e.g., by inhalation (see, e.g., U.S. Pat. nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of steroids useful in the treatment of inflammatory diseases, particularly asthma). These formulations for respiratory administration may be used alone or in combination with an inert carrier such as lactose, in the form of an aerosol or solution for an atomizer, or in the form of a fine powder for insufflation. In such cases, the particle diameter of the formulation will be less than 50 microns or less than 10 microns.

The compounds may be formulated for topical or local application, for example for topical application to the skin and mucous membranes, for example in the eye, in the form of gels, creams and lotions, for application to the eye or for intracisternal or intraspinal application. Topical administration is intended to be transdermal, as well as administration to the eye or mucosa, or inhalation therapy. Nasal solutions of the active compounds, alone or in combination with other pharmaceutically acceptable excipients, may also be administered.

These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01% to 10% isotonic solutions with appropriate salts, at a pH of about 5 to 7.

5.5.5 compositions for other routes of administration

Other routes of administration are also encompassed by the present invention, such as topical application, transdermal patches, and rectal administration.

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets with systemic effect. Rectal suppositories for use herein mean solid bodies for insertion into the rectum which melt or soften at body temperature releasing one or more pharmaceutically or therapeutically active ingredients. Pharmaceutically acceptable substances used in rectal suppositories are bases or carriers, and agents that raise the melting point. Examples of bases include cocoa butter (cocoa butter), glycerol-gelatin, polyethylene glycol (polyoxyethylene glycol), and suitable mixtures of fatty acid monoglycerides, fatty acid diglycerides, and fatty acid triglycerides. Combinations of substrates may be used. Agents that raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared by compression methods or molding. In some embodiments, the rectal suppository weighs about 2 to 3 grams.

Tablets and capsules for rectal administration are manufactured by the same method using the same pharmaceutically acceptable substances as preparations for oral administration.

5.5.6 sustained Release compositions

Active ingredients such as the compounds provided herein may be administered by controlled release means or delivery devices well known to those of ordinary skill in the art. Examples include, but are not limited to, U.S. Pat. nos. 3,845,770; 3,916,899; 3,536,809, respectively; 3,598,123, respectively; 4,008,719, respectively; 5,674,533, respectively; 5,059,595, respectively; 5,591,767, respectively; 5,120,548, respectively; 5,073,543, respectively; 5,639,476, respectively; 5,354,556, respectively; 5,639,480, respectively; 5,733,566; 5,739,108, respectively; 5,891,474, respectively; 5,922,356, respectively; 5,972,891, respectively; 5,980,945, respectively; 5,993,855, respectively; 6,045,830, respectively; 6,087,324, respectively; 6,113,943; 6,197,350, respectively; 6,248,363, respectively; 6,264,970, respectively; 6,267,981, respectively; 6,376,461, respectively; 6,419,961, respectively; 6,589,548, respectively; 6,613,358 and 6,699,500, each of which is incorporated herein by reference. Such dosage forms may be used to provide slow or controlled release of one or more active ingredients using, for example, hydroxypropylmethylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or combinations thereof, in varying proportions to provide the desired release profile. Suitable controlled release formulations known to those of ordinary skill in the art, including those described herein, can be readily selected for use with the active ingredients provided herein. Thus, provided compositions comprise single unit dosage forms suitable for oral administration, such as, but not limited to, tablets, capsules, gelcaps, and caplets, which are suitable for controlled release.

All controlled release drug products share a common goal: improving the treatment of the drug over that which can be achieved by the corresponding drug without controlled release. Ideally, the use of optimally designed controlled release formulations in medical treatment is characterized by the application of a minimum amount of drug to cure or control the condition in a minimum amount of time. Advantages of controlled release formulations include prolonged drug activity, reduced dosing frequency, and increased patient compliance. In addition, controlled release formulations may be used to affect the onset of action or other characteristics, such as blood levels of the drug, and thus may affect the occurrence of side effects (e.g., adverse effects).

Most controlled release formulations are designed to initially release a certain amount of the drug (active ingredient) which rapidly produces the desired therapeutic effect, and to gradually and continuously release other amounts of the drug to maintain this level of therapeutic or prophylactic effect over an extended period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a rate that will replace the amount of drug that is metabolized and expelled from the body. The controlled release of the active ingredient may be stimulated by various conditions, including but not limited to pH, temperature, enzymes, water, or other physiological conditions or compounds.

In some embodiments, the drug may be administered using intravenous infusion, implantable osmotic pumps, transdermal patches, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Sefton, CRC Crit. Ref. biomed Eng.14: 201 (1987); Buchwald et al, Surgery 88: 507 (1980); Saudek et al, N.Engl. J. Med.321: 574 (1989)). In another embodiment, polymeric materials may be used. In yet another embodiment, the Controlled Release system may be placed in the patient in a suitable location determined by a skilled artisan, i.e., so that only a small fraction of the systemic dose is required (see, e.g., Goodson, Medical Applications of Controlled Release, Vol.2, p.115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science 249: 1527-. The active ingredient may be dispersed in a solid internal matrix such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of acrylates and methacrylates, collagen, crosslinked polyvinyl alcohol, and crosslinked partially hydrolyzed polyvinyl acetate surrounded by an external polymeric film such as polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, neoprene, chlorinated polyethylene, polyvinyl chloride, polyvinyl, Vinyl chloride and vinyl acetate copolymers, vinylidene chloride, ethylene and propylene, polyethylene terephthalate ionomers, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymers, ethylene/vinyl acetate/vinyl alcohol terpolymers, and ethylene/ethyleneoxyethanol copolymers, which are insoluble in body fluids. Then, in the release rate controlling step, the active ingredient diffuses through the outer polymeric membrane. In such parenteral compositions, the percentage of active ingredient is highly dependent on its specific properties and the needs of the patient.

5.5.7 targeting agent

The compounds provided by the present invention, or pharmaceutically acceptable derivatives thereof, may also be formulated to target specific tissues, receptors, or other body regions of the patient to be treated. Many such targeting methods are well known to those skilled in the art. All such targeting methods are contemplated for use in the present compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874.

In one embodiment, liposomal suspensions, including tissue-targeting liposomes, such as tumor-targeting liposomes, may also be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as described in U.S. Pat. No. 4,522,811. Briefly, liposomes, such as multilamellar vesicles (MLV's), can be formed by drying egg phosphatidylcholine and brain phosphatidylserine (7: 3 molar ratio) on the inner wall of the flask. A solution of the compound provided by the invention in Phosphate Buffered Saline (PBS) lacking divalent cations was added and the flask was shaken until the lipid membrane was dispersed. The resulting vesicles were washed to remove the unencapsulated compounds, pelleted by centrifugation, and then resuspended in PBS.

5.5.8 dosage and unit dosage form

In human therapeutics, the physician will decide the dosage which will most suitably be considered in terms of prophylactic or therapeutic treatment and in terms of the age, weight, stage of disease and other factors which are characteristic of the patient being treated. Generally, the dosage will be about 1 to about 1000mg per day for an adult, or about 5 to about 250mg per day, or about 10 to 50mg per day. In some embodiments, the dose is from about 5 to about 400mg per day or from 25 to 200mg per day for each adult. Administration rates of about 50 to about 500mg per day are also contemplated.

In some embodiments, the amount of a compound or composition that will be effective in preventing or treating a liver disease or one or more symptoms thereof will vary depending on the nature and severity of the disease or condition, and the route of administration of the active ingredient. The frequency and dosage will also vary according to factors specific to each patient, depending on the particular treatment (e.g., therapeutic or prophylactic) being administered, the severity of the disorder, disease, or condition, the route of administration, and the age, body, weight, response, and past medical history of the patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Representative doses of the compositions include milligram or microgram amounts of the MMP inhibitor per kilogram of patient or sample weight (e.g., about 10 micrograms per kilogram to about 50 milligrams per kilogram, about 100 micrograms per kilogram to about 25 milligrams per kilogram, or about 100 micrograms per kilogram to about 10 milligrams per kilogram). In some embodiments, the dose administered to the patient is between 0.20mg/kg and 2.00mg/kg of patient body weight, or between 0.30mg/kg and 1.50mg/kg of patient body weight.

In some embodiments, for the conditions described herein, the recommended daily dosage of MMP inhibitors described herein ranges from about 0.1mg to about 1000mg per day, administered as a single once-a-day dose, or administered as separate doses over the course of a day. In one embodiment, the daily dose is administered twice daily in divided doses. Specifically, the daily dosage range should be from about 10mg to about 200mg per day, more specifically, between about 10mg and about 150mg per day, or even more specifically, between about 25 and about 100mg per day. In some cases, it may be necessary to use dosages of the active ingredient outside the scope of the present disclosure, as will be apparent to those of ordinary skill in the art. In addition, the clinician or treating physician will have an understanding of how and when to interrupt, adjust or terminate therapy in connection with the patient's response.

Different therapeutically effective amounts may be applied to different diseases and conditions, as will be readily appreciated by one of ordinary skill in the art. Similarly, amounts sufficient to prevent, control, treat or ameliorate such a condition, but insufficient to cause or sufficient to reduce the adverse effects associated with the compounds described herein, are also included in the aforementioned amounts and frequency schedules of administration. Furthermore, when a patient is administered multiple doses of a compound described herein, all doses need not be the same. For example, the dosage administered to a patient can be increased to enhance the prophylactic or therapeutic effect of the compound, or the dosage administered to a patient can be decreased to reduce one or more side effects experienced by the particular patient.

In one embodiment, the dose of a compound of the invention administered to prevent, treat, manage or ameliorate a disorder or one or more symptoms thereof in a patient is 0.1mg/kg, 1mg/kg, 2mg/kg, 3mg/kg, 4mg/kg, 5mg/kg, 6mg/kg, 10mg/kg, or 15mg/kg of the patient's body weight or more. In another embodiment, the dose of a compound provided herein that prevents, treats, manages or ameliorates the disorder, or one or more symptoms thereof, administered in a patient is a unit dose of 0.1mg to 200mg, 0.1mg to 100mg, 0.1mg to 50mg, 0.1mg to 25mg, 0.1mg to 20mg, 0.1mg to 15mg, 0.1mg to 10mg, 0.1mg to 7.5mg, 0.1mg to 5mg, 0.1 to 2.5mg, 0.25mg to 20mg, 0.25 to 15mg, 0.25 to 12mg, 0.25 to 10mg, 0.25mg to 7.5mg, 0.25mg to 5mg, 0.5mg to 2.5mg, 1mg to 20mg, 1mg to 15mg, 1mg to 12mg, 1mg to 10mg, 1mg to 7.5mg, 1mg to 5mg, or 1mg to 2.5 mg.

In some embodiments, treatment or prevention can be initiated with one or more loading doses of MMP inhibitor and/or caspase inhibitor provided herein, followed by one or more maintenance doses. In such embodiments, the loading dose may be, for example, from about 60 to about 400mg per day or from about 100 to about 200mg per day for one to five weeks. The loading dose may be followed by one or more maintenance doses. In another embodiment, each maintenance dose may independently be about 0.1mg to about 200mg per day, in one embodiment between about 5mg and about 150mg per day, in another embodiment between about 10 and about 80mg per day, in another embodiment between about 10mg to about 200mg per day, in another embodiment between about 25mg and about 150mg per day, or in yet another embodiment between about 25 and about 80mg per day. The maintenance dose may be administered daily, and may be administered as a single dose or as divided doses.

In some embodiments, a dose of an MMP inhibitor provided herein can be administered to achieve a stable concentration of the active ingredient in the blood or serum of the patient. The stable concentration may be determined by measurement techniques available to the skilled person, or may be based on physical characteristics of the patient, such as height, weight and age. In some embodiments, a sufficient amount of a compound provided herein is administered to achieve a stable concentration in the blood or serum of a patient of about 300 to about 4000ng/mL, about 400 to about 1600ng/mL, or about 600 to about 1200 ng/mL. The loading dose may be administered for one to five days to achieve a stable blood or serum concentration of about 1200 to about 8000ng/mL or about 2000 to about 4000 ng/mL. Maintenance doses can be administered to achieve a stable concentration in the blood or serum of a patient of about 300 to about 4000ng/mL, about 400 to about 1600ng/mL, or about 600 to about 1200 ng/mL.

In some embodiments, the same compound may be administered repeatedly, and may be administered at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months apart. In other embodiments, the same prophylactic or therapeutic agent can be administered repeatedly, and can be administered at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months apart.

In certain aspects, the invention provides a unit dose comprising a compound or a pharmaceutically acceptable derivative thereof in a form suitable for administration. Such forms are described in detail above. In some embodiments, a unit dose contains 1 to 1000mg, 5 to 250mg, or 10 to 50mg of the active ingredient. In particular embodiments, the unit dose comprises about 1,5, 10, 25, 50, 100, 125, 250, 500, or 1000mg of the active ingredient. Such unit doses can be prepared according to techniques familiar to those skilled in the art.

5.5.9 to name a few

The compound or pharmaceutically acceptable derivative may be packaged as an article of manufacture comprising packaging material, a compound provided herein or a pharmaceutically acceptable derivative thereof for treating, preventing or ameliorating one or more symptoms associated with a liver disease, and a label indicating that the compound or pharmaceutically acceptable derivative thereof is for treating, preventing or ameliorating one or more symptoms of a liver disease.

Articles of manufacture provided by the present invention include packaging materials. Packaging materials for use in packaging pharmaceutical products are well known to those skilled in the art. See, for example, U.S. patent nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment. The invention includes various formulations of the provided compounds and compositions.

5.6 evaluation of Compound Activity

The biological activity of the compounds can be demonstrated by methods known to those skilled in the art. For example, Neil Kaplowitz, a mouse model of acute Liver Injury is described in Mechanisms in Liver Injury and engineering Therapeutics, published by American Association for the Study of Liver Diseases (2006), which is incorporated herein by reference in its entirety.

TNF- α is a liver injury-inducing cytokine involved in a variety of acute and chronic liver diseases, such as chronic HCV and acute liver failure. One representative in vivo model for testing pharmacological agents for TNF- α induced damage is the mouse TNF- α/D-Gal liver damage model. In this model, mice were treated with TNF- α/D-Gal and compounds were administered to assess their ability to protect the liver from injury. The compound is administered prior to, concurrently with, or after treatment with TNF- α/D-Gal, and then for a period of about 6 hours. Allowing this model to last for 6 hours was used to examine the improvement in survival resulting from compound treatment.

A variety of outcome measurements are used to make this assessment. One of these is a measure of the level of liver enzyme ALT in the blood. Elevated ALT levels are routinely observed in the blood of patients with various liver diseases. ALT measurements are a very common and relevant clinical test for the extent of liver disease in patients. Another measurement includes a gross assessment and histological assessment of liver damage. The degree of liver damage can be graded by preparing and evaluating liver samples for microscopic observation by examination by a trained observer. In some embodiments, the liver injury can be severe enough to be lethal. In some embodiments, the compounds of the invention have a protective effect on TNF- α/D-Gal induced liver damage as measured by these parameters. In some embodiments, the compounds of the invention have a protective effect on Fas-induced liver injury as measured by these parameters. In some embodiments, the compounds provided herein are shown to reduce liver injury and liver fibrosis in a common bile duct ligation model.

Other models of liver injury include the LPS/D-Gal model, the alpha-Fas-induced liver injury model, and the ConA model of liver injury. These models are also associated with human disease. These three models are complementary to each other.

In some embodiments, the compounds provided herein are shown to inhibit HCV replication in an HCV replicon assay.

6. Combination therapy

In some embodiments, the MMP inhibitors provided herein are administered in combination with one or more second agents known to treat liver diseases. The dosage of the second agent used in the combination therapy is known in the art. In some embodiments, in the combination therapies provided herein, lower doses are used than those already or currently being used to prevent or treat liver diseases such as hepatitis b or hepatitis c. The recommended dose of the second medicament may be obtained by the knowledge of the skilled person. For those second agents approved for clinical use, recommended dosages are as set forth in, for example, Schiff's Diseases Of the Liver version 10 (2006), Lippincott, Williams and Wilkins, Hardman et al, eds., 1996, Goodman & Gilman's the pharmaceutical basic Of the Basis Of Therapeutics ninth edition, Mc-Graw-Hill, New York; physician's Desk Reference (PDR) 57 th edition, 2003, Medical Economics Co., Inc., Montvale, NJ, which are incorporated herein by Reference in their entirety.

In various embodiments, the treatments (e.g., a compound provided herein and a second agent) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, a therapeutic agent, and a second agent, all of the treatment are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 7 hours apart, about 8, 72 hours apart to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours apart. In some embodiments, two or more treatments are administered within a single patient visit.

In some embodiments, the compound provided herein and the second agent are administered cyclically. Cycling therapy involves the administration of a first treatment (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second treatment (e.g., a second prophylactic or therapeutic agent) for a period of time, followed by the administration of a third treatment (e.g., a third prophylactic or therapeutic agent) for a period of time, and so forth, and repeating such sequential administration, i.e., cycling, in order to reduce the development of resistance to one of the agents, avoid or reduce the side effects of one of the agents, and/or increase the efficacy of the treatment.

In some embodiments, the compounds provided herein and the second agent are administered to a patient, e.g., a mammal, e.g., a human, in such an order and within such time intervals that the compounds provided herein can act together with the other agents to provide an increased benefit over administering them otherwise. For example, the second active agent may be administered at the same time or sequentially in any order at different time points; however, if not administered at the same time, they should be administered close enough in time to provide the desired therapeutic or prophylactic effect. In one embodiment, the present invention provides that the compound and the second active agent exert their effects in partially overlapping times. Each second active agent may be administered separately in any suitable form and by any suitable route. In other embodiments, the compounds provided herein are administered prior to, concurrently with, or after the administration of the second active agent.

In some embodiments, the compounds provided herein and the second active agent are administered in a cycle of less than about 3 weeks, about once every two weeks, about once every 10 days, or about once every week. A cycle may comprise administering a compound provided herein and a second agent by infusion within about 90 minutes per cycle, within about 1 hour per cycle, and within about 45 minutes per cycle. Each cycle may comprise at least 1 week of rest, at least 2 weeks of rest, and at least 3 weeks of rest. The number of cycles of administration is from about 1 to about 12 cycles, more typically from about 2 to about 10 cycles, and more typically from about 2 to about 8 cycles.

In some embodiments, the same agent may be administered repeatedly, and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or 6 months. In other embodiments, the administration of a compound provided by the invention and a second agent are separated by about 2 to 4 days, by about 4 to 6 days, by about 1 week, by about 1 to 2 weeks, or by more than 2 weeks.

In other embodiments, the course of treatment is administered to the patient concurrently, i.e., separate doses of the second agent are administered separately, but within a time interval such that the compounds provided herein can act in conjunction with the second agent. For example, one ingredient may be administered once a week, in combination with other ingredients that may be administered once every two weeks or once every three weeks. In other words, the dosing regimens are performed simultaneously, even if the treatments are not administered simultaneously or during the same day.

The second agent may act additively or synergistically with the compounds provided herein. In one embodiment, the compounds provided herein are administered simultaneously with one or more second agents in the same pharmaceutical composition. In another embodiment, the compounds provided herein are administered simultaneously with one or more second agents in different pharmaceutical compositions. In yet another embodiment, the compounds provided herein are administered before or after the administration of the second agent. Alternatively, the compound provided by the invention and the second agent are administered by the same or different routes of administration, e.g., oral and parenteral. In some embodiments, when a compound provided herein is administered concurrently with a second agent that potentially causes an adverse side effect, including but not limited to toxicity, the second agent may advantageously be administered at a dose below the threshold that causes the adverse side effect.

In some embodiments, the compounds provided herein are administered in combination with a second agent. In further embodiments, the second agent is administered in combination with two second agents. In still further embodiments, the second agent is administered in combination with two or more second agents.

In combination therapy, effective doses of two or more agents are administered together, while in alternating or sequential stepwise therapy, effective doses of each agent are administered sequentially or sequentially. The prescribed dosage will depend on absorption, inactivation, and excretion rates of the drug, as well as other factors known to those skilled in the art. It should be noted that dosage values will also vary with the severity of the condition requiring relief. It will be further understood that for any particular patient, the specific dosage regimen and schedule will be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions.

In some embodiments, the methods provided herein comprise administering MMP inhibitors described herein, e.g., Intron A, peginterferon alpha-2 a (pyroxin R), peginterferon alpha-2 a + ribavirin (pyroxin and Copegus, see, e.g., Hoofnagle et al, N.ene.J.Med.355: 23), lamivudine, adefovir, entecavir, emtricitabine (FTC), telbivudine (L-dT), toltabine (VaI-LdC), elvucitabine (L-Fd4C), cladribine, Racivir, BAM 205, NOV-205(BAM 205), HepeX-B, amdoxovir (DAPD), ANA 380(LB80380), Lafutovir (Revavuvir), EHT 899, Padavir (Revavuv), Cor 899, Adrianfu (I-B, EP), Cordavu-S231, HBE-S231, and Copegus, in combination with other agents, MIV 210, SpecifEx-HepB, Pentacept (L-3' -FD4C), Bay41-4109INTM-191 or VX-950 (telaprevir).

In some embodiments, the second agent is selected from the following:

protease inhibitors: examples include Medivir HCV protease inhibitors (Medivir/Tobotec); ITMN-191(InterMune), SCH 503034(Schering), and VX950 (Vertex). Further examples of protease Inhibitors include substrate-based NS3 protease Inhibitors (Attwood et al, antibacterial peptide derivatives, PCT WO 98/22496, 1998; Attwood et al, antibacterial chemistry and chemistry 1999, 10, 259-273; Attwood et al, Preparation and use of amino derivatives as anti-viral agents, German patent publication DE 19914474; Tung et al, Inhibitors of serproteases, particulate peptides C bacteria NS3 protease, PCT WO98/17679, including alpha ketoamides and hydrazinoureas, and Inhibitors that terminate in electrophiles such as boronic acids or phosphonates (Llinas-Bruet et al, polypeptides C peptides derivatives, PCT WO 46 07734/46 07734); non-substrate based NS3 protease inhibitors such as 2, 4, 6-trihydroxy-3-nitro-benzamide derivatives (Sudo K. et al, Biochemical and Biophysical Research Communications, 1997, 238, 643-647; Sudo K. et al, Antiviral Chemistry and chemotherapeutics, 1998, 9, 186), including RD3-4082 and RD3-4078, the former having a 14 carbon chain substitution on the amide and the latter having a p-phenoxyphenyl group; and Sch 68631, phenanthrenequinones, HCV protease inhibitors (Chu M. et al Tetrahedron Letters 37: 7229-7232, 1996).

SCH 351633 isolated from the fungus Penicillium griseofulvum was identified as a protease inhibitor (Chu M. et al, Bioorganic and Medicinal Chemistry Letters 9: 1949-1952). Eglin c isolated from leeches is a potent inhibitor of several serine proteases, such as s.griseus proteases a and B, alpha-chymotrypsin, chymotrypsin and subtilisin. Qasim m.a. et al, Biochemistry 36: 1598-1607, 1997.

U.S. patents disclosing protease inhibitors for the treatment of HCV include, for example, U.S. patent No. 6,004,933 to Spruce et al, which discloses a class of cysteine protease inhibitors that inhibit HCV endopeptidase 2; zhang et al, U.S. Pat. No. 5,990,276, which discloses synthetic inhibitors of hepatitis C Virus NS3 protease; U.S. patent numbers 5,538,865 to Reyes et al; WO 02/008251 from Corvas International, and US 7,169,760, US2005/176648, WO02/08187, and WO 02/008256 from Schering. HCV inhibitor tripeptides are disclosed in U.S. Pat. Nos. 6,534,523, 6,410,531 and 6,420,380 to Boehringer Ingelheim and WO 02/060926 to Bristol Myers Squibb. Diaryl peptides as NS3 serine protease inhibitors of HCV are disclosed in WO 02/48172 and US6,911,428 by Schering corporation. Imidazolidinones as NS3 serine protease inhibitors of HCV are disclosed in WO02/08198 and US6,838,475 of Schering, Inc., and WO 02/48157 and US6,727,366 of Bristol Myers Squibb. U.S. patent 7,109,172 to VertexPharmaceuticals; 6,909,000, respectively; 6,617,390, respectively; 6,608,067, respectively; 6,265,380 and International publication No. WO98/17679, and WO02/48116 to Bristol Myers Squibb also disclose HCV protease inhibitors. Further examples of HCV protease inhibitors are disclosed in U.S. patent nos. 7,153,848 to intermone; 7,138,376, respectively; 7,135,462, respectively; 7,132,504, respectively; 7,112,601, respectively; and U.S. publication No. 2007/0010455; 2006/0276511, respectively; 2006/0257980, respectively; 2006/0258720, respectively; 2006/0252715, respectively.

Thiazolidine derivatives showing corresponding inhibition in reverse phase HPLC tests with NS3/4A fusion protein and NS5A/5B substrate (Sudo K. et al, Antiviral Research, 1996, 32, 9-18), in particular the compound RD-1-6250, which has a fused cinnamoyl moiety substituted by a long alkyl chain, RD46205 and RD 46193;

in Kakiuchi N.et al J.EBS Letters 421, 217-220; and thiazolidines and benzanilides as indicated in analytical biochemistry, 1997, 247, 242-246, of Takeshita N.et al;

phenanthrenequinone, Sch 68631, isolated from the fermentation broth of species of Streptomyces, which has activity against proteases in SDS-PAGE and autoradiography tests (Chu M. et al, Tetrahedron Letters, 1996, 37, 7229-;

helicase inhibitors (Diana G.D. et al, Compounds, compositions and methods for characterization of hepatitis C, U.S. Pat. No. 5,633,358; Diana G.D. et al, Piperidinedictives, pharmaceutical compositions of and the use of the same in the treatment of hepatitis C, PCT WO 97/36554);

nucleotide polymerase inhibitors and gliotoxins (Ferrari R. et al Journal of Virology, 1999, 73, 1649-1654), as well as the natural product cerulenin (Lohmann V. et al Virology, 1998, 249, 108-118);

interfering RNA (iRNA) -based antiviral agents, including short interfering RNA (siRNA) -based antiviral agents, such as Sirna-034 and International patent publication Nos. WO/03/070750 and WO2005/012525, as well as other antiviral agents described in U.S. patent publication No. US 2004/0209831.

An antisense phosphorothioate oligodeoxynucleotide (S-ODN) complementary to a sequence extension in the 5 'non-coding region (NCR) of the virus (Alt M. et al, Hepatology, 1995, 22, 707-717), or nucleotide 326-388, which comprises the 3' end of the NCR and nucleotide 371-388, located in the core coding region of the HCV RNA (Alt M. et al, Archives of Virology, 1997, 142, 589-599; Galderisis U. et al, Journal of Cellular Physiology, 1999, 181, 251-257);

inhibitors of IRES-dependent translation (Ikeda N et al, Agent for the Prevention and translation of hepatitis C, Japanese patent publication JP-08268890; Kai Y. et al, Prevention and translation of viral diseases, Japanese patent publication JP-10101591);

ribozymes such as nuclease resistant ribozymes (Maccjak, d.j. et al, Hepatology 1999, 30, abstract 995) and those disclosed in U.S. patent No. 6,043,077 to Barber et al and U.S. patent nos. 5,869,253, 5,610,054 to Draper et al; also provided are

In international publication nos. WO 01/90121 and WO 01/92282; WO 01/32153; WO 01/60315; WO 02/057425; WO 02/057287; WO 02/18404; WO 01/79246; nucleoside analogues described in WO 02/32920 and WO 02/48165. Some U.S. patents and patent applications disclosing the use of nucleoside analogs (which may be used as a second agent for treating hepatitis c virus) include: US 7,202,224, 7,125,855, 7,105,499 and 6,777,395 of Merck & co; US 2006/0040890, 2005/0038240, 2004/0121980, 6,846,810, 6,784,166 and 6,660,721 to Roche; US 2005/0009737, US 2005/0009737, 7,094,770 and 6,927,291 of Pharmasset limited.

PCT publication No. WO 99/43691 entitled "2 '-fluoronecroses" at the university of Emory discloses the use of certain 2' -Fluoronucleosides to treat HCV.

Other compounds include 1-amino-alkylcyclohexane (U.S. Pat. No. 6,034,134 to Gold et al), alkyl lipids (U.S. Pat. No. 5,922,757 to Chojkier et al), vitamin E and other antioxidants (U.S. Pat. No. 5,922,757 to Chojkier et al), squalene, amantadine, bile acids (U.S. Pat. No. 5,846,964 to Ozeki et al), N- (phosphonoacetyl) -L-aspartic acid (U.S. Pat. No. 5,830,905 to Diana et al), benzenedicarboxamides (U.S. patent No. 5,633,388 to Diana et al), polyadenylic acid derivatives (U.S. patent No. 5,496,546 to Wang et al), 2 ', 3' -dideoxyinosine (U.S. patent No. 5,026,687 to yarchon et al), benzimidazoles (U.S. patent No. 5,891,874 to Colacino et al), plant extracts (U.S. patent No. 5,837,257 to Tsai et al, U.S. patent No. 5,725,859 to ome et al, and U.S. patent No. 6,056,961), and piperidines (U.S. patent No. 5,830,905 to Diana et al).

Any other compound currently in preclinical or clinical development for the treatment of hepatitis C virus may be used in combination with the compounds provided herein. In some embodiments, compounds that can be used in combination with MMP inhibitors described herein include: interleukin-10 from Schering-Plough, IP-501 from Interneuron, Merimebodib (VX-497) from Vertex, amantadine from Endo Labs Solvay(Symmetrel), HEPTAZYME by RPIXTL-002 of XTL, HCV/MF59 of Chiron, CIVACIR of NABI(immunoglobulin from hepatitis C), LEVOVIRIN from ICN/RibapharmICN/Ribapharm VIRAMIDINEDada of Sci Clone(thymosin alpha-1), thymosin + PEG interferon of Sci Clone, CEPLENE of Maxim(histamine dihydrochloride), VX 950/LY 570310 from Vertex/Eli Lilly, ISIS 14803 from Isis Pharmaceutical/Elan, JTK 003 from AKROS Pharma, BILN-2061 from Boehringer Ingelheim, Cellcept from Roche (mycophenolate mofetil), beta-tubulin inhibitor T67 from Tularik, therapeutic vaccine for E2 from Innogenetics, FK788 from Fujisawa Healthcare, IdB 1016 (Siliphosphos, oral Silybum-phospholipid complex), RNA replication inhibitor from Virorpha/Wyeth (VP50406), therapeutic vaccine for Intercell, therapeutic vaccine for Epimune/Genencor, IRES inhibitor for Anyss, ANA and ANA 245 from Analyss, immunotherapy (Therapa), therapeutic inhibitor for Epimune/Genencorber, therapeutic vaccine for Cotex protease, therapeutic inhibitor for Chemicals, therapeutic inhibitor for Schlumb, therapeutic inhibitor for RNA from Tarcex protease, therapeutic RNA polymerase, therapeutic inhibitor for RNA from Tarcex polymerase, therapeutic RNA inhibitor for RNA from Tarcex polymerase, inhibitor for RNA from Akron, protease inhibitors of Chiron/Meivir, antisense therapy to AVI BioPharma, antisense therapy to Hybridon, hemofilters from Aethlon Medical, therapeutic vaccines from Merix, protease inhibitors of Bristol-Myers Squibb/axs, therapeutic vaccine for Tripep, Chron-VacC, UT 231B from Unitedherapeutics, protease, helicase and polymerase inhibitors from Genelabs Technologies, IRES inhibitors from Immunol, R803 from Rigel Pharmaceuticals, dried Renjin from InterMune(Interferon. alpha. con-1), OMNIFERON from Viragen(Natural interferons), ALBUFERON from Humangenome SciencesREBIF from Ares-Serono(Interferon beta-1 a), omega interferon from Biomedicine, InterMune interferon gamma, interferon tau, and interferon gamma-1 b.

In one embodiment, one or more compounds provided herein can be administered in combination or alternation with currently available or currently under development therapies for hepatitis c. In one embodiment, one or more compounds provided herein can be administered in combination or alternation with anti-hepatitis C virus interferon, e.g., Intron AInterferon alpha-2 b) and pirocinPegylated interferon alfa-2 a); roferon ARecombinant interferon alpha-2 a), and sichuanjinA consensus interferon; interferon alpha con-1), polyethylene glycol-IntronPEG interferon alpha-2 b) and pirocinPegylated interferon alfa-2 a).

In one embodiment, the anti-hepatitis C virus interferon is ganfujin, IL-29 (polyethylene glycol interference)Lambda), R7025 (Maxy-. alpha.), Belerofon, oral interferon alpha, BLX-883(Locteron), omega interferon, multiferon, medusa interferon, Albuferon or REBIF

In one embodiment, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus polymerase inhibitor, such as ribavirin, viramidine, NM283(valopicitabine), R7128/PSI-6130, R1626, HCV-796, or R1479.

In some embodiments, one or more compounds provided herein can be administered in combination with ribavirin and an anti-hepatitis C virus interferon, e.g., Intron AInterferon alpha-2 b) and pirocinPegylated interferon alfa-2 a); roferon ARecombinant interferon alpha-2 a), and sichuanjinA consensus interferon; interferon alpha con-1), polyethylene glycol-IntronPEG interferon alpha-2 b) and pirocinPegylated interferon alfa-2 a).

In some embodiments, RO-113-0830 is administered in combination with an anti-hepatitis C virus interferon, e.g., Intron AInterferon alpha-2 b) and pirocinPegylated interferon alfa-2 a); roferon ARecombinant interferon alpha-2 a), and sichuanjinA consensus interferon; interferon alpha con-1), polyethylene glycol-IntronPEG interferon alpha-2 b) and pirocinPegylated interferon alfa-2 a). In some embodiments, RO-113-0830 is administered in combination with ribavirin. In some embodiments, RO-113-0830 is administered in combination with ribavirin and an anti-hepatitis C virus interferon, e.g., Intron AInterferon alpha-2 b) and pirocinPegylated interferon alfa-2 a); roferon ARecombinant interferon alpha-2 a), and sichuanjinA consensus interferon; interferon alpha con-1), polyethylene glycol-IntronPEG interferon alpha-2 b) and pirocinPegylated interferon alfa-2 a).

In one embodiment, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus protease inhibitor, such as an ITMN-191, SCH 503034, VX950(telaprevir), or Mesivir HCV protease inhibitor.

In one embodiment, one or more compounds provided herein can be administered in combination or alternation with a vaccine against hepatitis C virus, such as TG4040, PeviproTM, CGI-5005, HCV/MF59, GV1001, IC41, or INNO0101 (E1).

In one embodiment, one or more compounds provided herein can be combined with an anti-hepatitis C virus monoclonal antibody such as AB68 or XTL-6865 (formerly HepX-C); or polyclonal antibodies against hepatitis C virus, such as cicavir, administered in combination or alternation.

In one embodiment, one or more compounds provided herein can be administered in combination or alternation with an anti-hepatitis C virus immunomodulator, e.g., daphneThymalfasin), NOV-205, or Oglufanide.

In one embodiment, one or more compounds provided herein can be administered in combination or alternation with Dougimel, Doxorubicin, PI-88, amantadine, JBK-122, VGX-410C, MX-3253(Ceglosivir), Suvus (BIVN-401 or virostat), PF-03491390 (formerly IDN-6556), G126270, UT-231B, DEBIO-025, EMZ702, ACH-0137171, MitoQ, ANA975, AVI-4065, Bavituxinab (Tarvacin), Alinia (nitazoxanide), or PYN 17.

It is recognized that resistant variants of HBV may appear after prolonged treatment with antiviral agents. Drug resistance is most typically produced by mutation of genes encoding enzymes used in the viral life cycle, most typically DNA polymerase in the case of HBV. Recently, it has been demonstrated that the efficacy of a drug against HBV infection can be prolonged, enhanced or reestablished by administering the compound in combination or alternation with a second or and third antiviral compound that induces mutations different from those induced by the primary drug. Alternatively, the pharmacokinetics, biodistribution or other parameters of the drug may be altered by such combination or alternation therapy. In general, combination therapy is generally preferred over alternating therapy because it results in multiple stresses on the simultaneous presence of viruses.

By administering two or more of these compounds in combination or alternation, the anti-hepatitis B virus activity of the compounds provided by the present invention can be increased. Alternatively, for example, one or more of the compounds provided herein may be administered in combination or alternation with any other known anti-hepatitis B virus agent, such as entecavir, cis-2-hydroxymethyl-5- (5-fluorocytosin-1-yl) -1, 3-oxathiolane, preferably substantially in the form of the (-) -optical isomer ("FTC", see WO 92/14743); the (-) -enantiomer of cis-2-hydroxymethyl-5- (cytosin-1-yl) -1, 3-oxathiolane (3 TC); β -D-1, 3-dioxolane purine nucleosides as described in U.S. Pat. nos. 5,444,063 and 5,684,010; β -D-dioxolane nucleosides such as β -D-dioxolanyl-guanine (DXG), β -D-dioxolanyl-2, 6-diaminopurine (DAPD), and β -D-dioxolanyl-6-chloropurine (ACP); L-FDDC (5-fluoro-3 '-thia-2', 3 '-dideoxycytidine), 3' -fluoro-modified L-enantiomer of β -2 '-deoxynucleoside 5' -triphosphate, carbopol, interferon, penciclovir and famciclovir, L-FMAU, famciclovir, penciclovir, BMS-200475, bis pom PMEA (adefovir dipivoxil); lobucavir, ganciclovir, ribavirin, INTM-191, VX-950(telaprevir), or EC showing less than 15 micromolar in 2.2.15 cells₅₀Any other compound of value; or a prodrug or pharmaceutically acceptable salt thereof. Several other examples of anti-HBV agents are provided in U.S. application publication No. 20050080034, which is incorporated by reference in its entirety.

In another embodiment, the compounds provided herein are administered in combination or alternation with immunomodulators or other modulators of pharmaceutical activity of viral replication, including biological materials such as proteins, peptides, oligonucleotides, or gamma globulin, including but not limited to interferons, interleukins, or antisense oligonucleotides of genes that express or modulate hepatitis b replication.

Any alternate method of providing treatment to a patient may be used. A non-limiting example of an alternating pattern includes administering an effective amount of one agent for 1-6 weeks followed by administering an effective amount of a second agent for 1-6 weeks. The alternating schedule may include periods of no treatment. Combination therapy typically involves the simultaneous administration of two or more active agents in a dose-effective ratio.

The compounds provided herein may also be administered in combination with antibiotics, other antiviral compounds, antifungal agents, or other agents for the treatment of secondary infections.

It should be understood that the foregoing detailed description and accompanying examples are illustrative only and are not to be construed as limiting the scope of the subject matter. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including but not limited to those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, formulations and/or methods of use provided herein, may be made without departing from the spirit and scope of the invention. U.S. patents and publications cited herein are incorporated by reference.

7. Examples of the embodiments

7.1 preparation of RO-113-0830

2, 7-dioxa-spiro [3.5] nonan-1-one (10.8g) can be prepared as described in U.S. Pat. No. 5,932,595, dissolved in N, N-dimethylformamide (95mL) and added slowly over a period of 10-15 minutes to a solution containing the sodium salt of 4- (4-chlorophenoxy) thiophenol, generated by addition of sodium hydride powder (2.14g, 89.2mmol) to a solution of 4- (4-chlorophenoxy) thiophenol (15.83g, 66.8mmol) in N, N-dimethylformamide (19mL) at 0 deg.C and stirring for 30 minutes, followed by stirring for an additional 15 minutes. The resulting slurry was heated to 40 ℃, stirred for 5 minutes, tert-butanol (2mL) was added and the mixture was cooled to room temperature over 20 minutes. Most of the N, N-dimethylformamide was removed in vacuo, the pH was adjusted to 9.2, and the resulting slurry was diluted with 30% diethyl ether-hexane (120mL) and filtered. The filter cake was washed with an additional portion of ether (3 times 70mL), acidified to pH 3.5 with 2N hydrochloric acid solution and extracted with dichloromethane (4X 350 mL). The combined organic layers were dried over magnesium sulfate and concentrated in vacuo. The solid residue was recrystallized from a minimum amount of dichloromethane-hexane to provide pure 4- [4- (4-chlorophenoxy) phenylthiomethyl ] tetrahydropyran-4-carboxylic acid.

7.2 in vivo evaluation of RO-113-0830

The in vivo efficacy of RO-113-0830 was evaluated in two well-established models of liver injury using male C57B1/6 mice (Simonsen laboratories). Mice were allowed to acclimate for at least three days.

In the TNF-. alpha.liver injury model, D-galactosamine (D-Gln) (800mg/kg) and TNF. alpha. (20 or 40. mu.g/kg) were injected intraperitoneally. RO-113-0830(0.001-30mg/kg) was administered orally by gavage 30 minutes prior to the insult. Six hours later, the animals were anesthetized with sodium pentobarbital (50mg/kg abdominal cavity) and bled by cardiac puncture. Plasma ALT activity was detected using a kit from Sigma-Aldrich. RO-113-0830 dose-dependently reduced plasma ALT activity in a TNF- α model. Mean ED of 4 studies₅₀The value was 0.26. + -. 0.08 mg/kg.

To test the benefit on survival, the TNF- α model, i.p., mice were left to survive 24 hours after injury by intraperitoneal injection of D-galactosamine (D-Gln) (800mg/kg) and TNF- α (20 or 40 μ g/kg). All the pathogenic mice were euthanized with 125mg/kg sodium pentobarbital in the abdominal cavity. The mean 24-hour survival rates for the 3 studies were 27 + -7.3% and.55 + -7.6% (p ═ 0.03), respectively, in TNF α/D-Gln control mice and RO-113-0830-treated mice.

In a Fas-driven liver injury model, an activating antibody to Fas (Jo-2) was administered intravenously. Six hours later, the animals were anesthetized with sodium pentobarbital (50mg/kg abdominal cavity) and bled by cardiac puncture. Plasma ALT activity was detected using a kit from Sigma-Aldrich. RO-113-0830(10 mg/kg; oral) significantly reduced the Fas-induced elevation of plasma ALT activity, with an average of 50% reduction in both studies (p < 0.05 per study).

The results of these studies indicate that, in some embodiments, RO-113-0830 is protective in the presence of two important pro-inflammatory cytokines involved in liver disease. Reduction in liver injury and reduction in inflammation were measured by reduction in plasma ALT levels compared to control animals. ALT is a clinically relevant marker of liver injury and is routinely used to assess the extent of ongoing liver injury and inflammation in patients. In addition, RO-113-0830 showed survival benefits after TNF- α administration.

7.3 inhibition of HCV replication in the replicon assay

In this study, the Huh7 human hepatoma cell line (21-5 cell line) was used, see Pietschmann, T.et al, J.Virol.76, 2002, 4008-. The tests were performed as described by Pietschmann, t.

The effect of RO-113-0830 at six semilog concentrations (each in quadruplicate) was tested in an HCV RNA replicon antiviral assessment assay. Human interferon alpha-2 b was included as a positive control compound for each test. The sub-confluent cultures of the ET line were seeded into 96-well plates dedicated for cell number (cytotoxicity) or antiviral activity assays, and the next day drugs were added to the appropriate wells. After 72 hours the cells were treated while they were still subconfluent. HCV RNA replicon levels and drug toxic concentrations that reduced the number of cells (as indicated by host cell ribosomal RNA (rRNA) levels) were assessed by TaqMan RT-PCR. Calculation of EC₅₀(concentration inhibiting 50% viral replication), IC₅₀(concentration to reduce cell viability by 50%) and SI₅₀(Selectivity index: IC)₅₀/EC₅₀) The value is obtained.

RO113-0830 dose-dependently inhibits HCV replication to 50% inhibition (EC) at a concentration of 70nM₅₀). IC of cytotoxicity in this assay₅₀The value is about 25 μm, thus the selectivity Index (IC)₅₀/EC₅₀) About 350 f. These data indicate that in some embodiments, RO113-0830 achieves a potent effect in inhibiting hepatitis c virus replication at a dose that does not affect cell viability.

7.4 in vitro study in common bile duct ligation model

The common bile duct ligation model is a good characteristic model of liver fibrosis. Briefly, 6 to 8 weeks old C57/BL6 wild-type mice were ligated with common Bile Duct (BDL) for 14 days. Sham operated wild type mice were used as controls. RO113-0830 or CMC (carboxymethyl cellulose) was administered by gavage at a dose of 10mg/kg body weight once daily. Hepatocyte apoptosis was quantified by TUNEL assay and activated caspase 3/7 immunofluorescence. Liver damage was assessed by histopathology and the amount of bile infarctions. Liver fibrosis was assessed by sirius red staining and quantitative morphometry. Real-time Polymerase Chain Reaction (PCR) was used to measure mRNA transcription of collagen 1 α (I) and α -smooth muscle actin.

After day BDL14, wild type mice treated with RO113-0830 had a 3-fold reduction in TUNEL and a 5-fold reduction in caspase 3/7-positive hepatocytes (p < 0.01) compared to vehicle-treated animals. Hepatohistology detection BDL wild-type animals treated with RO113-0830 had > 70% less biliary infarction compared to vehicle-treated BDL mice. Hepatic transcription of alpha-smooth muscle actin (astrocyte activation marker) and collagen I was increased 6 and 8 fold in BDL14 day mice compared to sham operated controls. These transcribed mrnas were reduced by > 60% in RO113-0830 treated BDL animals compared to vehicle treated BDL animals. Sirius red staining of hepatic collagen was also reduced by 3-fold in BDL wild type mice treated with RO 113-0830. Finally, after 14 days of BDL, total animal survival was also significantly increased in the group receiving the active drug (p < 0.05). These data indicate that, in some embodiments, liver injury and liver fibrosis are reduced following treatment with the MMP inhibitor RO 113-0830.

The embodiments described above are merely exemplary and those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific compounds, materials, and methods. All such equivalents are considered to be within the scope of the subject matter of the claims of this application and are encompassed by the following claims.

Claims (39)

1. A method of treating a liver disease selected from the group consisting of alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, primary biliary cirrhosis, hepatic ischemia reperfusion injury, viral hepatitis b, viral hepatitis c, and alcoholic hepatitis, the method comprising administering a matrix metalloproteinase inhibitor, wherein the matrix metalloproteinase inhibitor is selected from the group consisting of:

XL784 and pharmaceutically acceptable derivatives thereof, with the proviso that when the MMP inhibitor is ONO-4817, the liver disease is not hepatic ischemia reperfusion injury.

2. The method of claim 1, wherein the matrix metalloproteinase inhibitor is selected from:

and pharmaceutically acceptable derivatives thereof.

3. The method of claim 1, wherein the matrix metalloproteinase inhibitor is:

or a pharmaceutically acceptable derivative thereof.

4. The method of claim 1, wherein the matrix metalloproteinase inhibitor is:

or a pharmaceutically acceptable derivative thereof.

5. The method of claim 1, wherein the matrix metalloproteinase inhibitor is:

or a pharmaceutically acceptable derivative thereof.

6. The method of any one of claims 1-5, wherein the liver disease is an acute liver disease.

7. The method of any one of claims 1-5, wherein the liver disease is chronic liver disease.

8. The method of any one of claims 1-7, wherein the matrix metalloproteinase inhibitor is administered to a patient that has been previously treated with other drugs for liver disease.

9. The method of any one of claims 1-7, wherein the matrix metalloproteinase inhibitor is administered to a patient being treated with other drugs for liver disease.

10. The method of claim 8, wherein the patient has experienced a failed treatment for a liver disease.

11. The method of any one of claims 1-10, wherein the liver disease is selected from the group consisting of alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis, cirrhosis, and primary biliary cirrhosis.

12. The method of any one of claims 1-10, wherein the liver disease is viral hepatitis b.

13. The method of any one of claims 1-10, wherein the liver disease is viral hepatitis c.

14. The method of claim 13, wherein the matrix metalloproteinase inhibitor is administered to a patient who has undergone treatment for failure of hepatitis C.

15. The method of any one of claims 1-10, wherein the liver disease is alcoholic hepatitis.

16. The method of any one of claims 1-10, wherein the liver disease is non-alcoholic fatty liver disease.

17. The method of any one of claims 1-10, wherein the liver disease is non-alcoholic steatohepatitis.

18. The method of any one of claims 1-10, wherein the liver disease is liver fibrosis.

19. The method of claim 18, wherein the liver fibrosis is caused by hepatitis, chemical exposure, biliary obstruction, autoimmune disease, blood outflow disorders from the liver, cardiac and vascular disorders, alpha 1-antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage disorders, diabetes, malnutrition, Wilson's disease, or hemochromatosis.

20. The method of any one of claims 1-10, wherein the disease is cirrhosis.

21. The method of claim 20, wherein the cirrhosis is caused by alcohol abuse.

22. The method of claim 20, wherein the cirrhosis is caused by hepatitis, chemical exposure, biliary obstruction, autoimmune disease, blood outflow from the liver, cardiac and vascular disorders, alpha 1-antitrypsin deficiency, high blood galactose levels, high blood tyrosine levels, glycogen storage disorders, diabetes, malnutrition, Wilson's disease, or hemochromatosis.

23. The method of any one of claims 1-10, wherein the disease is primary biliary cirrhosis.

24. The method of any one of claims 1-10, wherein the disease is hepatic ischemia reperfusion injury.

25. A method of reducing elevated liver enzyme levels, the method comprising administering a matrix metalloproteinase inhibitor, wherein the matrix metalloproteinase inhibitor is selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

26. The method of claim 25, wherein the liver enzyme is alanine transaminase or aspartate transaminase.

27. The method of claim 25 or 26, wherein the elevated liver enzyme level is reduced by about 100% to about 1%.

28. The method of claim 25 or 26, wherein the elevated liver enzyme level is reduced by at least 99%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, at least 5%, at least 2%, or at least 1%.

29. The method of any one of claims 25-28, wherein the matrix metalloproteinase inhibitor is

Or a pharmaceutically acceptable derivative thereof.

30. A method of inhibiting the signaling cascade of TNF- α comprising administering a matrix metalloproteinase inhibitor,

wherein the matrix metalloproteinase inhibitor is selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

31. The method of claim 30, wherein the matrix metalloproteinase inhibitor is

Or a pharmaceutically acceptable derivative thereof.

32. A method of reducing liver damage associated with liver disease comprising administering a matrix metalloproteinase inhibitor, wherein the matrix metalloproteinase inhibitor is selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

33. A method of inhibiting the signaling cascade of α -Fas comprising administering a matrix metalloproteinase inhibitor, wherein the matrix metalloproteinase inhibitor is selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

34. A method of inhibiting excessive apoptosis in hepatocytes comprising administering a matrix metalloproteinase inhibitor, wherein the matrix metalloproteinase inhibitor is selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

35. A method of inhibiting hepatitis c virus replication in a cell infected with hepatitis c virus comprising administering a matrix metalloproteinase inhibitor, wherein the matrix metalloproteinase inhibitor is selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

36. A method of inhibiting hepatitis c virus replication in a patient infected with hepatitis c virus comprising administering to the patient a matrix metalloproteinase inhibitor selected from:

XL784 and pharmaceutically acceptable derivatives thereof.

37. The method of claim 35 or 36, wherein the matrix metalloproteinase inhibitor is

38. The method of claim 13, further comprising administering a therapeutically effective amount of a second agent.

39. The method of claim 38, wherein the second agent is selected from anti-hepatitis c virus interferon, ribavirin, or a combination thereof.