WO2017184412A1 - Purine cannabinoid agonist for treating nonalcoholic steatohepatitis and fibrosis - Google Patents

Purine cannabinoid agonist for treating nonalcoholic steatohepatitis and fibrosis Download PDF

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Publication number
WO2017184412A1
WO2017184412A1 PCT/US2017/027306 US2017027306W WO2017184412A1 WO 2017184412 A1 WO2017184412 A1 WO 2017184412A1 US 2017027306 W US2017027306 W US 2017027306W WO 2017184412 A1 WO2017184412 A1 WO 2017184412A1
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Prior art keywords
methyl
purine
piperazin
pyran
tetrahydro
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PCT/US2017/027306
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French (fr)
Inventor
Zhiming Ding
Bei Betty ZHANG
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Eli Lilly And Company
Lilly China Research And Development Co., Ltd.
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Priority claimed from PCT/CN2016/079541 external-priority patent/WO2017181317A1/en
Priority claimed from PCT/CN2017/072622 external-priority patent/WO2018137176A1/en
Application filed by Eli Lilly And Company, Lilly China Research And Development Co., Ltd. filed Critical Eli Lilly And Company
Publication of WO2017184412A1 publication Critical patent/WO2017184412A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • Nonalcoholic steatohepatitis is a liver disease with an etiological constellation characterized by macrovesicular hepatic steatosis, inflammation hepatocyte ballooning and fibrosis.
  • Nonalcoholic steatohepatitis occurs in people who drink little or no alcohol and is often comorbid with obesity, type II diabetes, elevated levels of cholesterol, and triglycerides.
  • Nonalcoholic steatohepatitis may lead to cirrhosis and liver failure. It has been established that patients with nonalcoholic steatohepatitis are more likely to develop cirrhosis, and have a higher risk of cardiovascular mortality, and hepatocyte carcinoma.
  • the stage of disease can be defined, for example, by the nonalcoholic steatohepatitis CRN (Clinical Research Network) fibrosis staging which measures the amount and pattern of nonalcoholic steatohepatitis fibrosis, as well as parenchymal architectural remodeling in a patient.
  • Nonalcoholic steatohepatitis is
  • liver biopsy typically diagnosed in a human patient using liver biopsy.
  • the present invention provides a pharmaceutical treatment for nonalcoholic steatohepatitis comprising administering an effective amount of the compound, 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine to a patient.
  • Compounds of WO 2010/080306 are stated to be agonists of the cannabinoid receptor 2 (CB2 receptor) in vitro and useful for treatment or prevention of pain.
  • CB2 receptor cannabinoid receptor 2
  • the present invention provides a method for treating nonalcoholic steatohepatitis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a patient.
  • the present invention provides a method for treating nonalcoholic steatohepatitis and fibrosis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2- methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a patient.
  • the present invention provides a method for treating nonalcoholic steatohepatitis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine to a patient.
  • the present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin- 1 -yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or pharmaceutically acceptable salt thereof, for use in treating nonalcoholic steatohepatitis in a patient.
  • the present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin- 1 -yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or pharmaceutically acceptable salt thereof, for use in treating nonalcoholic steatohepatitis and fibrosis in a patient.
  • the present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, for use in treating nonalcoholic steatohepatitis in a patient.
  • the present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin- 1 -yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or pharmaceutically acceptable salt thereof, for use in treating hepatic fibrosis in a mammal.
  • the present invention provides a pharmaceutical composition for use in the treatment of nonalcoholic steatohepatitis comprising 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
  • the present invention provides a pharmaceutical composition for use in the treatment of nonalcoholic steatohepatitis and fibrosis comprising 8-(2-Chloro-phenyl)-2- methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
  • the present invention provides the use of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nonalcoholic steatohepatitis.
  • the present invention provides the use of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nonalcoholic steatohepatitis and fibrosis.
  • the present invention provides the use of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of hepatic fibrosis.
  • the present invention provides a method for treating hepatic fibrosis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl- piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a mammal.
  • the compound, 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin- l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, has the following structural formula:
  • treating refers to restraining, slowing, or stopping the progression or alleviating the severity of the stated condition to be treated.
  • the term "effective amount” refers to the amount or dose of 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine, or a pharmaceutically acceptable salt thereof which upon single or multiple dose administration to the patient, provides the desired effect in the patient. It will be understood that the amount of active agent actually administered will be determined by a physician, in light of the relevant circumstances.
  • patient refers to a mammal in need of treatment for
  • nonalcoholic steatohepatitis In a preferred embodiment, the patient is a mammal.
  • mammal is a human. In a preferred embodiment the mammal is a human in need of treatment for nonalcoholic steatohepatitis and fibrosis.
  • 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4- yl)-9H-purine is preferably formulated as a pharmaceutical composition administered by any route which makes the compound bioavailable. Most preferably, such composition is for oral administration.
  • Such pharmaceutical compositions and processes for preparing the same are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006).
  • mice Male C57BL/6N mice are fed with D09100301 diet (Research Diets, 40% fat, 2% cholesterol, 24% fructose, 3H diet) for 150 days. Each mouse is then singly housed after 5 days of acclimation period. Plasma alanine aminotransferase (ALT) and CK18 (cytokeratin 18) are measured. After one week of acclimation, the mice are randomized into groups by their ALT, CK18 and body weight.
  • D09100301 diet Research Diets, 40% fat, 2% cholesterol, 24% fructose, 3H diet
  • CK18 cytokeratin 18
  • Animals of each group are administrated either vehicle (0.5% methylcellulose (MC) + 0.25% Tween 80 in distilled water) or the compound of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine (at a 1, 3, 10, and 30 mg/kg dose) once daily in a volume of 5 ml/kg for 11 weeks.
  • vehicle 0.5% methylcellulose (MC) + 0.25% Tween 80 in distilled water
  • 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine at a 1, 3, 10, and 30 mg/kg dose
  • mice treated with the compound of 8-(2-Chloro-phenyl)- 2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine for 76 days 2 hours after the last dose.
  • Compound levels in the plasma are analyzed by mass spectroscopy. The results are listed below in Table 1. Treated mice exhibit a dose- dependent increase in plasma compound levels.
  • the animals are sacrificed and their livers excised. Two sections of the left and right lobes are fixed in neutral buffered 10% formalin. Liver tissue slides are stained with hematoxylin and eosin (H&E), Sirius red, and Masson's Trichrome to prepare slides for pathological analysis. All specimens are examined microscopically and scored as a modified Brunt score system. Scores are based on the grading scheme and end-points as described in Brunt E. M, et al. , "Histopathology of nonalcoholic fatty liver disease," World J. of Gastroenterol, 2010, 16(42), 5286-5296. Group means are then calculated for each individual end-point.
  • mice that received 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine exhibit a significant decrease in hepatic inflammation
  • mice that received 3 mg/kg and higher doses of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine exhibit a significant decrease in macrovesicular vaculation and perisinusoidal fibrosis
  • mice that received 10 and 30 mg/kg of 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine also exhibit a significant decrease in portal fibrosis.
  • Plasma Levels of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine Dose Number of Plasma Compound (mg/kg) Animals Level (ng/ml)
  • Nonparametric test is applied to compare scores between compound treatment groups and vehicle group. Scores for left and right laterals are compared separately.
  • HFD high fat diet
  • mice that received 30 mg/kg of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l- yl)-9-(tetrahydro-pyran-4-yl)-9H-purine exhibit a significant decrease in ALT and AST after 33 weeks of compound treatment.
  • mice After 20 weeks of treatment, mice are sacrificed (un-fasted). The liver is isolated and total liver weight is determined. The medial lobe is isolated, fixed in formalin and embedded in paraffin for histological evaluation. Formalin-fixed and paraffin-embedded cross- sections (3 ⁇ ) are stained with haematoxylin and eosin and are scored blindly by a board-certified pathologist using an adapted grading method for human nonalcoholic steatohepatitis (Liang et al, "Establishment of a General NAFLD Scoring System for Rodent Models and Comparison to Human Liver Pathology," PLoS ONE, 9(12), 2014, 1- 17).
  • fibrosis is assessed histochemically by Picro-Sirius Red staining (Chroma, WALDECK-Gmbh, Miinster, Germany). The development of fibrosis is assessed by a pathologist to gain insight into the distribution pattern of the collagen and to quantify the percentage of pericellular fibrosis specifically.
  • mice treated with the compound of 8- (2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine is provided in Table 5.
  • the results indicate that the mice that received 30 mg/kg of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine exhibit a significant decrease in hepatic inflammation, macrovesicular vacuolation and fibrosis.
  • ANOVA test is applied to compare scores between compound treatment groups and vehicle group.
  • IU refers to international units
  • ANOVA test is applied to compare scores between compound treatment groups and vehicle group.
  • the total collagen content is determined by quantitative analysis of
  • results from this method can be useful to support that 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine significantly reduces liver fibrosis and total collagen in the nonalcoholic steatohepatitis model at a 30 mg/kg /day food mixture dosing.

Abstract

The present invention provides a method for treating nonalcoholic steatohepatitis comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-1-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a patient.

Description

PURINE CANNABINOID AGONIST FOR TREATING NONALCOHOLIC STEATOHEPATITIS AND
FIBROSIS
Treatment for Nonalcoholic Steatohepatitis and Fibrosis
Nonalcoholic steatohepatitis is a liver disease with an etiological constellation characterized by macrovesicular hepatic steatosis, inflammation hepatocyte ballooning and fibrosis. Nonalcoholic steatohepatitis occurs in people who drink little or no alcohol and is often comorbid with obesity, type II diabetes, elevated levels of cholesterol, and triglycerides. Nonalcoholic steatohepatitis may lead to cirrhosis and liver failure. It has been established that patients with nonalcoholic steatohepatitis are more likely to develop cirrhosis, and have a higher risk of cardiovascular mortality, and hepatocyte carcinoma.
This non-alcoholic, non-viral cirrhosis is, in fact, among the top causes of liver
transplantation.
Nonalcoholic steatohepatitis is a progressive disease characterized by the
development of liver fibrosis. The stage of disease can be defined, for example, by the nonalcoholic steatohepatitis CRN (Clinical Research Network) fibrosis staging which measures the amount and pattern of nonalcoholic steatohepatitis fibrosis, as well as parenchymal architectural remodeling in a patient. Nonalcoholic steatohepatitis is
typically diagnosed in a human patient using liver biopsy.
Currently, there is no approved pharmaceutical medicament specifically for the treatment of nonalcoholic steatohepatitis. Current nonalcoholic steatohepatitis patient recommendations include weight loss, diet, and exercise. There is a need for
pharmaceutical medicaments to offer additional treatment options for patients suffering from nonalcoholic steatohepatitis.
The present invention provides a pharmaceutical treatment for nonalcoholic steatohepatitis comprising administering an effective amount of the compound, 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine to a patient.
The compound, 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine is disclosed in WO 2010/080306 as Example 189.
Compounds of WO 2010/080306 are stated to be agonists of the cannabinoid receptor 2 (CB2 receptor) in vitro and useful for treatment or prevention of pain.
The present invention provides a method for treating nonalcoholic steatohepatitis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a patient.
The present invention provides a method for treating nonalcoholic steatohepatitis and fibrosis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2- methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a patient.
The present invention provides a method for treating nonalcoholic steatohepatitis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine to a patient.
The present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin- 1 -yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or pharmaceutically acceptable salt thereof, for use in treating nonalcoholic steatohepatitis in a patient.
The present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin- 1 -yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or pharmaceutically acceptable salt thereof, for use in treating nonalcoholic steatohepatitis and fibrosis in a patient.
The present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, for use in treating nonalcoholic steatohepatitis in a patient.
The present invention provides the compound 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin- 1 -yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or pharmaceutically acceptable salt thereof, for use in treating hepatic fibrosis in a mammal.
The present invention provides a pharmaceutical composition for use in the treatment of nonalcoholic steatohepatitis comprising 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
The present invention provides a pharmaceutical composition for use in the treatment of nonalcoholic steatohepatitis and fibrosis comprising 8-(2-Chloro-phenyl)-2- methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. The present invention provides the use of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nonalcoholic steatohepatitis.
The present invention provides the use of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of nonalcoholic steatohepatitis and fibrosis.
The present invention provides the use of 8-(2-Chloro-phenyl)-2-methyl-6-(4- methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of hepatic fibrosis.
The present invention provides a method for treating hepatic fibrosis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl- piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a mammal.
The compound, 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin- l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, has the following structural formula:
Figure imgf000004_0001
The artisan can readily prepare the compound using the methods set forth in WO 2010/080306. The term "treating" (or "treat" or "treatment") as used herein refers to restraining, slowing, or stopping the progression or alleviating the severity of the stated condition to be treated.
As used herein, the term "effective amount" refers to the amount or dose of 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine, or a pharmaceutically acceptable salt thereof which upon single or multiple dose administration to the patient, provides the desired effect in the patient. It will be understood that the amount of active agent actually administered will be determined by a physician, in light of the relevant circumstances.
As used herein, "patient" refers to a mammal in need of treatment for
nonalcoholic steatohepatitis. In a preferred embodiment, the patient is a mammal.
In a preferred embodiment, "mammal" is a human. In a preferred embodiment the mammal is a human in need of treatment for nonalcoholic steatohepatitis and fibrosis.
8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4- yl)-9H-purine, is preferably formulated as a pharmaceutical composition administered by any route which makes the compound bioavailable. Most preferably, such composition is for oral administration. Such pharmaceutical compositions and processes for preparing the same are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy (D.B. Troy, Editor, 21st Edition, Lippincott, Williams & Wilkins, 2006).
Study of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro- pyran-4-yl)-9H-purine, in Mouse Model of Nonalcoholic steatohepatitis and Fibrosis
Induced by 3H Diet
Male C57BL/6N mice are fed with D09100301 diet (Research Diets, 40% fat, 2% cholesterol, 24% fructose, 3H diet) for 150 days. Each mouse is then singly housed after 5 days of acclimation period. Plasma alanine aminotransferase (ALT) and CK18 (cytokeratin 18) are measured. After one week of acclimation, the mice are randomized into groups by their ALT, CK18 and body weight. Animals of each group are administrated either vehicle (0.5% methylcellulose (MC) + 0.25% Tween 80 in distilled water) or the compound of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine (at a 1, 3, 10, and 30 mg/kg dose) once daily in a volume of 5 ml/kg for 11 weeks. Blood is collected from mice treated with the compound of 8-(2-Chloro-phenyl)- 2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine for 76 days 2 hours after the last dose. Compound levels in the plasma are analyzed by mass spectroscopy. The results are listed below in Table 1. Treated mice exhibit a dose- dependent increase in plasma compound levels.
At the completion of the study, the animals are sacrificed and their livers excised. Two sections of the left and right lobes are fixed in neutral buffered 10% formalin. Liver tissue slides are stained with hematoxylin and eosin (H&E), Sirius red, and Masson's Trichrome to prepare slides for pathological analysis. All specimens are examined microscopically and scored as a modified Brunt score system. Scores are based on the grading scheme and end-points as described in Brunt E. M, et al. , "Histopathology of nonalcoholic fatty liver disease," World J. of Gastroenterol, 2010, 16(42), 5286-5296. Group means are then calculated for each individual end-point. The following endpoints are used to characterize the fast food model of NASH in mice as modified from NASH endpoints (See Brunt, E. M. "Histopathology of nonalcoholic fatty liver disease," Clin Liver Dis., 2009, 13, 533-544 and Brunt, E. M, et al, "Nonalcoholic steatohepatitis: A proposal for grading and staging the histological lesions", Am J Gastroenterology, 1999, 94(9), 2467-2474.
Histopathological analysis of the livers from the mice treated with the compound of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)- 9H-purine is provided in Table 2. The results indicate that all the mice that received 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine exhibit a significant decrease in hepatic inflammation; mice that received 3 mg/kg and higher doses of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine exhibit a significant decrease in macrovesicular vaculation and perisinusoidal fibrosis; mice that received 10 and 30 mg/kg of 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine also exhibit a significant decrease in portal fibrosis.
Table 1
Plasma Levels of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine Dose Number of Plasma Compound (mg/kg) Animals Level (ng/ml)
Vehicle 15 -
1 15 38±1
3 15 104+3
10 14 340+11
30 15 595±33
Data are presented as mean+SEM (SEM refers to standard error of mean).
Table 2
Liver Histopathological Analysis for 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl- piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine
Figure imgf000007_0001
1 Nonparametric test is applied to compare scores between compound treatment groups and vehicle group. Scores for left and right laterals are compared separately.
Data are presented as mean+SEM.
* p<0.05; **p<0M; *** /?<0.001
LDLr-/- Leiden Mice Fed High Fat Diet as a Model of NASH and Hepatic Fibrosis
Male LDLr-/- (low density lipoprotein receptor knockout mice) Leiden mice are fed a high fat diet (HFD) during a 20 week run-in period. After the run-in period mice are matched into 3 groups of 15 mice based on body weight (primary matching parameter), cholesterol, triglycerides, glucose and insulin measured at t=20 weeks. The animals are then fed with high fed diet (HFD, control), 10 mg/kg/day or 30 mg/kg/8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine, mixed in HFD. Blood is collected at different time points before and after compound treatment for ALT and AST (aspartate aminotransferase) measurements. The results of biochemical analysis are provided in Tables 3 and 4. The results indicate that mice that received 30 mg/kg of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l- yl)-9-(tetrahydro-pyran-4-yl)-9H-purine exhibit a significant decrease in ALT and AST after 33 weeks of compound treatment.
After 20 weeks of treatment, mice are sacrificed (un-fasted). The liver is isolated and total liver weight is determined. The medial lobe is isolated, fixed in formalin and embedded in paraffin for histological evaluation. Formalin-fixed and paraffin-embedded cross- sections (3 μιη) are stained with haematoxylin and eosin and are scored blindly by a board-certified pathologist using an adapted grading method for human nonalcoholic steatohepatitis (Liang et al, "Establishment of a General NAFLD Scoring System for Rodent Models and Comparison to Human Liver Pathology," PLoS ONE, 9(12), 2014, 1- 17). Briefly, two cross-sections/mouse are examined and the level of microvesicular and macrovesicular steatosis are determined relative to the liver area analyzed (expressed as a percentage). Hepatic inflammation is assessed by counting the number of inflammatory foci per field at a lOOx magnification (view size 3.1 mm2) in five non-overlapping fields per specimen, expressed as total number of foci. Fibrosis is assessed histochemically by Picro-Sirius Red staining (Chroma, WALDECK-Gmbh, Miinster, Germany). The development of fibrosis is assessed by a pathologist to gain insight into the distribution pattern of the collagen and to quantify the percentage of pericellular fibrosis specifically.
Histological analysis of the liver from the mice treated with the compound of 8- (2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine is provided in Table 5. The results indicate that the mice that received 30 mg/kg of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine exhibit a significant decrease in hepatic inflammation, macrovesicular vacuolation and fibrosis.
Table 3
Alanine Aminotransferase Changes in LDLr Knockout Mice after Treatment with 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine
ALT IU/L
Time (weeks on Diet)
Dose (mg/kg) 15 20 27 33 39
HFD 267+31 383±36 330±33 379±29 219±29
10 277+33 447+35 357+32 426±23 399±27 30 219+29 391+40 251+26 263+22* 244+23*
ANOVA test is applied to compare scores between compound treatment groups and vehicle group.
Data are presented as mean+SEM.
* /?<0.05; **/x0.01; *** /?<0.001
IU refers to international units
Table 4
Aspartate Aminotransferase Changes in LDLr Knockout Mice after Treatment with 8-(2- Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine
Figure imgf000009_0001
ANOVA test is applied to compare scores between compound treatment groups and vehicle group.
Data are presented as mean+SEM.
* p<0.05; **/x0.01; *** /?<0.001
Table 5
Liver Histopathological Analysis for 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl- piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine
Figure imgf000009_0002
The total collagen content is determined by quantitative analysis of
hydroxyproline in liver homogenate using a commercial colorimetric assay (QZBtotcol; Quickzyme Biosciences, Leiden, the Netherlands). Results from this method can be useful to support that 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine significantly reduces liver fibrosis and total collagen in the nonalcoholic steatohepatitis model at a 30 mg/kg /day food mixture dosing.

Claims

A method for treating nonalcoholic steatohepatitis, comprising administering an effective amount of 8-(2-Chloro-phenyl)-2- methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine, or a pharmaceutically acceptable salt thereof, to a patient. The method of Claim 1 wherein 8-(2-Chloro-phenyl)-2-methyl-6- (4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, is the free base.
The method as claimed by either of Claims 1 or 2 wherein the patient is a mammal.
The method as claimed by any one of Claims 1 to 3 wherein the patient is a human.
A method for treating nonalcoholic steatohepatitis and fibrosis, comprising administering an effective amount of 8-(2-Chloro- phenyl) -2-methyl- 6- (4-methyl-piperazin- 1 -yl) -9- (tetrahydro-pyran- 4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a patient.
The method of Claim 5 wherein 8-(2-Chloro-phenyl)-2-methyl-6- (4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, is the free base.
A method for treating hepatic fibrosis comprising administering an effective amount of 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl- piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, to a mammal.
8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for use in the treatment of nonalcoholic steatohepatitis.
8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine for use according to Claim 8. 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for use in the treatment of nonalcoholic steatohepatitis and fibrosis.
8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl-piperazin-l-yl)-9- (tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, for use in the treatment of hepatic fibrosis. A pharmaceutical composition for use in the treatment of nonalcoholic steatohepatitis comprising 8-(2-Chloro-phenyl)-2- methyl-6-(4-methyl-piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H- purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient. A pharmaceutical composition for use in the treatment of nonalcoholic steatohepatitis and fibrosis comprising 8-(2-Chloro- phenyl) -2-methyl- 6- (4-methyl-piperazin- 1 -yl) -9- (tetrahydro-pyran- 4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
A pharmaceutical composition for use in the treatment of hepatic fibrosis comprising 8-(2-Chloro-phenyl)-2-methyl-6-(4-methyl- piperazin-l-yl)-9-(tetrahydro-pyran-4-yl)-9H-purine, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier, diluent, or excipient.
25
PCT/US2017/027306 2016-04-18 2017-04-13 Purine cannabinoid agonist for treating nonalcoholic steatohepatitis and fibrosis WO2017184412A1 (en)

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Citations (3)

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EP1725223A2 (en) * 2004-03-09 2006-11-29 Inserm Use of antagonists of the cb1 receptor for the manufacture of a composition useful for the treatment of hepatic deseases
WO2010080306A1 (en) 2008-12-18 2010-07-15 Eli Lilly And Company Purine compounds
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