TREATMENT OF FATTY LIVER DISEASE

The present invention relates to a method of treating fatty liver disease in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a compound of Formula (I) 5′-N1N2N3N4N5N6-N7N8-dCm-dGc-N11N12N13N14N15N16-N17N18-3′. The fatty liver disease can be non-alcoholic fatty liver disease, such as simple fatty liver disease or NASH. The invention also relates to the use of adiponectin as a biomarker for identifying subjects more likely to respond to treatment with the compound on Formula (I) 5′-N1N2N3N4N5N6-N7N8-dCm-dGc-N11N12N13N14N15N16-N17N18-3′ and for assessing response of the subject during treatment with the compound of Formula (I) 5′-N1N2N3N4N5N6-N7N8-dCm-dGc-N11N12N13N14N15N16-N17N18-3′.

BACKGROUND OF THE INVENTION

Alcoholic and non alcoholic fatty liver disease (also known as hepatosteatosis) is a prevalent liver condition that occurs when lipids accumulate in liver cells. The accumulation of lipids causes cellular injury, sensitization of the liver to further injuries and damage to the hepatic microvascular circulation. The etiology of fatty liver disease is associated with excessive alcohol consumption, metabolic disorders, dietary conditions, exposure to certain chemicals and medications and complications of pregnancy (e.g., preeclampsia).

Fatty liver disease is a major health burden worldwide. The prevalence of non-alcoholic fatty liver disease (NAFLD) ranges from 15% to 37% of the population and is considered the most common liver disease worldwide. Moreover, NAFLD is also believed to affect as many as 3-10% of obese children. NAFLD can progress to a more advanced liver disease such as nonalcoholic steatohepatitis (NASH), a condition characterized by liver inflammation and damage, often accompanied by fibrosis or cirrhosis of the liver which can further lead to end stage liver disease and primary liver cancer. NASH has a prevalence of 3-10% of the general population.

Currently, there are no approved treatments for NAFLD and NASH. In general, current therapies include healthy lifestyle and non-specific metabolic modulators.

SUMMARY OF THE INVENTION

The invention relates to a method of treating fatty liver disease. The method comprising administering to a subject in need thereof a therapeutically effect amount of the compound of Formula (I) or a pharmaceutically acceptable salt thereof.

In one embodiment, the fatty liver disease is non-alcoholic fatty liver disease (NAFLD).

In a particular embodiment, the NAFLD is simple fatty liver disease.

In another embodiment, the NAFLD is non-alcoholic steatohepatitis (NASH).

In yet another embodiment the fatty liver disease is alcoholic liver disease (ALD).

In another embodiment, the present invention relates to the use of a compound of Formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for treating fatty liver disease.

In yet another embodiment, the present invention relates to the compound of Formula (I) or a pharmaceutically acceptable salt thereof for use in treating fatty liver disease.

In yet another embodiment, the present invention relates to a method of identifying the likelihood of a fatty liver disease in a subject to be responsive to administration of a compound of Formula (I), wherein the method comprises: a) obtaining or providing a plasma sample from a subject having a fatty liver disease;b) measuring the plasma adiponectin level in the sample; andc) comparing the plasma adiponectin level to a control level of adiponectin; wherein if the plasma adiponectin level is less than the control level the fatty liver disease is identified as being more likely to be responsive to the administration of the compound of Formula (I).

In a particular embodiment, the control level of adiponectin is 38 μg/mL

In a further embodiment, the present invention relates to a method of assessing the efficacy of a compound represented by Formula (I) in treating fatty liver disease in a subject in need thereof, the method comprising:a) detecting in a subject plasma sample at a first point in time the level of adiponectin;b) repeating step a) during at least one subsequent point in time after administration of the compound represented by Formula (I); andc) comparing the levels detected in steps a) and b), wherein an increased level of adiponectin relative to at least one subsequent subject plasma sample, indicates that the compound of Formula (I) treats the fatty liver disease in the subject.

DETAILED DESCRIPTION OF THE INVENTION

Compounds for Administration

The Compounds described herein for the treatment of fatty liver disease (e.g., NASH) are 18-mer 5′-to-3′ oligonucleotides represented by structural formula (I):

or a pharmaceutically acceptable salt thereof.

In structural formula (I):

dCmis a 5-methyl-cytosine 2′-deoxoriboside (“m5C-dRib,” “m5Cyt-dRib,” or “m5Cyt-dRibf”) of the following structural formula:

dGcis a 7-deaza-guanine 2′-deoxoriboside (“c7G-dRib,” “c7Gua-dRib,” or “c7Gua-Ribf”) of the following structural formula:

any two adjacent ribonucleosides are covalently linked by either a phosphodiester or a phosphorothioate linker, as represented by the following structural formulas:

It is understood that each phosphorothioate bond introduces a chiral center designated as either “Sp” or “Rp.” Unless otherwise indicated, the compounds of the present invention that include a phosphorothioate linker can include either Sp or Rp stereoisomer. In one embodiment, all linkers in the compounds of structural formula (I) are Sp phosphorothioate linkers.

In a first example embodiment of an oligonucleotide of structural formula (I), N7is a guanine 2′-methoxy-riboside (“G-Rib2Me,” “Gua-Rib2Me,” or “Gua-Ribf2Me”), represented by the following structural formula:

In a second example embodiment, N8is a uracil 2′-methoxy-riboside (“U-Rib2Me,” “Ura-Rib2Me,” or “Ura-Ribf2Me”) represented by the following structural formula:

In a third example embodiment, N17is a guanine 2′-methoxy-riboside. In a fourth example embodiment, N18is a uracil 2′-methoxy-riboside.

In a fifth example embodiment of an oligonucleotide of structural formula (I), N7is a guanine 2′-methoxy-riboside, N8is a uracil 2′-methoxy-riboside, N17is a guanine 2′-methoxy-riboside, and N18is a uracil 2′-methoxy-riboside.

In a sixth example embodiment of an oligonucleotide of structural formula (I), N7is cytosine 2′-deoxoriboside (“dC” or “dCyd”), N2is thymine 2′-deoxoriboside (“dT” or “dThd”), N3is adenine 2′-deoxoriboside (“dA” or “dAdo”), N4is thymine 2′-deoxoriboside, N5is cytosine 2′-deoxoriboside, and N6is thymine 2′-deoxoriboside. The values and example values of N7, N8, N17, and N18are as described above with respect to the first through fifth example embodiments of an oligonucleotide of structural formula (I).

In a seventh example embodiment of an oligonucleotide of structural formula (I), N11is thymine 2′-deoxoriboside, N12is thymine 2′-deoxoriboside, N13is cytosine 2′-deoxoriboside, N14is thymine 2′-deoxoriboside, N15is cytosine 2′-deoxoriboside, and N16is thymine 2′-deoxoriboside. The values and example values of N1through N8, and N17and N18are as described above with respect to the first through sixth example embodiments of an oligonucleotide of structural formula (I).

In an eighth example embodiment of an oligonucleotide of structural formula (I), the oligonucleotide is represented by the following structural formula:

In a ninth example embodiment of an oligonucleotide of structural formula (I), the oligonucleotide is represented by the following structural formula:

(III)5′-dC-Sp-dT-Sp-dA-Sp-dT-Sp-dC-Sp-dT-Sp-(G-Rib2Me)-Sp-(U-Rib2Me)-Sp-dCm-Sp-dGc-Sp-dT-Sp-dT-Sp-dC-Sp-dT-Sp-dC-Sp-dT-Sp-(G-Rib2Me)-Sp-(U-Rib2Me)-3′
or a pharmaceutically acceptable salt thereof. In a specific aspect, the oligonucleotide represented by structural formula (III) in is in the form of a sodium salt represented by structural formula (IIIA):

In a tenth example embodiment, the oligonucleotide used in the method described herein in the sodium salt of the oligonucleotide represented by structural formula (III) and is commonly referred to in the art as Bazlitoran or IMO-8400. The molecular weight of the non-salt form of IMO-8400 is 5800.67 g/mol and the molecular formula of non-salt form of IMO-8400 is C179H233N52O101P17S17. A sodium salt of Bazlitoran also referred to herein as AVO010 is represented by the structural formula shown inFIG. 2. The IUPAC name for the compound represented by the structural formula depicted inFIG. 2is 5′-hydroxy-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′→5′)-2′-deoxy-P-thioadenylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′→5′)-2′-O-methyl-P-thioguanylyl-(3′→5′)-2′-O-methyl-P-thiouridylyl-(3′→5′)-2′-deoxy-P-thio-5-methyl-cytidylyl-(3′→5′)-2′-deoxy-P-thio-7-deaza-guanylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′-5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′→5′)-2′-deoxy-P-thiocytidylyl-(3′→5′)-2′-deoxy-P-thiothymidylyl-(3′→5′)-2′-O-methyl-P-thioguanylyl-(3′→5′)-3′-hydroxy-2′-O-methyluridine 17 sodium salt.

As used herein, the term “ribonucleoside” refers to a compound having the following structural formula:

wherein the Base can be any one of nitrogenous bases, such as pyrimidine- or purine-derived bases, and, for example, nucleobases adenine (A), uracil (U), guanine (G), thymine (T), and cytosine (C), each of which can be optionally modified. Unless specifically indicated otherwise, a ribonucleoside includes a 2′-hydroxyl. A 2′-deoxoribonucleoside includes a —CH2— group at the 2′-position.

Fatty Liver Disease

Fatty liver disease occurs when there is a buildup of excess fat in the liver. There are two main types of fatty liver disease. The first type of fatty liver disease is non-alcoholic fatty liver disease (NAFLD). The second type of fatty liver disease is alcoholic fatty liver disease (ALD).

NAFLD includes simple fatty liver and non-alcoholic steatohepatitis (NASH). Simple fatty liver means that there is excess fat in the liver, but inflammation is not present. NASH is much more serious than simple fatty liver. NASH means there is fat in the liver and also inflammation and in some instances damage to the liver cells. The inflammation and liver cell damage that happen with NASH cause serious problems such as fibrosis (scarring of the liver), cirrhosis (sever scarring of the liver) and liver cancer.

ALD includes alcoholic hepatitis and alcoholic cirrhosis.

In some embodiments, the method described herein can further comprise improving the NAS score in a subject. The NAS score can be improved by at least 30%.

A “therapeutically effective amount” of an oligonucleotide or salt thereof as described herein is an amount that when administered to a subject with a disease or condition, will have the intended therapeutic effect, e.g., alleviation, amelioration, palliation or elimination of one of more manifestations of the disease or condition in the subject. A therapeutically effective amount of the compound of Formula (I) can be from about 0.5 mg/kg to about 50 mg/kg. For example, from about 0.5 mg/kg to about 25 mg/kg and 0.5 mg/kg to about 20 mg/kg. Suitable doses include about 0.75 mg/kg, about 1.5 mg/kg, about 3 mg/kg and about 6 mg/kg. In a particular embodiment, the dose is 3 mg/kg. In another particular embodiment, the dose is 3 mg/kg and the dose is administered once per week.

As used herein, method of treating means amelioration, prevention or relief from the symptoms and/or effects associated with a disorder or condition. The disorder or condition is fatty liver disease. In one embodiment, the fatty liver disease is non-alcoholic fatty liver disease (NAFLD). In a specific embodiment, the NAFLD is simple fatty liver (steatosis). In another embodiment, the NAFLD is non-alcoholic steatohepatitis (NASH).

Pharmaceutical Compositions/Methods of Administration

The compositions and methods of the present invention can be utilized to treat a subject in need thereof. In certain embodiments, the subject is a mammal such as a human, or a non-human mammal. When administered to subject, such as a human, the composition or the compound is preferably administered as a pharmaceutical composition comprising, for example, a compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include, for example, aqueous solutions such as water or physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil, or injectable organic esters. In a specific embodiment, when such pharmaceutical compositions are for human administration, particularly for invasive routes of administration (i.e., routes, such as injection or implantation, that circumvent transport or diffusion through an epithelial barrier), the aqueous solution is pyrogen-free, or substantially pyrogen-free. The excipients can be chosen, for example, to effect delayed release of an agent or to selectively target one or more cells, tissues or organs. The pharmaceutical composition can be in dosage unit form such as tablet, capsule (including sprinkle capsule and gelatin capsule), granule, lyophilizate for reconstitution, powder, solution, syrup, suppository, injection or the like. The composition can also be present in a transdermal delivery system, e.g., a skin patch. The composition can also be present in a solution suitable for topical administration, such as an eye drop.

A pharmaceutical composition (preparation) can be administered to a subject by any of a number of routes of administration including, for example, orally (for example, drenches as in aqueous or non-aqueous solutions or suspensions, tablets, capsules (including sprinkle capsules and gelatin capsules), boluses, powders, granules, pastes for application to the tongue); absorption through the oral mucosa (e.g., sublingually); anally, rectally or vaginally (for example, as a pessary, cream or foam); parenterally (including intramuscularly, intravenously, subcutaneously or intrathecally as, for example, a sterile solution or suspension); nasally; intraperitoneally; subcutaneously; transdermally (for example as a patch applied to the skin); and topically (for example, as a cream, ointment or spray applied to the skin, or as an eye drop). The compound may also be formulated for inhalation. In certain embodiments, a compound may be simply dissolved or suspended in sterile water.

Methods of preparing these formulations or compositions include the step of bringing into association an active compound, such as a compound of the invention, with the carrier and, optionally, one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finely divided solid carriers, or both, and then, if necessary, shaping the product.

Formulations of the pharmaceutical compositions for administration to the mouth may be presented as a mouthwash, or an oral spray, or an oral ointment.

Alternatively or additionally, compositions can be formulated for delivery via a catheter, stent, wire, or other intraluminal device. Delivery via such devices may be especially useful for delivery to the bladder, urethra, ureter, rectum, or intestine.

For use in the methods of this invention, active compounds can be given per se or as a pharmaceutical composition containing, for example, 0.1 to 99.5% (more preferably, 0.5 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.

A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. By “therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired therapeutic effect. It is generally understood that the effective amount of the compound will vary according to the weight, sex, age, and medical history of the subject. Other factors which influence the effective amount may include, but are not limited to, the severity of the subject's condition, the disorder being treated, the stability of the compound, and, if desired, another type of therapeutic agent being administered with the compound of the invention. A larger total dose can be delivered by multiple administrations of the agent. Methods to determine efficacy and dosage are known to those skilled in the art (Isselbacher et al. (1996) Harrison's Principles of Internal Medicine 13 ed., 1814-1882, herein incorporated by reference).

In certain embodiments, compounds of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds.

In certain embodiments, conjoint administration of compounds of the invention with one or more additional therapeutic agent(s) provides improved efficacy relative to each individual administration of the compound of the invention or the one or more additional therapeutic agent(s). In certain such embodiments, the conjoint administration provides an additive effect, wherein an additive effect refers to the sum of each of the effects of individual administration of the compound of the invention and the one or more additional therapeutic agent(s).

This invention includes the use of pharmaceutically acceptable salts of compounds of the invention in the compositions and methods of the present invention. A “pharmaceutically acceptable salt” means any non-toxic salt that, upon administration to a recipient, is capable of providing, either directly or indirectly, a compound of this invention. A “pharmaceutically acceptable counterion” is an ionic portion of a salt that is not toxic when released from the salt upon administration to a recipient. In certain embodiments, contemplated salts of the invention include, but are not limited to, Na, Ca, K, Mg, Zn or other metal salts. In a particular embodiment, the salts of the invention (the salts of the compound represented by Formula (I)) is the sodium salt.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which this disclosure belongs. Although other compounds or methods can be used in practice or testing, certain preferred methods are now described in the context of the following preparations and schemes.

Experimental Methods

Efficacy of IMO-8400 in Diet Induced NAFLD/NASH Cynomolgus Monkeys

The purpose of this study was to determine the efficacy of IMO-8400 in diet induced NAFLD/NASH male cynomolgus monkeys following subcutaneous (SC) administration of IMO-8400 at 3 mg/kg once weekly for 12 consecutive weeks.

Twelve NAFLD/NASH male cynomolgus monkeys were identified based on liver histophathology and cfDNA levels. The animals were randomly divided into the following groups:Group 1: n=5; administered vehicle (sterile saline) by subcutaneous injection once weekly for 12 consecutive weeks.Group 2: n=7; administered IMO-8400 solution by subcutaneous injection once weekly for 12 consecutive weeks.

Results are presented in detail below and include the following observations:a) Adiponectin Levels: Adiponectin levels in 6 out of 7 animals administered IMO-8400 were noticeably elevated at week 12 as compared to pre-dose (baseline) levels.b) Body Weight: Compared to day 1, the mean animal body weight was decreased 3.27% in Group 1 (Control) and 10.87% in Group 2 (Treated) on day 84 (end of treatment).c) NAS Score: 4 of 7 animals in Group 2 (Treated) had a clinically meaningful 2-point or more reduction in their NAS Score. Only 1 of 5 animals in Group 1 (Control) had a 2-point reduction.

Dose Administration: Animals were weighed prior to dose administration on each day of dosing to calculate the actual dose volume. Subcutaneous injections were made on the animal's back using an approximately 26-gauge needle. All animals in Group 1 received subcutaneous administration of sterile saline once weekly for 12 weeks. The animals in Group 2 received subcutaneous administration of IMO-8400 solution once weekly for 12 weeks.

Plasma Preparation: Whole blood (˜10 mL) was collected from overnight fasted animals in the morning on Day 1-predose, Day 42 and Day 84. Samples were collected into commercial vacuum tubes containing EDTA-K2 as anticoagulant. The collected blood samples were placed on wet ice, and processed for plasma by centrifugation at approximately 4° C., 3000 g for 15 minutes within 30 minutes of collection. The plasma supernatant was stored at −60° C. until analysis.

NAS scores were evaluated by standard criteria as described in: Brunt E M, Kleiner D E, Wilson L A, Belt P, Neuschwander-Tetri B A; NASH Clinical Research Network (CRN). Nonalcoholic fatty liver disease (NAFLD) activity score and the histopathologic diagnosis in NAFLD: distinct clinicopathologic meanings. Hepatology 2011; 53: 810-820; and Kleiner D E, Brunt E M, Van Natta M, Behling C, Contos M J, Cummings O W, Ferrell L D, Liu Y C, Torbenson M S, Unalp-Arida A, Yeh M, McCullough A J, Sanyal A J; Nonalcoholic Steatohepatitis Clinical Research Network. Design and validation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41:1313-1321.

The chart below provides the framework for scoring.

Results showing Adiponectin levels for both the Control Group (Group 1) and the Treated Group (Group 2) are shown in Table 1 below.

Human Levels: 2-38 μg/ml Depending on Sex and BMI

Results showing NAS scoring for both the Control Group (Group 1) and the Treated Group (Group 2) are shown in TABLE 2 below.

Results of weight loss over the 12 week testing period for both the Control Group (Group 1) and the Treated Group (Group 2) are shown inFIG. 1.