Compositions and methods for treating non-alcoholic steatohepatitis
阅读说明：本技术 用于治疗非酒精性脂肪性肝炎的组合物和方法 (Compositions and methods for treating non-alcoholic steatohepatitis ) 是由 M·威廉姆斯 于 2019-07-03 设计创作，主要内容包括：千金藤碱、瑞吡司特、半酒石酸艾芬地尔、布罗曼坦、阿克他利、氯苯扎利、伊索拉定、伊曲茶碱、瑞帕地、贝米特尔用于治疗或预防对象的小叶炎症或非酒精性脂肪肝疾病或非酒精性脂肪性肝炎。(Cepharanthine, repirailast, ifenprodil hemitartrate, browman's, aclopril, clobenzaprine, issorafen, eltanophylline, rapado, bemidel for use in treating or preventing lobular inflammation or non-alcoholic fatty liver disease or non-alcoholic steatohepatitis in a subject.)
1. Use of cepharanthine in the treatment or prevention of lobular inflammation or non-alcoholic fatty liver disease in a subject.
2. The use of claim 1, wherein the amount of cepharanthine is 1-5mg/kg of subject.
3. The use of claim 2, wherein the amount of stephanine is about 2.25mg/kg of subject.
4. The use of claim 3, wherein the amount of stephanine is about 2.5mg/kg of subject.
5. Use of repiraast for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
6. The use of claim 5, wherein the amount of repirafelast is 1-20mg/kg of subject.
7. The use of claim 6, wherein the amount of repirafelast is 3-10mg/kg of subject.
8. The use of claim 7, wherein the amount of repirafelast is about 10mg/kg of subject.
9. Use of ifenprodil hemitartrate for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
10. Use according to claim 9, wherein the amount of ifenprodil hemitartrate is from 0.1 to 5mg/kg of subject.
11. Use according to claim 10, wherein the amount of ifenprodil hemitartrate is from 0.5 to 3mg/kg of subject.
12. Use according to claim 11, wherein the amount of ifenprodil hemitartrate is about 1mg/kg of subject.
13. Use of browman's coat for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
14. The use of claim 13, wherein the amount of browman's day is 0.5-5mg/kg of subject.
15. The use of claim 14, wherein the amount of browman's day is 1-2mg/kg of subject.
16. The use of claim 15, wherein the amount of browman's day is about 1.7mg/kg of subject.
17. Use of acrtalide in treating or preventing lobular inflammation or nonalcoholic fatty liver disease in a subject.
18. The use of claim 17, wherein the amount of acrtalide is 1-10mg/kg of subject.
19. The use of claim 18, wherein the amount of acrtalide is 2-7mg/kg of subject.
20. The use of claim 19, wherein the amount of acrtalide is 4-6mg/kg of subject.
21. The use of claim 20, wherein the amount of acrtalide is about 5mg/kg of subject.
22. Use of clobenzaprine in the treatment or prevention of lobular inflammation or non-alcoholic fatty liver disease in a subject.
23. The use of claim 22, wherein the amount of clobenzaprine is 1-10mg/kg of subject.
24. The use of claim 23, wherein the amount of clobenzaprine is 2-8mg/kg of subject.
25. The use of claim 24, wherein the amount of clobenzaprine is 3-6mg/kg of subject.
26. The use of claim 24, wherein the amount of clobenzaprine is about 4mg/kg of subject.
27. Use of issoprazole for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
28. The use of claim 27, wherein the amount of issorafenib is 0.02-0.5mg/kg of subject.
29. The use of claim 28, wherein the amount of issorafenib is 0.05-0.1mg/kg of subject.
30. The use of claim 29, wherein the amount of issorafenib is about 0.07mg/kg of subject.
31. Use of istradefylline in the treatment or prevention of lobular inflammation or non-alcoholic fatty liver disease in a subject.
32. The use of claim 31, wherein the amount of istradefylline is 0.1-4mg/kg of subject.
33. The use of claim 32, wherein the amount of istradefylline is 0.3-1.3mg/kg of subject.
34. The use of claim 33, wherein the amount of istradefylline is about 1.3mg/kg of subject.
35. Use of rapadid in the treatment or prevention of lobular inflammation or non-alcoholic fatty liver disease in a subject.
36. The use as claimed in claim 35, wherein the amount of rapadid is 1-10mg/kg of subject.
37. Use as claimed in claim 36, wherein the amount of rapadid is 3-8mg/kg of subject.
38. The use of claim 37, wherein the amount of rapadid is about 5mg/kg of subject.
39. The use as claimed in any one of claims 35 to 38, wherein a single dose of 10mg/kg rapadid is used prior to the use as claimed in any one of claims 44 to 46.
40. Use of bemidel for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
41. The use of claim 40, wherein the amount of bemidel is 5-50mg/kg of subject.
42. The use of claim 41, wherein the amount of bemidel is 12-25mg/kg of subject.
43. The use of claim 42, wherein the amount of bemidel is 8-17mg/kg of subject.
44. The use of claim 43, wherein the amount of bemidel is about 8mg/kg of subject.
45. The use of claim 44, wherein the amount of bemidel is about 17mg/kg of subject.
46. The use of any one of claims 1-45, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
47. The use of any one of claims 1-46, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis-derived hepatocellular carcinoma.
48. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of cepharanthine.
49. The method of claim 48, wherein 1 to 5mg cepharanthine is administered to the subject per kg of subject.
50. The method of claim 49, wherein about 2.25mg cepharanthine is administered to the subject per kg of subject.
51. The method of claim 50, wherein about 2.5mg cepharanthine is administered to the subject per kg of subject.
52. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of repiraast.
53. The method of claim 52, wherein 1 to 20mg of repirolast is administered per kg of subject.
54. The method of claim 53, wherein 3 to 10mg of repirolast is administered per kg of subject.
55. The method of claim 54, wherein about 10mg of repirafelast is administered per kg of subject.
56. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of ifenprodil hemitartrate.
57. A method according to claim 56, wherein 0.1 to 5mg ifenprodil hemitartrate is administered to the subject per kg of the subject.
58. A method according to claim 57, wherein 0.5 to 3mg ifenprodil hemitartrate is administered to the subject per kg of the subject.
59. The method of claim 58, wherein about 1mg ifenprodil hemitartrate is administered to the subject per kg of the subject.
60. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of browman's.
61. The method of claim 60, wherein 0.5 to 5mg of Blomaptan is administered to the subject per kg of the subject.
62. The method of claim 61, wherein 1.7 to 3.3mg of Blomaptan is administered to the subject per kg of subject.
63. The method of claim 62, wherein about 3.3mg of Blomaptan is administered to the subject per kg of the subject.
64. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of acrtalide.
65. The method of claim 64, wherein 1 to 10mg of acrtalide is administered to the subject per kg of the subject.
66. The method of claim 65, wherein 2 to 7mg of acrtalide is administered to the subject per kg of the subject.
67. The method of claim 66, wherein about 5mg of acrtalide is administered to the subject per kg of the subject.
68. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of clobenzaprine.
69. The method of claim 68, wherein 1 to 10mg of clobenzaprine is administered to the subject per kg of the subject.
70. The method of claim 69, wherein 2 to 8mg of clobenzaprine is administered to the subject per kg of the subject.
71. The method of claim 70, wherein the subject is administered about 4mg of clobenzaprine per kg of subject.
72. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of issorafenib.
73. The method of claim 72, wherein 0.02 to 0.5mg of isradine/kg of subject is administered to the subject.
74. The method of claim 73, wherein 0.05 to 0.1mg of issorafenib/kg of subject is administered to the subject.
75. The method of claim 74, wherein about 0.07mg of issorafenib/kg of subject is administered to the subject.
76. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of istradefylline.
77. The method of claim 76, wherein 0.1 to 4mg of istradefylline per kg of subject is administered to the subject.
78. The method of claim 77, wherein 0.3 to 1.3mg of istradefylline per kg of subject is administered to the subject.
79. The method of claim 78, wherein about 1.3mg of istradefylline per kg of subject is administered to the subject.
80. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of rapadid.
81. The method of claim 80, wherein 1 to 10mg rapadid/kg of subject is administered to the subject.
82. The method of claim 81, wherein 3 to 8mg rapadid/kg of subject is administered to the subject.
83. The method of claim 82, wherein about 5mg rapadid/kg of subject is administered to the subject.
84. A method for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject, the method comprising:
administering to the subject a therapeutically effective amount of bemidel.
85. The method of claim 84, wherein 5 to 50mg of bemidel/kg of subject is administered to the subject.
86. The method of claim 85, wherein 12 to 25mg of bemidel is administered to the subject per kg of the subject.
87. The method of claim 86, wherein about 17mg of bemidel/kg of subject is administered to the subject.
88. The method of any one of claims 48-87, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
89. The method of any one of claims 48-88, wherein non-alcoholic fatty liver disease is non-alcoholic steatohepatitis-derived hepatocellular carcinoma.
Technical Field
The invention relates to application of a compound for treating non-alcoholic fatty liver diseases, in particular to application of the compound for treating non-alcoholic fatty liver diseases, non-alcoholic fatty liver diseases and non-alcoholic steatohepatitis.
Background
Nonalcoholic fatty liver disease (NAFLD) encompasses a range of liver conditions characterized by the storage of excess fat in hepatocytes. As the name suggests, the reason for this is generally unrelated to alcohol consumption. This is different from alcoholic liver disease caused by heavy drinking. Most commonly, NAFLD is non-alcoholic simple fatty liver or non-alcoholic steatohepatitis (NASH).
Non-alcoholic fatty liver refers to a condition in which there is fat in the liver, but little or no inflammation or damage to hepatocytes. It does not usually progress to cause liver damage or complications.
NASH includes fatty liver and hepatitis. Although subjects with only non-alcoholic fatty liver disease are often asymptomatic, inflammation and hepatocyte injury caused by NASH may lead to liver fibrosis or scarring, and in severe cases may lead to cirrhosis (advanced scarring) or liver cancer. In this regard, common signs and symptoms of NASH and cirrhosis may include abdominal distension, vasodilation beneath the surface of the skin, enlargement of the male breast, and jaundice.
Factors that increase the risk of NASH in humans include: diabetes, obesity, high fructose diet, genetics and advanced age. Improving diet and exercise in humans is an effective method to control NAFLD and reduce insulin resistance, but there are currently no strategies for NAFLD or NASH, nor a therapy for each individual. Current drugs used to treat NAFLD and NASH may help to increase lipid loss and/or improve levels of biomedical markers, but once hepatic scarring begins, no drug is able to reverse or reduce NAFLD and NASH.
Mouse models of NAFLD and NASH are well-characterized experimental models of metabolic-induced liver injury that ultimately leads to spontaneous hepatic steatosis, a common feature of many chronic hepatitis diseases. In a dietary model, progression to fatty liver is highly predictable and reproducible, leading to insulin resistance and obesity-based steatosis (Ishii et al, Female idiopathic diabetic Torri obese rats developed non-alcoholic steatohepatitis-like liver injury (Femal specific diabetes Touretic fat rats, World J Gastroenterol, 21(30), 9067-78(2015), Kucera O and Cervinkova, Experimental model of rat non-alcoholic fatty liver disease (Experimental models of non-alcoholic fatty liver disease in rats), World J Gastroenterol, 20 (8326), 64-76, 2014, Talchari et al, non-alcoholic liver disease, model of fatty liver disease, 18, hepatic fibrosis, non-alcoholic liver disease, Animal model of fatty liver disease, 18, hepatic fibrosis, non-alcoholic liver disease, model of fatty liver disease, liver disease model of fatty liver disease, liver disease of human liver disease, No. 19, liver disease of alcoholic liver disease, 2300, liver disease of liver disease, No. 3-alcoholic liver disease, 3, 11, hepatic fibrosis, liver disease of fatty liver disease of human liver disease, No. 3,7, 3, hepatic fibrosis, Animal model, No. 1, 3.
One such model (termed the Stelic animal model or STAM) in mice NASH-derived hepatocellular carcinoma ("NASH-HCC") (referred to as the Stelic animal model or STAM)IM) In (b), subcutaneous streptozotocin ("STZ") exposure followed by a sustained high fat diet can lead to diabetes, severe steatosis, chronic lobular inflammation, cells within a short 6 to 16 weeksPeripheral fibrosis, cirrhosis and HCC that resembles the entire cascade of human NASH-HCC, including modest increases in transaminase and plasma lipids, the major markers of human NASH (Fujii M et al, "mouse model of non-alcoholic steatohepatitis-evidence of association between diabetes and hepatocellular carcinoma" (a multiple model for non-alcoholic steatohepatitis bearing evidence of association between diabetes and hepatocellular carcinoma), Med Mol morphology, 46(3):141-52 (2013)).
The present invention provides new uses of existing drugs, generally investigated as potential therapies for other pathologies, for the treatment and/or alleviation of NAFLD and NASH.
Disclosure of Invention
In one embodiment, the invention provides uses and methods of Cepharanthine (cephaloranthine) for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the invention provides the use and method of repirast (Repirinast) for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the present invention provides the use and method of Ifenprodil hemimitartrate for the treatment or prevention of lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the invention provides the use and method of Bromantane (Bromantane) for treating or preventing lobular inflammation or nonalcoholic fatty liver disease in a subject.
In another embodiment, the invention provides the use and method of Suplatast tosilate for the treatment or prevention of lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the present invention provides the use and method of acrtalide (Actarit) for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the present invention provides the use and method of clobenzarit (Lobenzarit) for treating or preventing lobular inflammation or nonalcoholic fatty liver disease in a subject.
In another embodiment, the present invention provides uses and methods of issorafen (Irsogladine) for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the invention provides uses and methods of Istradefylline (Istradefylline) for treating or preventing lobular inflammation or nonalcoholic fatty liver disease in a subject.
In another embodiment, the invention provides the use and method of rapadil for treating or preventing lobular inflammation or nonalcoholic fatty liver disease in a subject.
In another embodiment, the invention provides the use and method of bemidel (bezithal) for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject.
In another embodiment, the invention provides the use and method of cicodex (ceriviroc) for treating or preventing lobular inflammation or nonalcoholic fatty liver disease in a subject.
In a further aspect, the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
In an embodiment of the invention, the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis-derived hepatocellular carcinoma.
Drawings
Exemplary embodiments are shown in referenced figures of the drawings. The embodiments and figures disclosed herein are intended to be illustrative rather than restrictive.
Figure 1 shows the assessment of liver function and disease progression (in units/L) consisting of plasma alanine Aminotransferase (ALT) for each of the 12 study groups in the first study of C57BL/6 mice, consisting of a "vehicle" control group and 11 treatment groups including the positive control treatment group Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figures 2a, 2b, 2C and 2d show representative photomicrographs of HE-stained liver sections from each of 12 study groups of C57BL/6 mice, consisting of the "vehicle" control group and 11 treatment groups including the positive control treatment group Telmisartan (Telmisartan). The magnification of the upper picture is x 50. The magnification of the lower picture is x 200. The categories for each study group are listed above the top panel of each pair of panels.
Figure 3 shows NAFLD activity scores for each of 12 study groups of C57BL/6 mice (consisting of the "vehicle" control group and 11 treatment groups including the positive control treatment group Telmisartan (Telmisartan)). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 4 shows the steatosis score for each of 12 study groups of C57BL/6 mice, consisting of the "vehicle" control group and 11 treatment groups including the positive control treatment group Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 5 shows the leaflet inflammation score for each of 12 study groups of C57BL/6 mice, consisting of a "vehicle" control group and 11 treatment groups including a positive control treatment group of Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 6 shows the hepatocyte balloon degeneration (hepatocellular balloon) score for each of 12 study groups of C57BL/6 mice, consisting of the "vehicle" control group and 11 treatment groups including the positive control treatment group Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 7 shows representative micrographs of sirius red stained liver sections from each study group of C57BL/6 mice, consisting of the "vehicle" control group and browman's and istradefylline treated groups including the positive control treated group Telmisartan (Telmisartan). The magnification of the picture is x 200. The categories for each study group are listed above the top panel of each pair of panels.
Fig. 8 shows Sirius red-positive areas (Sirius red-positive areas) of each study group in the study group of C57BL/6 mice, consisting of the "vehicle" control group and browman's and istradefylline treatment groups including the positive control treatment group Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 9 shows the assessment of liver function and disease progression (in units/L) consisting of plasma alanine Aminotransferase (ALT) for each of 7 study groups (consisting of the normal (no NASH) group, the "vehicle" control group and 5 treatment groups including the positive control treatment group Telmisartan (Telmisartan)) in the second study of C57BL/6 mice. Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Fig. 10a, 10b and 10C show representative photomicrographs of HE-stained liver sections of each study group in 7 study groups of C57BL/6 mice, consisting of a normal (no NASH) group, a "vehicle" control group and 5 treatment groups including a positive control treatment group of Telmisartan (Telmisartan). The magnification of the upper picture is x 50. The magnification of the lower picture is x 200. The categories for each study group are listed above the top panel of each pair of panels.
Figure 11 shows NAFLD activity scores for each of 7 study groups of C57BL/6 mice, consisting of a normal (no NASH) group, a "vehicle" control group, and 5 treatment groups including a positive control treatment group of Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 12 shows the steatosis score for each of 7 study groups of C57BL/6 mice, consisting of the normal (no NASH) group, the "vehicle" control group, and the 5 treatment groups including the positive control treatment group Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 13 shows the leaflet inflammation score for each of 7 study groups of C57BL/6 mice, consisting of the normal (no NASH) group, the "vehicle" control group, and 5 treatment groups including the positive control treatment group, Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Figure 14 shows the hepatocyte ballooning degeneration score for each of 7 study groups of C57BL/6 mice, consisting of the normal (no NASH) group, the "vehicle" control group and 5 treatment groups including the positive control treatment group Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Fig. 15a and 15b show representative photomicrographs of sirius red-stained liver sections of each of 7 study groups of C57BL/6 mice, consisting of a normal (no NASH) group, a "vehicle" control group, and 5 treatment groups including a positive control treatment group of Telmisartan (Telmisartan). The magnification of the picture is x 200. The categories for each study group are listed above the top panel of each pair of panels.
Figure 16 shows the assessment of liver function and disease progression consisting of fibrotic regions (i.e., sirius red positive regions) in percent for 7 study groups of C57BL/6 mice, consisting of a normal (no NASH) group, a "vehicle" control group, and 5 treatment groups including a positive control treatment group of Telmisartan (Telmisartan). Mean values are expressed as ± SD, determined using Bonferroni multiple comparison test.
Detailed Description
The inventors have found that a number of pharmaceutical compounds approved for other pathologies can be used as active ingredients for inhibiting or alleviating hepatic steatosis, lobular inflammation and hepatocellular ballooning, and can be used for the prevention and/or treatment of NASH. In some embodiments, the mouse STAM found in NASH-HCCTMIn the model, the levels of hepatic steatosis, lobular inflammation and hepatocellular ballooning degeneration are inhibited or alleviated. Based on the experimental results described herein, it was shown that in certain embodiments, the compounds described herein will be useful in the prevention and/or treatment of NASH and/or NASH-derived HCC.
The examples and data below show the effect of inhibiting or alleviating hepatic steatosis, lobular inflammation and hepatocellular ballooning in two studies. In the first study, therapeutically effective amounts of 10 drug compounds were administered. In a second study, 5 pharmaceutical compounds were administered in therapeutically effective amounts. Pharmaceutical compounds (hereinafter "test agents") are approved for other conditions and are formulated with pharmaceutically acceptable carriers for delivery and absorption.
The currently proposed therapy for the treatment of NAFLD and NASH is the administration of the pharmaceutical compound telmisartan (Gitto et al, treatment of adult nonalcoholic steatohepatitis: present and future, gastrointestinal disease research practice (Gastroenterol Res practice), 2015: 732870 (2015); Paschos P and Tziomalos K, Inc.)Non-alcoholic fatty liver disease and kidney The plain-angiotensin system: therapeutic significance》(Nonalcoholic fatty liver disease and the renin- angiotensinsystem:Implications for treatment)，World journal of liver disease (World J Hepatol)),4(12): 327-31 (2012); musso G et al, "meta-analysis of randomized trials for the treatment of non-alcoholic fatty liver disease," Hepatology (Hepatology), 52 (1): 79-104 (2010); georgescu EF et al, "Angiotensin receptor blockers as a therapy for mild to moderate hypertension-associated nonalcoholic steatohepatitis" ((Angiotensin-receptor as therapy for small-to-modified hypertension), World J Gastroenterol, 15 (8)): 942-54(2009)), telmisartan was used as a positive control in the experimental examples described herein.
Telmisartan (2- (4- { [ 4-methyl-6- (1-methyl-1H-1, 3-benzodiazol-2-yl) -2-propyl-1H-1, 3-benzodiazol-1-yl ] methyl } phenyl) benzoic acid) is an angiotensin receptor blocker known in the art for the treatment of hypertension. The chemical structure of telmisartan is:
in the animal model of NASH-HCC described herein, a therapeutically effective amount can generally be calculated as the animal equivalent daily dose to the maximum daily dose in humans.
Use of cepharanthine
Stephanine ((14S,27R) -22, 33-dimethoxy-13, 28-dimethyl-2, 5,7, 20-tetraoxa)-13, 28-diaza-octacyclo [25.6.2.216,19.13,10.121,25.04,8.014,39.031,35]Trinexadecyl (nonalconta) -1(33),3,8,10(39),16,18,21(36),22,24,31,34, 37-dodecene (dodecaene)) are known in the art as anti-inflammatory and anti-tumor compounds for the treatment of radiation-induced leukopenia, idiopathic thrombocytopenic purpura, alopecia areata and pityrosporum alopecia, xerostomia, sarcoidosis, refractory anemia, and various cancer-related conditions. The chemical structure of stephanine is as follows:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with Cepharanthine (cephaloranthine) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In one embodiment, the amount of stephanine used is 0.5 to 10mg/kg of subject. In a preferred embodiment, the amount of stephanine used is 1 to 4mg/kg of subject. In other preferred embodiments, the amount of stephanine used is about 2.25mg/kg of subject. In other preferred embodiments, the amount of stephanine used is about 2.5mg/kg of subject.
Stephanine or a pharmaceutically acceptable variant thereof may be administered to a subject orally, intravenously, or in a manner known in the art. Cepharanthine or a pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of repirolast
Repiraast (7, 8-dimethyl-4, 5-dioxo-5, 6-dihydro-4H-pyrano [3,2-c ] quinoline-2-carboxylic acid 3-methylbutyl ester) is known in the art as an antihistamine. The chemical structure of repirolast is:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with repirast (Repirinast) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of repirafelast used is between 2 and 50mg/kg of subject/day. In a preferred embodiment, the amount of repirafelast used is between 5 and 10mg/kg of subject/day. In other preferred embodiments, the amount of repirafelast used is about 10mg/kg of subject/day.
The repirast or a pharmaceutically acceptable variant thereof may be administered to the subject orally, intravenously or in a manner known in the art. The repirakast or pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of Ifenprodil
Ifenprodil (4- [2- (4-benzylpiperidin-1-yl) -1-hydroxypropyl ] phenol) is known in the art as a selective NMDA receptor (glutamate) antagonist. The chemical structure is as follows:
in some embodiments tested in the examples herein, Ifenprodil hemimitartrate (4- [2- (4-benzylpiperidin-1-ium-1-yl) -1-hydroxypropyl ] phenol; 2,3, 4-trihydroxy-4-oxobutyrate) having the structure below is used
In one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with Ifenprodil hemimitartrate (Ifenprodil hemimitartrate) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of ifenprodil used is from 0.1 to 5mg/kg of subject/day. In a preferred embodiment, ifenprodil is used in an amount of 0.5 to 3mg/kg of subject/day. In other preferred embodiments, the amount of ifenprodil used is about 1mg/kg of subject/day.
Ifenprodil hemitartrate, or a pharmaceutically acceptable variant thereof, may be administered to a subject orally, intravenously, or in a manner known in the art. Ifenprodil hemitartrate or a pharmaceutically acceptable variant thereof may also be administered together with one or more pharmaceutically acceptable excipients.
Use of browman's essential
Bloomantan (N- (4-bromophenyl) adamantan-2-amine) is an atypical psychostimulant of the adamantane (adamantine) family and anxiolytic known in the field of neurasthenia treatment. The chemical structure of browman's is:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with Bromantane (Bromantane) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of browman's day used is from 0.8 to 5mg/kg of subject/day. In a preferred embodiment, the amount of browman's day used is from 1.7 to 3.3mg/kg of subject/day. In other preferred embodiments, the amount of browman's day used is about 1.7mg/kg of subject/day.
The browman's or pharmaceutically acceptable variant thereof may be administered to a subject orally, intravenously, or in a manner known in the art. Bloomantan or a pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of Sulfolast (Sullast Tosylate)
Sulfast ((3- { [4- (ethoxy-2-hydroxypropoxy) phenyl ] amino } -3-oxopropyl) (dimethyl) sulfonium 4-methylbenzenesulfonate) is a Th2 cytokine inhibitor known in the art as an antiallergic. The chemical structure of the suplatast tosilate is as follows:
in one aspect, the invention provides the use and method of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with Suplatast tosilate or a pharmaceutically acceptable salt thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of suplatast tosilate used is from 1 to 10mg/kg of subject/day. In a preferred embodiment, the amount of suplatast tosilate used is from 2 to 8mg/kg of subject/day. In another preferred embodiment, the amount of suplatast used is from 4.5 to 5.4mg/kg of subject/day. In other preferred embodiments, the amount of suplatast used is about 5mg/kg of subject/day.
The suplatast or a pharmaceutically acceptable salt thereof may be administered to the subject orally, intravenously or in a manner known in the art. Sulfaast or a pharmaceutically acceptable salt thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of acrtalide
Actarit ((4-acetamidophenyl) acetic acid) is a disease modifying antirheumatic agent known in the art for the treatment of rheumatoid arthritis. The chemical structure of acrtalide is:
in one aspect, the invention provides uses and methods for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with acltalit (Actarit) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of acrilex used is from 3.75 to 6.25mg/kg of subject/day. In a preferred embodiment, the amount of acrilex used is from 4.16 to 5.83mg/kg of subject/day. In another preferred embodiment, the amount of acrilex used is from 4.58 to 5.42mg/kg of subject/day. In other preferred embodiments, the amount of acrilex used is about 5mg/kg of subject/day.
The acrtalide or a pharmaceutically acceptable variant thereof can be administered to the subject orally, intravenously, or in a manner known in the art. The acrtalide or pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of clobenzaprine
Clobenzaprine (2- [ (2-carboxyphenyl) amino ] -4-chlorobenzoic acid) is a known immunomodulator in the art for the treatment of arthritis. The chemical structure of the clobenzaprine is as follows:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with clobenzarit (Lobenzarit) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of clobenzaprine used is 1 to 10mg/kg of subject/day. In a preferred embodiment, the amount of clobenzaprine used is from 2 to 7mg/kg of subject/day. In another preferred embodiment, the amount of clobenzaprine used is from 3 to 5mg/kg of subject/day. In other preferred embodiments, the amount of clobenzaprine used is about 4mg/kg of subject/day.
The clobenzaprine or pharmaceutically acceptable variant thereof may be administered to the subject orally, intravenously, or in a manner known in the art. The clobenzaprine or pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of Ixodine
Issorafen (6- (2, 5-dichlorophenyl) -1,3, 5-triazine-2, 4-diamine) is a phosphodiesterase inhibitor known in the art as a mucosal protective drug for the treatment of peptic ulcer disease and acute gastritis. The chemical structure of the isradine is as follows:
in one aspect, the invention provides uses and methods for treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with issorafen (Irsogladine) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In embodiments, the amount of issorafen used is 0.0416 to 0.125mg/kg of subject/day. In a preferred embodiment, the amount of issorafenib used is between 0.066 and 0.1mg/kg of subject/day. In other preferred embodiments, the amount of issorafenib used is about 0.08mg/kg of subject/day. In other preferred embodiments, the amount of issorafenib used is about 0.07mg/kg of subject/day.
The isradine or pharmaceutically acceptable variant thereof can be administered to the subject orally, intravenously, or in a manner known in the art. The isradine or pharmaceutically acceptable variant thereof can also be administered with one or more pharmaceutically acceptable excipients.
Use of istradefylline
Istradefylline (8- [ (E) -2- (3, 4-dimethoxyphenyl) vinyl ] -1, 3-diethyl-7-methyl-3, 7-dihydro-1H-purine-2, 6-dione) is a selective A2A receptor antagonist known in the art for the treatment of dyskinesia in parkinson's disease. The chemical structure of the istradefylline is as follows:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with Istradefylline (Istradefylline) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In an embodiment, the amount of istradefylline used is from 0.1 to 5mg/kg of subject/day. In a preferred embodiment, the amount of istradefylline used is from 0.3 to 1.3mg/kg of subject/day. In other preferred embodiments, the amount of istradefylline used is about 1.3mg/kg of subject/day.
The istradefylline or a pharmaceutically acceptable variant thereof can be administered to a subject orally, intravenously, or in a manner known in the art. The istradefylline or a pharmaceutically acceptable variant thereof can also be administered with one or more pharmaceutically acceptable excipients.
Use of rapadid
Rapadi (N, N-diethyl-5-methyl- [1,2,4] triazolo [1,5-a ] pyrimidin-7-amine) is known in the art as a vasodilator, antiplatelet agent and platelet-derived growth factor antagonist. The chemical structure of rapadid is:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with rapidil or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In embodiments, the amount of rapadid used is from 4.16 to 5.83mg/kg of subject/day. In a preferred embodiment, the amount of rapadid used is from 4.58 to 5.41mg/kg of subject/day. In other preferred embodiments, the amount of rapadid used is about 5mg/kg of subject/day. In another embodiment, a single dose of 10mg/kg rapadi is administered to a subject prior to administration of 5mg/kg rapadi to the subject.
Rapadid or a pharmaceutically acceptable variant thereof may be administered to a subject orally, intravenously, or in a manner known in the art. Rapadid or a pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Use of Bemidel
Bemidel (2-ethylsulfonyl-1H-benzimidazole) is a synthetic activity protector (activirotector), antioxidant and antimutator known in the art and is often used to improve body functions. The chemical structure of bemidel is:
in one aspect, the invention provides uses and methods of treating or preventing lobular inflammation or non-alcoholic fatty liver disease in a subject with bezier (bezithyl) or a pharmaceutically acceptable variant thereof. The non-alcoholic fatty liver disease may be NASH or NASH-HCC.
In embodiments, the amount of bemidel used is from 0.5 to 30mg/kg of subject/day. In a preferred embodiment, the amount of bemidel used is from 1 to 25mg/kg of subject/day. In other preferred embodiments, the amount of bemidel used is from 5 to 20mg/kg of subject/day. In other preferred embodiments, the amount of bemidel used is about 8mg/kg of subject/day. In other preferred embodiments, the amount of bemidel used is about 17mg/kg of subject/day.
The bemidel or a pharmaceutically acceptable variant thereof may be administered to the subject orally, intravenously, or in a manner known in the art. The bemidel or a pharmaceutically acceptable variant thereof may also be administered with one or more pharmaceutically acceptable excipients.
Are used in combination
In another aspect, the invention provides uses and methods for treating or preventing NASH or NASH-HCC in a subject using one or more of Cepharanthine (cephalorantine), repirast (reprinast), Ifenprodil Hemitartrate (Ifenprodil hemimitarate), bromantan (Bromantane), Suplatast (Suplatast tosilate), acrtalci (Actarit), clobenzarit (Lobenzarit), isradine (Irsogladine), Istradefylline (isotrafylline), and rapadil (Trapadil) in combination. In another aspect, the invention provides uses and methods for treating or preventing NASH or NASH-HCC in a subject using a combination of: one or more of Cepharanthine (Cepharanthine), repirastine (repiraast), Ifenprodil Hemitartrate (Ifenprodil hemimitartrate), browman (Bromantane), Suplatast (Suplatast tosilate), Actarit (Actarit), cyclobenzarit (Lobenzarit), isradine (Irsogladine), istrine (Istradefylline) and rapadil (Trapadil), and one or more of a cholesterol-lowering drug, a diabetes drug, a blood pressure lowering drug or vitamin E.
Examples of cholesterol lowering drugs used in combination include: atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin, pitavastatin (Livalo), pravastatin (pravastatin), rosuvastatin calcium (codex), simvastatin (zuelan (Zocor)) and nicotinic acid, Alirocumab (airocumab) (prant), eloreuptab (evocumolub) (rebalofen (retathatha)), alikumab (plementol) and eloukumab (rexab). Examples of diabetes drugs used in combination include insulin. Examples of the antihypertensive drugs used in combination include: anti-stress agents, calcium channel blockers, ACEI inhibitors, angiotensin II receptor blockers, diuretics, and beta blockers. Examples of known angiotensin II receptor antagonists include: angiotensin I receptor subtype antagonists and angiotensin II receptor subtype antagonists. Suitable angiotensin II receptor antagonists include: losartan (losartan) and valsartan (valsartan). Suitable calcium channel blockers include: for example, verapamil, diltiazemNicardipine, nifedipine, amlodipine, felodipine, nimodipine and bepridil (bepridil). Diuretics include: for example, furosemide (furosemide), chlorothiazide (diuril), amiloride, and hydrochlorothiazide (hydrodiuril). Losartan, candesartan, telmisartan, valsartan, olmesartan (olmes)artan), irbesartan (irbesartan), etc. can be used as the hypotensive agent.
The term "effective amount" as used herein refers to an amount of active ingredient sufficient to confer the desired prophylactic or therapeutic effect in the subject being treated. In one aspect, the amount used to inhibit or alleviate hepatic steatosis, lobular inflammation, hepatocellular ballooning degeneration or NASH-derived HCC ameliorates or reduces one or more symptoms, conditions or progression thereof. In some embodiments, symptoms, pathology, or progression are determined and assessed using methods known in the art that measure various indicators associated with disease progression, such as analyzing liver sections by immunohistochemical staining.
In some embodiments, an effective amount is determined by one of skill in the art, e.g., assessing the route and frequency of administration, body weight, and species of the subject receiving the pharmaceutical compound. In some embodiments, an effective amount of a pharmaceutical compound is formulated with a pharmaceutically acceptable carrier and administered to a subject.
The term "pharmaceutically acceptable" as used herein means that the carrier is known in the art to be compatible with the pharmaceutical compound and also safe for the subject to be treated. In some embodiments, a pharmaceutically acceptable carrier is determined by one of skill in the art, e.g., to assess the solubility of a pharmaceutical compound in the carrier.
Exemplary embodiments of the present invention are further described with reference to the following examples, which are intended to be illustrative in nature and not limiting. In the first study, therapeutically effective amounts of 10 drug compounds were administered. In a second study, 5 pharmaceutical compounds were administered in therapeutically effective amounts.
Example 1
Materials and methods
Male newborn C57BL/6 mice were used. All mice were from pathogen-free 14-day pregnant mice obtained from Japan SLC corporation (Japan SLC, Inc.).
Murine STAM for NASH-HCC according to methods previously described and known in the artTMModel (Takakura et al, Characterization of non-alcoholic steatohepatitis, Anticancer Res, 34 (9): 4849-55 (2014); Fujii et al (2013)). Male mice were NASH induced after 4 weeks (day 28 ± 2) of age by a single subcutaneous injection of 200 μ g streptozotocin (STZ, Sigma, missouri, usa) 2 days after birth and with continuous feeding of a high fat diet ad libitum (CLEA Japan Inc, tokyo, Japan).
Following induction of NASH, mice were randomized into 12 separate study groups of 8 mice (42 ± 2 days) of 6 weeks of age each based on body weight the day before treatment began.
Mice were given once daily oral treatment from 6 weeks of age plus one day (day 43 ± 2, day 1 of treatment) to 9 weeks of age (hereinafter referred to as "treatment period") on a randomized basis. Mice in 11 of 12 study groups (hereinafter "treatment groups") were treated individually with unique pharmaceutical compounds formulated with a pharmaceutically acceptable carrier. Each of the 11 study groups received 1 of 10 test reagents or telmisartan as a positive control. The pharmaceutically acceptable carrier in all groups was 0.5% carboxymethylcellulose (CMC). Mice in the remaining study groups (hereinafter "vehicle control group") were treated with the same pharmaceutically acceptable vehicle alone without the active ingredient. Individual body weights were measured daily during the treatment period. Mice were also monitored daily for survival, clinical signs and behavior.
TABLE 1
Group of
Once daily oral dosage mg/kg
1
Normal (without NASH)
Is free of
2
Telmisartan (+)
10
3
Vehicle (-)
N/A
4
Cepharanthine
12
5
Repirolast
60
6
Ifenprodil hemitartrate
30
7
Broumantan
20
8
Sulfast tosilate
60
9
Actalid
60
10
Clobenzaprine
48
11
Ixodine
1
12
Istradefylline
18
13
Rapadid
60
In each case, 10mL/kg of 0.5% CMC (with or without the active ingredient as shown in table 1) was administered orally from 6 weeks of age to 9 weeks of age until sacrifice. One exception is rapadi, which is administered orally at a dose of 120mg/kg on day 0 and then 60mg/kg daily starting on day 1 of treatment. All mice were subsequently sacrificed at 9 months of age. Blood samples were taken from all mice and livers of each mouse were removed for analysis.
The dose selected for animal studies was determined by: the known maximum daily human dose is divided by the average adult body weight (about 60-70kg) to give the mg/kg dose for the human. This number is then multiplied by 12, which can be converted to mouse doses based on a conventional dosage table. See, Nair and Jacob, J Basic clean Pharm, 2016 3 months-2016 5 months, 7(2): 27-31.
The following measurements and evaluations were performed for each mouse.
Weight: body weights of all mice were measured daily throughout the treatment period.
Blood sample collection and biochemical analysis: blood was collected from all sacrificed mice and frozen for further analysis or transport.
Plasma alanine Aminotransferase (ALT): alanine Aminotransferase (ALT) in plasma of each blood sample was analyzed by FUJI DRI CHEM (Fujifilm), as an indicator of liver function and disease progression.
Liver sample collection, biochemical analysis and histological analysis: livers were removed from all sacrificed mice and frozen for further analysis or transport.
Liver weight: after removal, the livers of all sacrificed mice were weighed in grams.
Histopathology of the liver: the removed liver was fixed in formalin and embedded in paraffin, and then a cross section (4 μm) was prepared.
Evaluation of steatohepatitis: hematoxylin and eosin (H & E) staining of liver cross sections was performed using standard techniques for histological assessment of hepatic steatosis, lobular inflammation and hepatocellular ballooning. The severity of steatohepatitis in each liver cross section is expressed as the NAFLD activity score as follows: in 3 randomly selected H & E stained liver cross sectional areas, 0 (normal), 1-2(NAFLD), 3-4 (critical) or at least 5(NASH), steatosis assessments were performed at 50-fold magnification and inflammation and ballooning degeneration assessments were performed at 200-fold magnification. NAFLD activity score is an unweighted sum of: 1) hepatic steatosis score (0-3); 2) leaflet inflammation score (0-2); 3) hepatocyte ballooning degeneration score (0-2).
Sirius red staining was also performed on the cross sections of the livers of the vehicle group as well as the browman, eltarerin and telmisartan treated groups using standard techniques for histological estimation of the percentage of fibrotic regions. For quantitative analysis of the fibrotic region, brightfield images of sirius red stained sections around the central vein were captured at 200-fold magnification using a digital camera (DFC 295; Leica, germany) and positive regions in 5 fields/section were measured using ImageJ software (national institute of health).
Statistical analysis
The values are arithmetic averages. Two-tailed variance (two-sample unequal variance) student's T-test was used to compare the study group with the positive control group. P values <0.05 were considered statistically significant after Bonferroni post hoc statistical correction analysis (Bonferroni post-hoc correction analysis) on multiple groups (Bonferroni multiple comparison test). In this case, the correction factor is 12, corresponding to the number of study groups. The selected P-value classifications and statistical significance levels, as well as their corresponding classification scores, are shown in table 2, by conventional practice.
Table 2: p value Classification statistical significance level
P value
Statistical description
Classification scoring
P≥0.05
Is not significant
NS
0.01≤P<0.05
Is remarkable in that
*
0.001≤P<0.01 0.01
Is very remarkable
**
P<0.001
Is extremely remarkable
***
Results
Liver function assessment: liver function and disease progression were assessed by plasma ALT as previously described. The results are summarized in FIG. 1.
Evaluation of steatohepatitis: steatohepatitis and disease progression were assessed by liver cross-section H & E-staining as previously described. Representative micrographs of HE-stained liver sections for each of the 12 study groups are shown in figures 2a-2 d.
NAFLD activity scores for each study group consisted of the mean NAFLD activity scores for all mice in each study group. The score was determined based on the steatosis score, the lobular inflammation score, and the hepatocyte balloon-like degeneration score for each animal. The results are summarized in FIGS. 3-6.
Steatohepatitis and disease progression were also assessed by sirius red staining of the liver cross sections, as previously described. Representative micrographs of sirius red stained liver sections of the vehicle, bloomant, eltanopine, and telmisartan study groups are shown in fig. 7. The results are shown in FIG. 8.
Statistically significant reductions in NAFLD activity scores were observed for browman and istradefylline treated groups as well as telmisartan positive controls relative to vehicle controls. A statistically significant reduction in hepatocyte ballooning-like degeneration scores was observed for the browman, isradine and istradefylline treated groups relative to vehicle controls.
The bloomantan and eltanopine groups showed a significant reduction in the fibrotic region (sirius red positive region) compared to the vehicle group. The fibrotic regions tended to be reduced in the telmisartan group compared to the vehicle group. Liver sections of the vehicle group showed increased collagen deposition in the central peripheral region of the liver lobules.
Statistically significant and clinically relevant composite reductions in NAFLD activity scores (especially browman's and eltarepine) indicate that these compounds are useful for the prevention and/or treatment of NASH and its sequelae.
Example 2
Materials and methods
Male newborn C57BL/6 mice were used as described previously. All mice were from pathogen-free 14-day pregnant mice obtained from Japan SLC corporation (Japan SLC, Inc.).
Murine STAM for NASH-HCC according to methods previously described and known in the artTMModel (Takakura et al, Characterization of non-alcoholic steatohepatitis, Anticancer research (Anticancer Res), 34 (9): 4849-55 (2014); Fujii et al (2013)). Male mice were NASH induced after 4 weeks (day 28 ± 2) of age by a single subcutaneous injection of 200 μ g streptozotocin (STZ, Sigma, missouri, usa) 2 days after birth and with continuous feeding of a high fat diet ad libitum (CLEA Japan Inc, tokyo, Japan).
Following induction of NASH, mice were randomized into 7 separate study groups of 8 mice (42 ± 2 days) of 6 weeks of age each based on the body weight of the mice the day before treatment began.
Mice were given once daily oral treatment from 6 weeks plus one day (day 43 ± 2, treatment day 1) to 9 weeks of age, randomized one day after. Mice in 6 of 7 study groups were treated with therapeutic amounts of the pharmaceutical compounds alone, formulated with a pharmaceutically acceptable carrier. Each of these 6 study groups received 1 of 4 test reagents or telmisartan as a positive control. The pharmaceutically acceptable carrier in all groups was 0.5% CMC. Mice in the remaining study groups (vehicle control group) were treated with the same pharmaceutically acceptable vehicle alone without the active ingredient. Individual body weights were measured daily during the treatment period. Mice were also monitored daily for survival, clinical signs and behavior.
TABLE 3
Group of
Once daily oral dosage mg/kg
1
Normal (without NASH)
Is free of
2
Telmisartan (+)
10
3
Vehicle (-)
Is free of
4
Broumantan
20
5
Broumantan
40
6
Bemite
200
7
Ceco Villoro
30
In each case, 10mL/kg of 0.5% CMC (with or without active ingredient as shown in table 3) was administered orally from 6 weeks of age to 9 weeks of age. All mice were subsequently sacrificed at 9 months of age. Blood samples were taken from all mice and livers of each mouse were removed for analysis.
The dose selected for animal studies was determined by: the known maximum daily human dose is divided by the average adult body weight (about 60-70kg) to give the mg/kg dose for the human. This number is then multiplied by 12, which can be converted to mouse doses based on a conventional dosage table. See, Nair and Jacob, J Basic clean Pharm, 2016 3 months-2016 5 months, 7(2): 27-31.
The following measurements and evaluations were performed for each mouse.
Weight: body weights of all mice were measured daily throughout the treatment period.
Blood sample collection and biochemical analysis: blood was collected from all sacrificed mice and frozen for further analysis or transport.
Plasma alanine Aminotransferase (ALT): alanine Aminotransferase (ALT) in plasma of each blood sample was analyzed by FUJI DRI CHEM (FUJI DRI film corporation, japan) as an index of liver function and disease progression.
Liver sample collection, biochemical analysis and histological analysis: livers were removed from all sacrificed mice and frozen for further analysis or transport. After removal, the livers of all sacrificed mice were weighed in grams.
Histopathology of the liver: the removed liver was fixed in formalin and embedded in paraffin, and then a cross section (4 μm) was prepared.
Evaluation of steatohepatitis: hematoxylin and eosin (H & E) staining of liver cross sections was performed using standard techniques for histological assessment of hepatic steatosis, lobular inflammation and hepatocellular ballooning. The severity of steatohepatitis in each liver cross section is expressed as the NAFLD activity score as follows: in 3 randomly selected H & E stained liver cross sectional areas, 0 (normal), 1-2(NAFLD), 3-4 (critical) or at least 5(NASH), steatosis assessments were performed at 50-fold magnification and inflammation and ballooning degeneration assessments were performed at 200-fold magnification. NAFLD activity score is an unweighted sum of: 1) hepatic steatosis score (0-3); 2) leaflet inflammation score (0-2); 3) hepatocyte ballooning degeneration score (0-2).
Sirius red staining was also performed on liver cross sections of the treatment groups using standard techniques for histological evaluation of the percentage of fibrotic regions. For quantitative analysis of the fibrotic region, brightfield images of sirius red stained sections around the central vein were captured at 200-fold magnification using a digital camera (DFC 295; Leica, germany) and positive regions in 5 fields/section were measured using ImageJ software (national institute of health).
Statistical analysis
The values are arithmetic averages. Two-tailed variance (two-sample unequal variance) student's T-test was used to compare the study group with the positive control group. P values <0.05 were considered statistically significant after Bonferroni post hoc statistical correction analysis (Bonferroni post-hoc correction analysis) on multiple groups (Bonferroni multiple comparison test). In this case, the correction factor is 7, corresponding to the number of study groups. The selected P-value classifications and statistical significance levels, as well as their corresponding classification scores, are shown in table 2 (above) by conventional practice.
Results
Liver function assessment: liver function and disease progression were assessed by plasma ALT as previously described. The results are summarized in FIG. 9.
Evaluation of steatohepatitis: steatohepatitis and disease progression were assessed by liver cross-section H & E-staining as previously described. Representative micrographs of HE-stained liver sections for each of the 7 study groups are shown in figures 10a-10 c.
NAFLD activity scores for each study group consisted of the mean NAFLD activity scores for all mice in each study group. The score was determined based on the steatosis score, the lobular inflammation score, and the hepatocyte balloon-like degeneration score for each animal. The results are summarized in FIGS. 11-14.
Statistically significant reductions in NAFLD activity scores were observed for the high browman and cilovalol treatment groups as well as the telmisartan positive control relative to the vehicle control. Statistically significant reductions in steatosis scores were observed for the browman's high treated group as well as the telmisartan positive control relative to the vehicle control.
Clearly, all study groups (including low blomann's, high blomann's, bemidel and ceco verlo groups) showed a significant reduction in fibrotic regions (sirius red positive regions) compared to the vehicle group. The bemidel group showed a statistically significant decrease in the fibrotic area. The fibrotic regions of the telmisartan group also tended to be reduced compared to the vehicle group.
Statistically significant and clinically relevant compound reductions in NAFLD activity scores (particularly high browman's, bemidel and cicovitine) indicate that these compounds are useful in the prevention and/or treatment of NASH and its sequelae.
Conclusion
In summary, administration of a number of pharmaceutical compound test agents approved for other pathologies (especially browman's, eltanopine and cicovitine) show improvement in reducing NAFLD activity scores, hepatic steatosis, lobular inflammation and hepatocyte ballooning degeneration, and are useful in the prevention and/or treatment of NASH. In particular, the group treated with 40mg/kg of bloomant and 18mg/kg of itratheophylline, followed by 30mg/kg of cilovallor, improved better.
Oral telmisartan, 10mg/kg, also showed improvement in NAFLD activity score, hepatic steatosis, lobular inflammation and hepatocellular ballooning compared to the vehicle group.
Throughout the description, specific details are set forth in order to provide a more thorough understanding to persons skilled in the art. However, well known elements may not have been shown or described in detail to avoid unnecessarily obscuring the disclosure. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense.
While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all modifications, permutations, additions and sub-combinations as are generally consistent with the broadest interpretation of the present specification.