GHRH or analogues thereof for the treatment of liver disease

文档序号：1835801 发布日期：2021-11-12 浏览：8次中文

阅读说明：本技术 用于治疗肝脏疾病的ghrh或其类似物 (GHRH or analogues thereof for the treatment of liver disease ) 是由 S·K·格林斯彭于 2020-03-27 设计创作，主要内容包括：本申请涉及用于利用GHRH分子,例如反式-3-己烯酰基-GHRH-((1-44))-NH-(2)或其药学上可接受的盐,预防或治疗非酒精性脂肪肝(NAFL)、非酒精性脂肪性肝炎(NASH)和/或肝纤维化,减缓其进展和/或降低受试者,例如HIV感染受试者的肝癌风险的新方法。所述受试者可能具有特定的病理特征,例如肝纤维化、至少约10％的肝脏脂肪分数(HFF)、至少约30U/L的血清丙氨酸转氨酶(ALT)水平和/或分数至少为4或5的NAFLD活动性评分(NAS)。(The present application relates to methods for utilizing GHRH molecules, such as trans-3-hexenoyl-GHRH (1‑44) ‑NH 2 Or a pharmaceutically acceptable salt thereof, for preventing or treating non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH) and/or liver fibrosisNovel methods of slowing their progression and/or reducing the risk of liver cancer in a subject, e.g., an HIV-infected subject. The subject may have a particular pathological characteristic, such as liver fibrosis, a liver fat fraction (HFF) of at least about 10%, a serum alanine Aminotransferase (ALT) level of at least about 30U/L, and/or a NAFLD Activity Score (NAS) of at least 4 or 5.)

1. A method for preventing or treating non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH) in a subject in need thereof, comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has: (i) by proton magnetic resonance spectroscopy (¹H MRS) a liver fat fraction (HFF) of at least about 10%; (ii) a serum alanine Aminotransferase (ALT) level of at least about 30U/L; (iii) a NAFLD Activity Score (NAS) of at least 3, 4 or 5 as measured by a NAS clinical research network (NAS CRN) scoring system; (iv) liver fibrosis; or (v) any combination of (i) to (iv).

2. The method of claim 1, wherein the subject has an HFF of at least about 10%.

3. The method of claim 1, wherein the subject has at least about 15% or at least about 20% HFF.

4. A method according to one of claims 1 to 3, wherein the subject's HFF is reduced by at least 4% (absolute reduction).

5. The method of any one of claims 1-4, wherein the subject has a serum ALT level of at least about 30U/L.

6. The method of any one of claims 1-4, wherein the subject has a serum ALT level of at least about 35U/L.

7. The method of any one of claims 1 to 6, wherein the subject has a NAS of at least 6.

8. The method of claim 7, wherein the subject has a NAS of at least 7.

9. The method of any one of claims 1 to 8, wherein the subject has liver fibrosis.

10. The method of any one of claims 1 to 9, wherein the subject has a Human Immunodeficiency Virus (HIV) infection.

11. The method according to any one of claims 1 to 10, wherein the subject has a Body Mass Index (BMI) of at least about 25.

12. The method of claim 11, wherein the subject has a BMI of at least about 30.

13. A method according to any one of claims 1-12, wherein the method comprises administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

14. The method of claim 13, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

15. The method according to any one of claims 1 to 14, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

16. The method of claim 15, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or itThe pharmaceutically acceptable salt is administered in a daily dose of about 1mg to about 2 mg.

17. A method according to any one of claims 1 to 16, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

18. A method according to any one of claims 1 to 17, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

19. The method of claim 18, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

20. The method of claim 18, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

21. Trans-3-hexenoyl-GHRH for use in preventing or treating non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH) in a subject_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has: (i) by proton magnetic resonance spectroscopy (¹H MRS) a liver fat fraction (HFF) of at least about 10%; (ii) a serum alanine Aminotransferase (ALT) level of at least about 30U/L; (iii) a NAFLD Activity Score (NAS) of at least 4 or 5 as measured by a NAS clinical research network (NAS CRN) scoring system; or (iv) any combination of (i) to (iii).

22. trans-3-hexenoyl-GHRH for use according to claim 21_(1-44)-NH₂Or it may be pharmaceuticallyThe received salt, wherein the HFF of the subject is at least about 10%.

23. trans-3-hexenoyl-GHRH for use according to claim 21_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 15% or at least about 20%.

24. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 23_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is reduced by at least 4% (absolute reduction).

25. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 24_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 30U/L.

26. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 24_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 35U/L.

27. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 26_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAS of at least 6.

28. trans-3-hexenoyl-GHRH for use according to claim 27_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAS of at least 7.

29. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 28_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has liver fibrosis.

30. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 29_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Human Immunodeficiency (HIV) infection.

31. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 31_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Body Mass Index (BMI) of at least about 25.

32. trans-3-hexenoyl-GHRH for use according to claim 31_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a BMI of at least about 30.

33. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 33_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

34. trans-3-hexenoyl-GHRH for use according to claim 33_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

35. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 34_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

36. trans-3-hexenoyl-GHRH for use according to claim 37_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

37. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 36_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

38. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 37_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

39. trans-3-hexenoyl-GHRH for use according to claim 38_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

40. trans-3-hexenoyl-GHRH for use according to claim 38_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

41. A method for (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject, comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof.

42. The method of claim 41, wherein the subject has a fibrosis score of at least 1C before treatment.

43. The method of claim 41, wherein the subject has a fibrosis score of at least 2 before treatment.

44. A method according to any one of claims 41 to 43, wherein the subject has an HFF of at least about 10%.

45. The method of claim 44, wherein the HFF in the subject is at least about 15%.

46. The method of claim 45, wherein the HFF in the subject is at least about 20%.

47. The method of any one of claims 41-46, wherein the subject has a serum ALT level of at least about 30U/L.

48. The method of any one of claims 41-46, wherein the subject has a serum ALT level of at least about 35U/L.

49. The method of any one of claims 41 to 48, wherein the subject has a NAFLD Activity Score (NAS) of at least 2 as measured by a NAS clinical research network (NAS CRN) scoring system.

50. The method of claim 49, wherein the subject has a NAS of at least 3.

51. The method of any one of claims 41-50, wherein the subject has a Human Immunodeficiency Virus (HIV) infection.

52. The method according to any one of claims 41-51, wherein the subject has a Body Mass Index (BMI) of at least about 25.

53. The method of claim 52, wherein the subject has a BMI of at least about 30.

54. A method according to one of claims 41-53, wherein the method comprises administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

55. The method of claim 54, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

56. A method according to any one of claims 41 to 55, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

57. The method of claim 56, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

58. A method according to any one of claims 41 to 57, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

59. A method according to any one of claims 41 to 58, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

60. The method of claim 59, wherein in said pharmaceutical compositionThe trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

61. The method of claim 59, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

62. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject.

63. Trans-3-hexenoyl-GHRH used according to claim 62_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a fibrosis score of at least 1C prior to treatment.

64. Trans-3-hexenoyl-GHRH used according to claim 62_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a fibrosis score of at least 2 prior to treatment.

65. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 64_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 10%.

66. Trans-3-hexenoyl-GHRH used according to claim 65_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 15%.

67. Trans-3-hexenoyl-GHRH used according to claim 65_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein of the subjectThe HFF is at least about 20%.

68. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 67_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 30U/L.

69. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 67_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 35U/L.

70. Trans-3-hexenoyl-GHRH used according to any one of claims 62 to 69_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAFLD Activity Score (NAS) of at least 2 as measured by a NAS clinical research network (NAS CRN) scoring system.

71. trans-3-hexenoyl-GHRH for use according to claim 70_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAS of at least 3.

72. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 71_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Human Immunodeficiency (HIV) infection.

73. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 72_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Body Mass Index (BMI) of at least about 25.

74. trans-3-hexenoyl-GHRH for use according to claim 73_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a BMI of at least about 30.

75. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 74_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

76. trans-3-hexenoyl-GHRH for use according to claim 75_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

77. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 76_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

78. trans-3-hexenoyl-GHRH for use according to claim 77_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

79. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 78_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

80. trans-3-hexenoyl-GHRH for use according to any one of claims 62 to 79_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof in a packageA pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

81. trans-3-hexenoyl-GHRH for use according to claim 80_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

82. trans-3-hexenoyl-GHRH for use according to claim 80_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

83. The method of any one of claims 1 to 20 and 41 to 61, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

84. The method of claim 83, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

85. trans-3-hexenoyl-GHRH for use according to any one of claims 21 to 40 and claims 62 to 82_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

86. trans-3-hexenoyl-GHRH for use according to claim 85_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

87. A method for reducing the risk of liver cancer in a subject with non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH), comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof.

88. A method according to claim 87, wherein the method comprises administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

89. The method of claim 88, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

90. The method of any one of claims 87 to 89 wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

91. The method of claim 90, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

92. The method of any one of claims 87 to 91, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

93. The method of any one of claims 87 to 92, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a composition comprising at least one pharmaceutically acceptable saltA pharmaceutical composition of acceptable excipients.

94. The method of claim 93, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

95. The method of claim 93, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

96. The method of any one of claims 87 to 95, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

97. The method of claim 96, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

98. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in reducing the risk of liver cancer in a subject having non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH).

99. trans-3-hexenoyl-GHRH for use according to claim 98_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

100. trans-3-hexenoyl-GHRH for use according to claim 99_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

101. trans-3-hexenoyl-GHRH for use according to any one of claims 98 to 100_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

102. trans-3-hexenoyl-GHRH for use according to claim 101_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

103. trans-3-hexenoyl-GHRH for use according to any one of claims 98 to 102_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

104. trans-3-hexenoyl-GHRH for use according to any one of claims 98 to 103_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

105. trans-3-hexenoyl-GHRH for use according to claim 104_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

106. Use according to claim 105trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

107. trans-3-hexenoyl-GHRH for use according to any one of claims 98 to 106_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

108. trans-3-hexenoyl-GHRH for use according to claim 107_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

Technical Field

The present disclosure relates generally to the treatment of liver diseases, such as non-alcoholic fatty liver disease (NAFL), non-alcoholic steatohepatitis (NASH), and liver fibrosis.

Background

NAFL is defined as the excess storage of triglycerides in hepatocytes (steatosis) and is generally characterized by the resulting inflammation, cellular swelling and damage, and fibrosis. Significant changes in this area have led to NASH. Nonalcoholic fatty liver disease (NAFLD) may develop into fibrosis and eventually cirrhosis, an increasingly important cause of end-stage liver disease in the general population, and it has also been studied among HIV-infected persons (PLWH; Rockstroh JK., "Current HIV/AIDS report (Current HIV/AIDS Rep)" 2017; 14: 47-53; Vodkin I et al, "nutritional pharmacology and therapeutics (animal Pharma Ther)" 2015; 41: 368-78). NAFL/NASH on HIVThe prevalence is higher in patients and tends to progress faster than the general population. NAFLD is at CD4, compared to many HIV-associated complications that worsen with increasing severity of HIV disease⁺T cell counts are higher and among the obese HIV patients more likely to be more common and are associated with central obesity (Guaraldi G et al, clinical infectious disease (Clin infectious disease) 2008; 47: 250-7; van der Poorten D et al, Hepatology (2008; 48: 449-57; Maurice JB et al, AIDS 2017; 31: 1621-32). In PLWH, obesity, abdominal fat accumulation and increased visceral fat are common, even with newer antiretroviral drugs (Lake JE et al, clinical infectious diseases 2017; 64: 1422-9). Despite vitamin E (Sanyal AJ., N Engl J Med 2010; 362: 1675-85; Lavine JE et al, J.Am. (JAMA) 2011; 305: 1659-68; Sato K et al, Nutrition 2015; 31:923-30) and pioglitazone⁷Has been shown to improve the histological features of NASH in the general population, but there is no proven drug therapy for NAFLD or NASH in HIV infected persons. A product, aramchol, an oral stearoyl-CoA-desaturase 1 inhibitor known to reduce liver fat content in patients with primary NAFLD, was evaluated in patients with HIV-associated NAFLD without significant effect on this particular patient population (Ajmera et al, hepatology, 23 months 4.2019, doi: 10.1002/hep.30674) [ electronic Pre-printing publication])。

Liver fibrosis is caused by chronic damage to the liver and accumulation of extracellular matrix (ECM) proteins, which are characteristic of most types of chronic liver disease. Advanced liver fibrosis can lead to cirrhosis, liver failure and portal hypertension, often requiring liver transplantation. There is currently no effective treatment that can effectively target liver fibrosis associated with NAFL/NASH.

There is therefore a need for new therapies for the treatment of NAFLD, NASH and/or liver fibrosis, particularly in HIV infected patients.

The present specification makes reference to a number of documents, the contents of which are incorporated herein by reference in their entirety.

Disclosure of Invention

The present disclosure relates generally to the treatment of liver diseases, such as non-alcoholic fatty liver disease (NAFLD). NAFLD refers to fatty liver not associated with alcohol consumption, further divided into non-alcoholic fatty liver disease (NAFL) with or without fibrosis and non-alcoholic steatohepatitis (NASH), and in one aspect relates to the use of Growth Hormone (GH) secretagogues, and more particularly to Growth Hormone Releasing Hormone (GHRH) or analogues thereof for the treatment (including prevention and/or treatment) of such liver diseases or associated conditions.

In various aspects and embodiments, the present disclosure provides the following items:

1. a method for preventing or treating non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH) in a subject in need thereof, comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has: (i) by proton magnetic resonance spectroscopy (¹H MRS) (or any other suitable method) a liver fat fraction (HFF) of at least about 5% or 10%; (ii) a serum alanine Aminotransferase (ALT) level of at least about 25 or 30U/L; (iii) a NAFLD Activity Score (NAS) of at least 3, 4 or 5 as measured by a NAS clinical research network (NAS CRN) scoring system; (iv) liver fibrosis; or (iv) any combination of (i) to (iv).

2. The method of clause 1, wherein the subject has an HFF of at least about 5% or 10%.

3. The method of clause 1, wherein the HFF of the subject is at least about 15%.

4. The method of clause 1, wherein the HFF of the subject is at least about 20%.

5. The method of any of clauses 1-4, wherein the subject has a serum ALT level of at least about 25 or 30U/L.

6. The method of any of clauses 1-4, wherein the subject has a serum ALT level of at least about 35U/L.

7. The method of any one of clauses 1 to 6, wherein the subject has a NAS of at least 6.

8. The method of clause 7, wherein the subject has a NAS of at least 7.

9. The method of any one of items 1 to 8, wherein the subject has liver fibrosis, e.g., stage 1A liver fibrosis or more severe liver fibrosis, preferably stage 1C or 2 liver fibrosis or more severe liver fibrosis.

10. The method of any one of items 1 to 9, wherein the subject has a Human Immunodeficiency Virus (HIV) infection.

11. The method of any of clauses 1-10, wherein the subject has a Body Mass Index (BMI) of at least about 25.

12. The method of clause 11, wherein the subject has a BMI of at least about 30.

13. The method according to any one of items 1 to 12, wherein the method comprises administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

14. The method of clause 13, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

15. The method of any one of clauses 1 to 14, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

16. The method of clause 15, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

17. The method of any one of clauses 1 to 16, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

18. The method of any one of clauses 1 to 17, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or it may be pharmaceuticallyThe acceptable salt is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

19. The method of clause 18, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

20. The method of clause 18, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

21. Trans-3-hexenoyl-GHRH for use in preventing or treating non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH) in a subject_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has: (i) by proton magnetic resonance spectroscopy (¹H MRS) a liver fat fraction (HFF) of at least about 5% or 10%; (ii) a serum alanine Aminotransferase (ALT) level of at least about 25 or 30U/L; (iii) a NAFLD Activity Score (NAS) of at least 5 as measured by a NAS clinical research network (NAS CRN) scoring system; or (iv) any combination of (i) to (iii).

22. Trans-3-hexenoyl-GHRH used according to item 21_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 5% or 10%.

23. Trans-3-hexenoyl-GHRH used according to item 21_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 15%.

24. Trans-3-hexenoyl-GHRH used according to item 21_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 20%.

25. trans-3-hexenoyl-GHRH for use according to any of items 21 to 24_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 25 or 30U/L.

26. According to items 21 to 24Trans-3-hexenoyl-GHRH used in either case_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 35U/L.

27. trans-3-hexenoyl-GHRH for use according to any of items 21 to 26_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAS of at least 6.

28. Trans-3-hexenoyl-GHRH used according to item 27_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAS of at least 7.

29. trans-3-hexenoyl-GHRH for use according to any of items 21 to 28_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has liver fibrosis, for example stage 1A liver fibrosis or more severe liver fibrosis, preferably stage 1C or 2 liver fibrosis or more severe liver fibrosis.

30. trans-3-hexenoyl-GHRH for use according to any of items 21 to 29_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Human Immunodeficiency (HIV) infection.

31. trans-3-hexenoyl-GHRH for use according to any of items 21 to 31_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Body Mass Index (BMI) of at least about 25.

32. Trans-3-hexenoyl-GHRH used according to item 31_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a BMI of at least about 30.

33. trans-3-hexenoyl-GHRH for use according to any of items 21 to 33_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

34. Trans-3-hexenoyl-GHRH used according to item 33_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

35. trans-3-hexenoyl-GHRH for use according to any of items 21 to 34_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

36. Trans-3-hexenoyl-GHRH used according to item 37_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

37. trans-3-hexenoyl-GHRH for use according to any of items 21 to 36_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

38. trans-3-hexenoyl-GHRH for use according to any of items 21 to 37_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

39. Trans-3-hexenoyl-GHRH used according to item 38_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

40. Trans-3-hexenoyl-GHRH used according to item 38_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

41. A method for (i) preventing or slowing down in a subjectA method of progression or progression of liver fibrosis or (ii) reducing liver fibrosis, comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof.

42. The method of clause 41, wherein the subject has a fibrosis score of at least 1C prior to treatment.

43. The method of clause 41, wherein the subject has a fibrosis score of at least 2 prior to treatment.

44. The method of any one of clauses 41-43, wherein the HFF of the subject is at least about 10%.

45. The method of clause 44, wherein the HFF of the subject is at least about 15%.

46. The method of clause 45, wherein the HFF of the subject is at least about 20%.

47. The method of any of clauses 41-46, wherein the subject has a serum ALT level of at least about 30U/L.

48. The method of any of clauses 41-46, wherein the subject has a serum ALT level of at least about 35U/L.

49. The method of any of items 41-48, wherein the subject has a NAFLD Activity Score (NAS) of at least 2 as measured by a NAS clinical research network (NAS CRN) scoring system.

50. The method of clause 49, wherein the subject has a NAS of at least 3.

51. The method of any one of items 41 to 50, wherein the subject has a Human Immunodeficiency Virus (HIV) infection.

52. The method of any of clauses 41-51, wherein the subject has a Body Mass Index (BMI) of at least about 25.

53. The method of clause 52, wherein the subject has a BMI of at least about 30.

54. The method of any of clauses 41-53, wherein the method comprises administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

55. The method of clause 54, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

56. The method of any of clauses 41 to 55, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

57. The method of clause 56, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

58. The method of any of clauses 41 to 57, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

59. The method of any of clauses 41 to 58, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

60. The method of clause 59, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

61. The method of clause 59, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

63. Trans-3-hexenoyl-GHRH used according to item 62_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, whereinThe subject had a fibrosis score of at least 1C prior to treatment.

64. Trans-3-hexenoyl-GHRH used according to item 62_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a fibrosis score of at least 2 prior to treatment.

65. trans-3-hexenoyl-GHRH for use according to any of items 62 to 64_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 10%.

66. Trans-3-hexenoyl-GHRH used according to item 65_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 15%.

67. Trans-3-hexenoyl-GHRH used according to item 65_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the HFF of the subject is at least about 20%.

68. trans-3-hexenoyl-GHRH for use according to any of items 62 to 67_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 30U/L.

69. trans-3-hexenoyl-GHRH for use according to any of items 62 to 67_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a serum ALT level of at least about 35U/L.

70. trans-3-hexenoyl-GHRH for use according to any of items 62 to 69_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAFLD Activity Score (NAS) of at least 2 as measured by a NAS clinical research network (NAS CRN) scoring system.

71. Trans-3-hexenoyl-GHRH used according to item 70_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a NAS of at least 3.

72. trans-3-hexenoyl-GHRH for use according to any of items 62 to 71_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof,wherein the subject has a Human Immunodeficiency (HIV) infection.

73. trans-3-hexenoyl-GHRH for use according to any of items 62 to 72_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a Body Mass Index (BMI) of at least about 25.

74. Trans-3-hexenoyl-GHRH used according to item 73_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has a BMI of at least about 30.

75. trans-3-hexenoyl-GHRH for use according to any of items 62 to 74_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

76. Trans-3-hexenoyl-GHRH used according to item 75_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

77. trans-3-hexenoyl-GHRH for use according to any of items 62 to 76_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

78. Trans-3-hexenoyl-GHRH used according to item 77_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

79. trans-3-hexenoyl-GHRH for use according to any of items 62 to 78_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

80. trans-3-hexenoyl-GHRH for use according to any of items 62 to 79_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

81. Trans-3-hexenoyl-GHRH used according to item 80_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

82. Trans-3-hexenoyl-GHRH used according to item 80_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

83. The method of any one of items 1 to 20 and items 41 to 61, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

84. The method of clause 83, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

85. trans-3-hexenoyl-GHRH for use according to any one of items 21 to 40 and items 62 to 82_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

86. Trans-3-hexenoyl-GHRH used according to item 85_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

87. A composition for reducing the risk of non-alcoholic fatty liver disease (NAFL) or non-alcoholic fatty liver disease (NAFL)A method of treating a subject having non-alcoholic steatohepatitis (NASH) at risk for developing liver cancer, comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof.

88. The method of clause 87, wherein the method comprises administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

89. The method of clause 88, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

90. The method of any one of clauses 87 to 89, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

91. The method of clause 90, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

92. The method of any one of clauses 87 to 91, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

93. The method of any one of clauses 87 to 92, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

94. The method of clause 93, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

95. The method of clause 93, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

96. According to itemItem 87 to 95, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

97. The method of clause 96, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

99. Trans-3-hexenoyl-GHRH used according to item 98_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

100. Trans-3-hexenoyl-GHRH used according to item 99_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

101. trans-3-hexenoyl-GHRH for use according to any of items 98 to 100_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

102. Trans-3-hexenoyl-GHRH used according to item 101_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

103. trans-3-hexenoyl-GHRH for use according to any of items 98 to 102_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereofAdministered by subcutaneous injection.

104. trans-3-hexenoyl-GHRH for use according to any of items 98 to 103_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

105. Trans-3-hexenoyl-GHRH used according to item 104_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

106. Trans-3-hexenoyl-GHRH used according to item 105_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein said trans-3-hexenoyl-GHRH in said pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

107. trans-3-hexenoyl-GHRH for use according to any of items 98 to 106_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

108. Trans-3-hexenoyl-GHRH used according to item 107_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

109. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for preventing or treating non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH) in a subject, wherein the subject has: (i) by proton magnetic resonance spectroscopy (¹H MRS) a liver fat fraction (HFF) of at least about 5% or 10%; (ii) at least about 25 or 30Serum alanine transaminase (ALT) levels of U/L; (iii) a NAFLD Activity Score (NAS) of at least 5 as measured by a NAS clinical research network (NAS CRN) scoring system; or (iv) any combination of (i) to (iii).

110. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in preventing or treating non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH) in a subject, wherein the subject has: (i) by proton magnetic resonance spectroscopy (¹H MRS) a liver fat fraction (HFF) of at least about 5% or 10%; (ii) a serum alanine Aminotransferase (ALT) level of at least about 25 or 30U/L; (iii) a NAFLD Activity Score (NAS) of at least 5 as measured by a NAS clinical research network (NAS CRN) scoring system; or (iv) any combination of (i) to (iii).

111. The use of clauses 109 or 110, wherein the subject has at least about 5% or 10% HFF.

112. The use of clauses 109 or 110, wherein the subject has an HFF of at least about 15%.

113. The use of clauses 109 or 110, wherein the subject has an HFF of at least about 20%.

114. The use of any of clauses 109 to 113, wherein the subject has a serum ALT level of at least about 25 or 30U/L.

115. The use of any of clauses 109 to 114, wherein the subject has a serum ALT level of at least about 35U/L.

116. The use of any one of clauses 109 to 115, wherein the subject has a NAS of at least 6.

117. The use of clause 116, wherein the subject has a NAS of at least 7.

118. The use according to any of items 109 to 117, wherein the subject has liver fibrosis, e.g. stage 1A liver fibrosis or more severe liver fibrosis, preferably stage 1C or 2 liver fibrosis or more severe liver fibrosis.

119. The use of any one of items 109-118, wherein the subject has a Human Immunodeficiency (HIV) infection.

120. The use of any of clauses 109-119, wherein the subject has a Body Mass Index (BMI) of at least about 25.

121. The use of clause 120, wherein the subject has a BMI of at least about 30.

122. The use according to any one of items 109 to 121, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

123. The use of clause 122, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

124. The use of any one of items 109 to 123, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

125. The use of clause 124, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

126. The use of any one of items 109 to 125, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

127. The use of any one of items 109 to 126, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

128. The use of clause 127, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

129. The use of clauses 127 or 128, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or pharmaceutically thereofAcceptable salt concentrations are from about 1mg/mL to about 8 mg/mL.

130. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject.

131. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject.

132. The use of clauses 130 or 131, wherein the subject has a fibrosis score of at least 1C before treatment.

133. The use of clauses 130 or 131, wherein the subject has a fibrosis score of at least 2 prior to treatment.

134. The use of any one of clauses 130 to 133, wherein the subject has an HFF of at least about 10%.

135. The use of clause 134, wherein the subject has an HFF of at least about 15%.

136. The use of clause 135, wherein the subject has an HFF of at least about 20%.

137. The use of any of clauses 130 to 136, wherein the subject has a serum ALT level of at least about 30U/L.

138. The use of any of clauses 130 to 137, wherein the subject has a serum ALT level of at least about 35U/L.

139. The use of any of items 130 to 138, wherein the subject has a NAFLD Activity Score (NAS) of at least 2 as measured by a NAS clinical research network (NAS CRN) scoring system.

140. The use of clause 139, wherein the subject has a NAS of at least 3.

141. The use of any of clauses 130 to 140, wherein the subject has a Human Immunodeficiency (HIV) infection.

142. The use of any of clauses 130-141, wherein the subject has a Body Mass Index (BMI) of at least about 25.

143. The use of clause 142, wherein the subject has a BMI of at least about 30.

144. The use of any of clauses 130 to 143, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

145. The use of clause 144, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

146. The use of any of clauses 130 to 145, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

147. The use of clause 146, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

148. The use of any of clauses 130 to 147, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

149. The use of any of clauses 130 to 148, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

150. The use of clause 149, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

151. The use of clauses 149 or 150, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

152. Trans form-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in reducing the risk of liver cancer in a subject having non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH).

153. trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for reducing the risk of liver cancer in a subject having non-alcoholic fatty liver disease (NAFL) or non-alcoholic steatohepatitis (NASH).

154. The use of clauses 152 or 153, wherein trans-3-hexenoyl-GHRH is used_(1-44)-NH₂A pharmaceutically acceptable salt of (a).

155. The use of clause 154, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

156. The use of any one of clauses 152 to 155, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4 mg.

157. The use of clause 156, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 2 mg.

158. The use of any one of items 152 to 157, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered by subcutaneous injection.

159. The use of any one of items 152 to 158, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in a pharmaceutical composition comprising at least one pharmaceutically acceptable excipient.

160. The use of clause 159, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 10 mg/mL.

161. The use of clauses 159 or 160, wherein the trans-3-hexenoyl-GHRH in the pharmaceutical composition_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, at a concentration of about 1mg/mL to about 8 mg/mL.

162. The use of any one of items 109 to 161, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 3, 6 or 9 months.

163. The use of clause 162, wherein the trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for at least 12 months.

Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

Drawings

In the drawings:

figure 1 is a diagram showing participant flow and reasons for excluding patients from the study.

Figure 2 is a table showing the effect of temorelin (tesamorelin) on liver fat, metabolism and immunological indices.

Fig. 3A and 3B are graphs depicting the change in absolute (fig. 3A) and relative (fig. 3B) liver fat content between baseline and 12 months, with p-values for comparison of changes between groups using the t-test.

Fig. 3C is a graph depicting the percent resolution of steatosis, defined as a 12 month liver fat fraction < 5%, with a P value for Pearson Chi-Square.

FIG. 3D is a graph depicting the change between baseline and 12 months for alanine Aminotransferase (ALT) at ALT ≧ 30U/L at baseline, with p-values for comparison of change between groups by t-test.

Fig. 4A is a graph depicting the percentage of patients with fibrosis progression at 12 months, with P values for pearson chi-square.

Figure 4B is a graph depicting the relationship between fibrosis change and NAS score change at 12 months, with P values for ANOVA.

FIG. 5 shows temsirolimus (trans-3-hexenoyl-GHRH)_(1-44)-NH₂(ii) a SEQ ID NO: 1) the structure of (1).

Detailed Description

The use of the terms "a" and "an" and "the" and similar referents in the context of describing subject matter (especially in the context of the following claims) is to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

The terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within these ranges are also incorporated into the specification as if they were individually recited herein.

Similarly, the general chemical structures herein having various substituents and the various groups recited for those substituents are intended as shorthand methods for referring individually to each molecule obtained by the combination of any group for any substituent. Each individual molecule is incorporated into the specification as if it were individually recited herein. Moreover, all subsets of the molecules within these general chemical structures are also incorporated into the specification as if they were individually recited herein.

All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language ("e.g.," such as ") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed.

In this context, the term "about" has its ordinary meaning. The term "about" is used to indicate that a value includes the inherent variation of error of the device or method used to determine the value, or includes values close to the stated value, for example, values within 10% of the stated value (or range of values).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

In the studies described herein, temsirolimus (trans-3-hexenoyl-GHRH) was shown_(1-44)-NH₂Acetate) significantly reduces and/or normalizes liver fat content and prevents liver fibrosis progression in HIV-infected subjects with NAFL/NASH with or without fibrosis. The results provide evidence that the beneficial effects of tesamorelin treatment are more pronounced in subjects with a higher NAFLD Activity Score (NAS) prior to treatment, i.e., subjects with more advanced or severe disease. Given that the fibrosis stage is the strongest predictor of mortality in NAFLD patients, and that a large proportion of HIV patients with NAFLD show fibrosis progression over 1 year, the higher NAS patients shown in this study have clinical significance in preventing liver fibrosis by treatment with temorelin. Furthermore, temorelin surprisingly has a more pronounced effect on reducing liver fat compared to visceral fat. Gene expression analysis showed that administration of tesamorelin was associated with upregulation of genes associated with oxidative phosphorylation and genes associated with a good prognosis of hepatocellular carcinoma (HCC) and downregulation of genes associated with liver inflammation, tissue repair, cell renewal and a poor prognosis of HCC.

In one aspect, the present disclosure provides a method for treating nonalcoholic fatty liver (NAFL) or nonalcoholic steatohepatitis (NASH) with or without fibrosis in a subject in need thereof, comprising: administering to the subject an effective amount of a GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, wherein the subject has: (i) toLiver fat fraction (HFF) as low as 5%, 6%, 7%, 8%, 9% or 10% (e.g. by proton magnetic resonance spectroscopy: (HFF))¹H MRS) assay); (ii) (iv) a serum alanine Aminotransferase (ALT) level of at least 25, 26, 27, 28, 29, or 30U/L; (iii) a NAS with a score of at least 1, 2, 3, 4, or 5, preferably at least 3, 4, or 5, as measured by a NAS clinical research network (NAS CRN) scoring system; (iv) liver fibrosis, such as stage 1A, 1B, 1C, 2, 3 liver fibrosis or more severe fibrosis; or (v) any combination of (i) to (iv).

In another aspect, the present disclosure provides a method for treating liver fat fraction (HFF) of at least about 5% or 10% (e.g., by proton magnetic resonance spectroscopy: (¹H MRS) in a subject, the method comprising: administering to the subject an effective amount of a GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is at least about 5% or 10% in treating HFF (e.g., by¹H MRS assay) in NAFL or NASH. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is used in the preparation of a medicament for treating HFF in an amount of at least about 5% or 10% (e.g., by¹H MRS assay) in NAFL or NASH in a subject. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, in treating HFF at least about 5% or 10% (e.g., by proton magnetic resonance spectroscopy: (¹H MRS) in NAFL or NASH in a subject.

In another aspect, the present disclosure provides a method for treating NAFL or NASH in a subject having a serum ALT level of at least about 25 or 30U/L, the method comprising: administering to the subject an effective amount of a GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexeneacyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in treating NAFL or NASH in a subject having a serum ALT level of at least about 25 or 30U/L. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for treating NAFL or NASH in a subject having a serum ALT level of at least about 25 or 30U/L. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in treating NAFL or NASH in a subject having a serum ALT level of at least about 25 or 30U/L.

In another aspect, the present disclosure provides a method for (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject having NAFL or NASH with or without fibrosis, the method comprising: administering to the subject an effective amount of a GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject having NAFL or NASH with or without fibrosis. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for (i) preventing or slowing the development or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject having NAFL or NASH with or without fibrosis. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutical preparation thereofThe above acceptable salt for use in (i) preventing or slowing the progression or progression of liver fibrosis or (ii) reducing liver fibrosis in a subject having NAFL or NASH with or without fibrosis.

In another aspect, the present disclosure provides a method for maintaining or reducing NAS in a subject having NAFL or NASH and at least 1, 2, 3, 4, or 5, preferably at least 3, 4, or 5, NAS as measured by a NAS CRN scoring system, the method comprising: administering to the subject an effective amount of a GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in maintaining or reducing the NAS in a subject having NAFL or NASH and having a NAS of at least 1, 2, 3, 4 or 5, preferably at least 3, 4 or 5 as measured by the NAS CRN scoring system. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for maintaining or reducing NAS in a subject having NAFL or NASH and having a NAS of at least 1, 2, 3, 4 or 5, preferably at least 3, 4 or 5 as measured by the NAS CRN scoring system. The present disclosure also provides GHRH molecules or pharmaceutically acceptable salts thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in maintaining or reducing NAS in a subject having NAFL or NASH with or without fibrosis and having a NAS of at least 1, 2, 3, 4 or 5, preferably at least 3, 4 or 5, as measured by the NAS CRN scoring system.

In another aspect, the present disclosure provides a method for reducing the risk or likelihood of liver cancer in a subject with NAFL or NASH, the method comprising: administering to the subject an effective amount of trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof. The disclosure also provides trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, use thereofTo reduce the risk or likelihood of liver cancer in a subject suffering from NAFL or NASH. The disclosure also provides trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, for use in, or in the manufacture of a medicament for, reducing the risk or likelihood of developing liver cancer in a subject suffering from NAFL or NASH. In one embodiment, the liver cancer is hepatocellular carcinoma (HCC).

In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 5%, 6%, 7%, 8%, 9%, or 10%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 11%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 12%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 13%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 14%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 15%. In one embodiment, the subject is treated, for example, by¹The HFF measured by HMRS is at least about 16%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 17%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 18%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 19%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 20%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 21%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 22%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 23%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 24%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 25%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 30%. In one embodiment, the subject is treated, for example, by¹H MRS has an HFF of at least about 35%.

In one embodiment, the method or use comprises identification, for example by¹H MRS is at least about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, or 35% of subjects.

In another embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least about 35% (relative reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least about 40% (relative reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least about 45% (relative reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least about 50% (relative reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least two-fold. In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or itAdministration of the pharmaceutically acceptable salt reduces HFF by at least three-fold. In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least four-fold. In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least five-fold.

In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least 4% or 5% (absolute reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least 6% (absolute reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least 7% (absolute reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least 8% (absolute reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least 9% (absolute reduction). In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces HFF by at least 10% (absolute reduction).

In one embodiment, the subject has a NAS of at least 1 as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of at least 2 as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of at least 3 as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of at least 4 as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of at least 5 as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of at least 6, e.g., as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of at least 7, e.g., as measured by the NAS CRN scoring system. In one embodiment, the subject has a NAS of 8, for example as measured by the NAS CRN scoring system. NAS calculated according to the NAS CRN scoring system includes a graded sum of steatosis (grade 0-3), hepatocyte swelling (grade 0-2) and lobular inflammation (grade 0-3) (Kleiner DE et al, hepatology 2005; 41: 1313-21). In one embodiment, the steatosis score is reduced by treatment. In one embodiment, the hepatocyte expansion score is reduced by treatment. In one embodiment, the leaflet inflammation score is reduced by treatment. In one embodiment, at least two of the steatosis score, the hepatocyte swelling score, and the lobular inflammation score are reduced by the treatment.

In one embodiment, the above method or use comprises identifying a subject as having a NAS of at least 1, 2, 3, 4, 5,6, 7, or 8 as measured by the NAS CRN scoring system.

In another embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, does not result in a significant change (i.e., stabilization or normalization) or decrease in NAS over time. In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, results in a decrease in NAS score. In one embodiment, the NAS score is reduced by at least 0.2. In another embodiment, the NAS score is reduced by at least 0.3. In another embodiment, the NAS score is reduced by at least 0.4. In another embodiment, the NAS score is reduced by at least 0.5. In another embodiment, the NAS score is reduced by at least 0.6. In another embodiment, the NAS score is reduced by at least 0.7. In another embodiment, the NAS score is reduced by at least 0.8. In addition toIn one embodiment, the NAS score is reduced by at least 0.9. In another embodiment, the NAS score is reduced by at least 1.0. In another embodiment, the NAS score is reduced by at least 2.0.

In one embodiment, the subject has a serum ALT level of at least about 30U/L. In one embodiment, the subject has a serum ALT level of at least about 35U/L. In another embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces serum ALT levels in the subject by at least about 10%, 15%, 20%, 25%, or 30%.

In one embodiment, the method or use described above comprises identifying a subject having a serum ALT level of at least about 25U/L, 30U/L, or 35U/L.

In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces inflammation and/or oxidative stress (e.g., levels of Reactive Oxygen Species (ROS)) in the liver of the subject. In one embodiment, the reduction in inflammation comprises a reduction in the activity of an inflammatory pathway (e.g., TNF- α, IL-6, and/or IL-2 pathway) or an inflammation-associated gene.

In one embodiment, the GHRH molecule or a pharmaceutically acceptable salt thereof, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, reduces tissue repair in the liver, for example reduces expression of genes involved in tissue repair, apoptosis and/or Epithelial Mesenchymal Transition (EMT), for example genes of the TGF- β pathway.

In one embodiment, the subject has a Human Immunodeficiency Virus (HIV) infection. In one embodiment, the subject is undergoing antiretroviral therapy.

In one embodiment, the treatment or use described herein has no or substantially no effect on low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C), or triglycerides in a subject. In one embodiment, the treatment or use described herein has no effect or substantially no effect on fasting glucose or hemoglobin A1c in the subject.

In one embodiment, the subject has a Body Mass Index (BMI) of at least about 25. In one embodiment, the subject has a BMI of at least about 26. In one embodiment, the subject has a BMI of at least about 27. In one embodiment, the subject has a BMI of at least about 28. In one embodiment, the subject has a BMI of at least about 29. In one embodiment, the subject has a BMI of at least about 30. In one embodiment, the subject has a BMI of at least about 31. In one embodiment, the subject has a BMI of at least about 32.

In one embodiment, the subject has liver fibrosis. In one embodiment, the above method or use comprises identifying a subject having liver fibrosis. In one embodiment, the subject has stage 1 (in another embodiment, stage 1A, 1B, or 1C) liver fibrosis. In one embodiment, the subject has stage 2 liver fibrosis. In one embodiment, the subject has stage 3 liver fibrosis. The fibrosis stage was determined using a scoring system designed by the NASH clinical research network Pathology Committee (Kleiner DE et al, hepatology 2005; 41: 1313-21):

no fibrosis at stage 0;

stage 1-perisinus or periportal fibrosis;

stage 1A: mild fibrosis, zone 3, perisinus;

stage 1B: moderate fibrosis, zone 3, perisinus;

and (3) stage 1C: portal vein only/periportal fibrosis;

stage 2-periantral and portal/periportal fibrosis;

stage 3 ═ bridging fibrosis;

stage 4 ═ cirrhosis.

In one embodiment, the subject has at least two of the following features (1) to (3): (1) HFF at least 15%, preferably at least 20%, 25% or 30%; (2) a NAS score of at least 3, preferably at least 4, 5 or 6; and (3) liver fibrosis, preferably stage 1C or 2 liver fibrosis or more severe liver fibrosis. In one embodiment, the subject has the features (1) to (3) described above. In one embodiment, the above method or use comprises identifying a subject having at least two or all of the above features (1) to (3).

As used herein, reference to preventing progression of liver fibrosis refers to the absence or substantial absence of progression of liver fibrosis following treatment with a GHRH molecule relative to the absence of such treatment. As used herein, reference to slowing the progression of liver fibrosis refers to the following: the progression of liver fibrosis may continue following treatment with the GHRH molecule, but at a reduced rate compared to the case where such treatment is not performed.

In one embodiment, the treatment or use described herein reduces the risk or likelihood of a subject developing liver cancer, e.g., hepatocellular carcinoma (HCC).

The term "GHRH molecule" as used in the context of the present disclosure includes, but is not limited to: human natural GHRH_(1-44)And fragments thereof (e.g., GHRH)_(1-40)、GHRH_(1-29)1-29 and 1-44 sequences), as well as any other fragments; GHRH from other species and fragments thereof; GHRH variants comprising amino acid substitutions, additions and/or deletions; a derivative or analog of GHRH, or a fragment or variant thereof, having an organic group or moiety coupled to the GHRH amino acid sequence, e.g., at the N-terminus, C-terminus, or side chain; and pharmaceutically acceptable salts of GHRH (human or from other species), as well as pharmaceutically acceptable salts of GHRH fragments, variants, analogs and derivatives. GHRH molecules of the present disclosure also include GHRH molecules currently known in the art, including but not limited to: GHRH bound to albumin (us patent No. 7,268,113); pegylated GHRH peptides (U.S. patent nos. 7,256,258 and 6,528,485); porcine GHRH (1-40) (U.S. patent No. 6,551,996); canine GHRH (U.S. patent application No. 2005/0064554); 1-29 to 1-44 amino acids in lengthGHRH variants of (us patent nos. 5,846,936, 5,696,089, 5,756,458 and 5,416,073, and us patent application nos. 2006/0128615 and 2004/0192593); and Pro⁰GHRH peptides and variants thereof (us patent No. 5,137,872).

GHRH analogs include those described in U.S. patent nos. 5,681,379 and 5,939,386, whose synthetic methods are also described. More specifically, these GHRH analogs are defined by the following formula a:

X-GHRH peptide (A)

Wherein the GHRH peptide is a peptide of formula B below (SEQ ID NO: 2):

A1-A2-Asp-Ala-Ile-Phe-Thr-A8-Ser-Tyr-Arg-Lys-A13-Leu-A15-Gln-Leu-A18-Ala-Arg-Lys-Leu-Leu-A24-A25-Ile-A27-A28-Arg-A30-A31-A32-A33-A34-A35-A36-A37-A38-A39-A40-A41-A42-A43-A44-R0 (B)

wherein the content of the first and second substances,

a1 is Tyr or His;

a2 is Val or Ala;

a8 is Asn or Ser;

a13 is Val or Ile;

a15 is Ala or Gly;

a18 is Ser or Tyr;

a24 is Gln or His;

a25 is Asp or Glu;

a27 is Met, Ile or Nle

A28 is Ser or Asn;

a30 is absent or is any amino acid, preferably Gln;

a31 is absent or is any amino acid, preferably Gln;

a32 is absent or is any amino acid, preferably Gly;

a33 is absent or is any amino acid, preferably Glu;

a34 is absent or is any amino acid, preferably Ser;

a35 is absent or is any amino acid, preferably Asn;

a36 is absent or is any amino acid, preferably Gln;

a37 is absent or is any amino acid, preferably Glu;

a38 is absent or is any amino acid, preferably Arg;

a39 is absent or is any amino acid, preferably Gly;

a40 is absent or is any amino acid, preferably Ala;

a41 is absent or is any amino acid, preferably Arg;

a42 is absent or is any amino acid, preferably Ala;

a43 is absent or is any amino acid, preferably Arg;

a44 is absent or is any amino acid, preferably Leu; and is

R0 is NH₂Or NH- (CH)₂)n-CONH₂And n is 1 to 12.

The X group is a hydrophobic tail anchored to the N-terminus of the peptide by an amide bond, the hydrophobic tail defining a backbone of 5 to 7 atoms. The main chain may be substituted by C_1-6Alkyl radical, C_3-6Cycloalkyl or C_6-12Aryl is substituted and the backbone comprises at least one rigid moiety attached to at least two atoms of the backbone. The rigid moiety being a double bond, a triple bond, a saturated or unsaturated C_3-9Cycloalkyl or C_6-12And (4) an aryl group.

In one embodiment, the X group is:

in one embodiment, in formula B, a30-a44 is: (a) deletion; (b) an amino acid sequence corresponding to positions 30-44 of native GHRH peptide (SEQ ID NO: 3); or (C) the amino acid sequence of (b) wherein 1 to 14 amino acids are deleted from the terminal C-terminus thereof.

In one embodiment, the GHRH peptide is a GHRH peptide comprising SEQ ID NO: 4.

In one embodiment, GHRH isIs (hexenoyl trans-3) hGHRH_(1-44)NH₂(SEQ ID NO: 1) or a pharmaceutically acceptable salt thereof. [ trans-3-hexenoyl group]hGHRH_(1-44)Amides (also known as temorelin and (hexenoyl trans-3) hGHRH_(1-44)NH₂) Is a synthetic human GHRH (hGHRH) analog comprising the hGHRH 44 amino acid sequence at which the hexenoyl moiety (C)₆Side chain) has been anchored to the amino-terminal tyrosine residue. [ trans-3-hexenoyl group]hGHRH_(1-44)The structure of the amide is shown in FIG. 5.

The term "pharmaceutically acceptable salt" refers to a GHRH molecule (e.g., trans-3-hexenoyl-GHRH)_(1-44)-NH₂) A salt which is pharmacologically acceptable and substantially non-toxic to a subject to which it is administered. More specifically, these salts retain the GHRH molecule (e.g., trans-3-hexenoyl-GHRH)_(1-44)-NH₂) And is formed from a suitable non-toxic organic or inorganic acid or base.

For example, such salts include GHRH molecules (e.g., trans-3-hexenoyl-GHRH)_(1-44)-NH₂) Acid addition salts which are sufficiently basic to form such salts. Such acid addition salts include acetate, adipate, alginate, lower alkanesulfonate (e.g., methanesulfonate, trifluoromethanesulfonate or ethanesulfonate), arylsulfonate (e.g., benzenesulfonate, 2-naphthalenesulfonate or toluenesulfonate (also known as tosylate)), ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cinnamate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerosulfonate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, bisulfate, 2-hydroxyethanesulfonate, itaconate, lactate, maleate, mandelate, methanesulfonate, nicotinate, nitrate, acetate, methanesulfonate, nicotinate, tosylate), ascorbate, mesylate, or mesylate, Oxalate, pamoate, pectate, perchlorate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, and the like,Succinate, sulfate, sulfonate, tartrate, thiocyanate, undecanoate, and the like.

In addition, for example, P.Stahl et al discusses acids commonly considered suitable for forming pharmaceutically useful salts from basic Pharmaceutical compounds (Camile G. (eds.) Handbook of Pharmaceutical salts, Properties, selections and uses (Handbook of Pharmaceutical salts, Properties, and uses) (2002) Soxhlet, Wiley-VCH; S.Berge et al, Journal of Pharmaceutical Sciences (1977)66(1) 1-19; P.Gould, Journal of International J.of pharmaceuticals (1986) 33201; Anderson et al, The Practice of Pharmaceutical Chemistry (The Practice of The Pharmaceutical Chemistry) (1996), American publications, New York, and The Special food administration (Book of Orange peels, Japan, and Ore.g., Japan).

Such salts can be readily formed by those skilled in the art using standard techniques. In fact, chemical modification of Pharmaceutical compounds (i.e., drugs) into salts is a well-known technique to Pharmaceutical chemists (see, e.g., H.Ansel et al, Pharmaceutical Dosage Forms and Drug Delivery Systems (6 th edition, 1995), pages 196 and 1456-1457). trans-3-hexenoyl-GHRH_(1-44)-NH₂Salts can be prepared, for example, by reacting trans-3-hexenoyl-GHRH_(1-44)-NH₂With an amount (e.g., equivalent amount) of an acid or base in a medium such as one in which a salt precipitates or in an aqueous medium and then lyophilized.

In one embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂The pharmaceutically acceptable salt of (a) is acetate.

In one embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is present in the pharmaceutical composition at a dose of about 1mg/ml to about 10 mg/ml. In another embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof at about 1mg/ml to 10mg/ml, preferably about 1mg/ml to 10mg/mlAbout 8mg/ml or about 4mg/ml to about 8mg/ml, e.g. about 1mg/ml, about 2mg/ml, about 3mg/ml, about 4mg/ml, about 5mg/ml, about 6mg/ml, about 7mg/ml or about 8mg/ml, is present in the pharmaceutical composition.

In one embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients.

As used herein, the term "pharmaceutically acceptable excipient" has its usual meaning in the art and refers to any ingredient that is itself an inactive ingredient (drug). Excipients include, for example, binders, lubricants, diluents, bulking agents (fillers), thickeners, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizers, sustained release agents, and other components. As used herein, "pharmaceutically acceptable excipient" refers to any excipient that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the subject, i.e., is a type of excipient and/or is used in an amount that is non-toxic to the subject. Excipients are well known in the art, and the present composition is not limited in these respects. In certain embodiments, the pharmaceutical composition comprises one or more excipients, including for example, but not limited to: one or more binders, thickeners, surfactants, diluents, slow release agents, colorants, flavoring agents, fillers, disintegrants/dissolution enhancers, lubricants, plasticizers, silica flow modifiers, glidants, anti-caking agents, anti-sticking agents, stabilizers, antistatic agents, swelling agents, and any combination thereof. One skilled in the art will recognize that a single excipient may perform more than two functions simultaneously, e.g., may act as both a binder and a thickener. The skilled person will also recognise that these terms are not necessarily mutually exclusive. Therapeutic formulations are prepared by mixing the active ingredient in the desired purity with one or more optional pharmaceutically acceptable carriers, excipients and/or stabilizers using standard methods known in the art. Excipients may be suitable for, e.g., intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic artery, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal, or pulmonary (e.g., in aerosol form) (see Remington: "Pharmaceutical Science and Practice (The Science and Practice of Pharmacy), written by Loyd V Allen, Jr 2012, 22 nd edition, uk Pharmaceutical press;" Handbook of pharmaceutic Excipients, written by Rowe et al, 7 th edition 2012, uk Pharmaceutical press "). In one embodiment, the pharmaceutical composition is an injectable composition. In one embodiment, the pharmaceutical composition comprises one or more excipients for subcutaneous administration/injection.

In one embodiment, the pharmaceutical composition comprises a bulking agent. As used herein, the term "bulking agent" refers to a compound used to provide sufficient or desired tonicity to a solution resulting from reconstitution of a lyophilized formulation. Preferably, the sufficient or desired tonicity of the solution is equal to or close to the isotonicity of the physiological fluid of the subject to which the solution is administered. For example, one or more sugars may be used as a leavening agent. As used herein, sugars include, but are not limited to, monosaccharides, oligosaccharides, and polysaccharides. Examples of suitable sugars include, but are not limited to, mannose, sorbose, xylose, maltose, lactose, sucrose, and dextran. Sugars also include sugar alcohols such as mannitol, inositol, galactitol, xylitol, and arabitol. Mixtures of sugars may also be used in accordance with the present disclosure. In one embodiment, the leavening agent is mannitol. For example, one or more amino acids, such as glycine, may be used as a leavening agent. The concentration of the bulking agent in the pharmaceutical composition is about 1% to about 10% (w/w) or about 2% to about 8% (w/w). In one embodiment, the concentration of the bulking agent in the pharmaceutical composition is about 3% to about 5% (w/w). In another embodiment, the concentration of the bulking agent in the pharmaceutical composition is about 4% (w/w).

In one embodiment, the pharmaceutical composition of the present disclosure may further comprise a surfactant. Typical examples of the surfactant include: sorbitol fatty acid esters such as sorbitol monocaprylate, sorbitol monolaurate, sorbitol monopalmitate; glycerin fatty acid esters such as glyceryl monocaprylate, glyceryl monomyristate, glyceryl monostearate; polysweetOil fatty acid esters such as decaglycerol monostearate, decaglycerol distearate, decaglycerol monolinoleate; polyoxyethylene sorbitan fatty acid esters such as polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monopalmitate, polyoxyethylene sorbitan trioleate, polyoxyethylene sorbitan tristearate; polyoxyethylene sorbitol fatty acid esters such as polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate; polyoxyethylene glycerin fatty acid esters such as polyoxyethylene glycerin monostearate; polyethylene glycol fatty acid esters such as polyethylene glycol distearate; polyoxyethylene alkyl ethers such as polyoxyethylene lauryl ether; polyoxyethylene polyoxypropylene alkyl ethers such as polyoxyethylene polyoxypropylene glycol ether, polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkylphenyl ethers such as polyoxyethylene nonylphenyl ether; polyoxyethylene hardened castor oil such as polyoxyethylene castor oil, polyoxyethylene hardened castor oil (polyoxyethylene hydrogenated castor oil); polyoxyethylene beeswax derivatives such as polyoxyethylene sorbitol beeswax; polyoxyethylene lanolin derivatives, such as polyoxyethylene lanolin; polyoxyethylene fatty acid amides such as polyoxyethylene stearic acid amide; having a structure of C_10-18Alkyl sulfates of alkyl groups such as sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate; having an average EO molar number of 2-4 and C_10-18Polyoxyethylene alkyl ether sulfates of alkyl groups, such as sodium polyoxyethylene lauryl sulfate; having a structure of C_8-18Alkyl sulfosuccinate salts of alkyl groups, such as sodium lauryl sulfosuccinate; lecithin; a glycerophospholipid; phosphosphingolipids, such as sphingomyelin; c_12-18Sucrose fatty acid esters of fatty acids.

In one embodiment, the surfactant is a nonionic surfactant. In another embodiment, the surfactant is a polyoxyethylene sorbitan alkyl ester. In yet another embodiment, the surfactant is polysorbate-20 (T20 or tween 20)^TM)。

In one embodiment, the pharmaceutical composition of the present disclosure may further comprise one or more stabilizing agents. As used herein, the term "stabilizer" is intended to mean a compound used to stabilize a therapeutic agent against physical, chemical, or biochemical processes that would reduce the therapeutic activity of the therapeutic agent. Suitable stabilizers are non-reducing sugars, including for example, but not limited to: sucrose (or cane sugar) and trehalose; and non-reducing polyols, including for example and without limitation: sorbitol, mannitol, maltitol, xylitol, glycols, glycerol and ethylene glycol. In one embodiment, the pharmaceutical composition comprises about 2% to about 10% (w/v) mannitol. In another embodiment, the pharmaceutical composition comprises about 2% to about 8% (w/v), about 3% to about 7% (w/v), about 4% to about 6% (w/v), or about 5% (w/v) mannitol.

In one embodiment, the pharmaceutical composition of the present disclosure comprises a non-reducing sugar. As used herein, "non-reducing sugar" refers to a sugar that does not contain a hemiacetal, e.g., a carbohydrate or sugar characterized by the formation of a glycosidic bond between the reducing ends of the sugar units, rather than between the reducing end of one sugar unit and the non-reducing end of another sugar unit. In another embodiment, the non-reducing sugar is trehalose or sucrose. In another embodiment, the non-reducing sugar is sucrose. In one embodiment, the concentration of the non-reducing sugar in the pharmaceutical composition of the present disclosure is about 0.1% to about 5% (w/w). In one embodiment, the concentration of the non-reducing sugar is from about 1% to about 3% (w/w). In another embodiment, the concentration of the non-reducing sugar is about 2% (w/w).

In one embodiment, the pharmaceutical composition of the present disclosure comprises a buffering agent, i.e. an agent that maintains the pH of the pharmaceutical composition close to a selected value. Examples of the buffer include acetate buffer, succinate buffer, citrate buffer, phosphate buffer, and histidine buffer. In one embodiment, the buffer is a histidine buffer. In one embodiment, the concentration of histidine in the pharmaceutical composition is from about 0.01% to about 1%, for example from about 0.05% to about 0.5% or from about 0.1% to about 0.3%. In another embodiment, the concentration of histidine sugars is about 0.15%.

In another embodiment, the amount of surfactant in the pharmaceutical composition of the present disclosure is from about 0.0001% to about 10% (w/w). In another embodiment, the amount of surfactant in the pharmaceutical composition of the present disclosure is from about 0.001% to about 5%, 1% or 0.1% (w/w) or from about 0.005% to about 0.05%. In yet another embodiment, the amount of surfactant in the pharmaceutical composition of the present disclosure is about 0.01% (w/w).

In one embodiment, the pharmaceutical composition of the present disclosure comprises an oligosaccharide, for example a cyclic oligosaccharide, such as cyclodextrin. As used herein, the term "cyclodextrin" refers to a family of cyclic oligosaccharides comprising macrocycles of glucopyranoside subunits (5 or more) linked by α -1,4 glycosidic bonds. Examples of cyclodextrins include alpha-cyclodextrin, alpha 0-cyclodextrin, and gamma-cyclodextrin, which contain 6, 7, and 8 glucopyranoside subunits, respectively, and their analogs (e.g., modified cyclodextrins). In one embodiment, the cyclodextrin is an α 1-cyclodextrin or a modified α 2-cyclodextrin. As used herein, a modified α 3-cyclodextrin refers to an α 4-cyclodextrin molecule in which one or more hydroxyl groups of one or more saccharide units can be modified (e.g., with an alkyl, alkenyl, or alkynyl group, or with a substituted alkyl, alkenyl, or alkynyl group). Thus, in embodiments, the α 5-cyclodextrin may be unmodified or unsubstituted, or may be modified or substituted. Thus, in another embodiment, the beta-cyclodextrin is a modified beta-cyclodextrin. As used herein, "modified β -cyclodextrin" refers to a modified β -cyclodextrin that is comprised at one or more hydroxyl groups of one or more saccharide units of the β -cyclodextrin, i.e., a group or moiety that is attached to one or more hydroxyl groups of one or more saccharide units of the β -cyclodextrin. Thus, in embodiments, the modified β -cyclodextrin is an alkyl-, alkenyl-, alkynyl, substituted alkyl-, substituted alkenyl, or substituted alkynyl- β -cyclodextrin (e.g., with hydroxyl substitution). In embodiments, alkyl, alkenyl or alkynyl is (C)₁-C₆) Alkyl, (C)₁-C₆) Alkenyl or (C)₁-C₆) Alkynyl. In another embodiment, the modified β -cyclodextrin is (C)₁-C₆) Alkyl beta-cyclodextrins ofAnother example is methyl- β -cyclodextrin (M- β -CD). In another embodiment, the modified β -cyclodextrin is hydroxy (C)₁-C₆) Alkyl β -cyclodextrin, and in another embodiment hydroxypropyl- β -cyclodextrin (HP- β -CD). In one embodiment, the cyclodextrin is present in the pharmaceutical composition at a concentration of about 2% to about 15% (w/v), in another embodiment about 2% to about 12.5% (w/v), such as about 2% to about 10% (w/v), about 2.5% to about 15% (w/v), about 2.5% to about 12.5% (w/v), about 2.5% to about 10% (w/v), about 5% to about 15% (w/v), about 5% to about 12.5% (w/v), about 5% to about 10% (w/v), about 7.5% to about 12.5% (w/v), about 7.5% to about 10% (w/v), about 5%, 7.5%, 10%, 12.5% or 15% (w/v), or about 10% (w/v).

In one embodiment, the pH of the pharmaceutical composition of the present disclosure is from about 4.5 to about 6.5, for example from about 5.0 to about 6.0. According to another embodiment, the pH of the pharmaceutical composition is about 5.0. According to another embodiment, the pH of the pharmaceutical composition is about 5.5. According to another embodiment, the pH of the pharmaceutical composition is about 6.0.

In one embodiment, the pharmaceutical composition of the present disclosure comprises a diluent, such as an aqueous solution. In another embodiment, the pharmaceutical composition comprises water (typically sterile water).

The pharmaceutical compositions of the present disclosure may also contain other diluents, solubilizers, excipients, pH adjusting agents, soothing agents, buffers, sulfur-containing reducing agents, antioxidants, and the like, if desired. For example, sulfur-containing reducing agents include N-acetylcysteine, N-acetylhomocysteine, lipoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and salts thereof, sodium thiosulfate, glutathione, methionine, and mercapto-containing compounds, such as thioalkanoic acids having 1 to 7 carbon atoms. Antioxidants include methionine, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, alpha-tocopherol, tocopheryl acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, tripentyl gallate, propyl gallate or chelating agents such as disodium Ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate. Other commonly used ingredients may also be contained, for example, inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium bicarbonate, and the like; and organic salts such as sodium citrate, potassium citrate, sodium acetate, and the like.

In one embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 8 mg. In another embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1mg to about 4mg, about 1mg to about 3mg, or about 1mg to about 2 mg. In another embodiment, the GHRH molecule, preferably trans-3-hexenoyl-GHRH_(1-44)-NH₂Or a pharmaceutically acceptable salt thereof, is administered in a daily dose of about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2.0 mg.

In other aspects, the present disclosure provides the use of the GHRH molecule or composition described above in achieving one or more of the biological/therapeutic effects noted herein, e.g., for ameliorating, slowing the progression of, stabilizing, reducing the severity of, preventing and/or treating a symptom, disease or condition described herein, or for the manufacture/manufacture of a medicament for ameliorating, slowing the progression of, stabilizing, reducing the severity of, preventing and/or treating a symptom, disease or condition described herein. In other aspects, the present disclosure provides the above compositions for use in ameliorating, slowing the progression of, stabilizing, reducing the severity of, preventing and/or treating a symptom, disease or condition described herein, or for use in the preparation/manufacture of a medicament for ameliorating, slowing the progression of, stabilizing, reducing the severity of, preventing and/or treating a symptom, disease or condition described herein.

As used herein, the term "treating" is defined as applying or administering a therapeutic agent to a subject, or to an isolated tissue or cell line of a subject having a disorder, disease, a symptom of a disorder or disease, or a predisposition for a disorder or disease, with the purpose of treating, curing, alleviating, delaying, alleviating, altering, remediating, ameliorating, improving, or affecting the disorder/disease, the symptom of the disorder/disease, or the predisposition for the disorder/disease.

In one embodiment, the treatment lasts for a period of at least 3, 6,9, or 12 months. In another embodiment, the treatment lasts for a period of at least 12 months.

In embodiments, the GHRH molecule is administered in an effective amount, for example, in a therapeutically effective amount or a prophylactically effective amount. "therapeutically effective amount" means an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, e.g., to achieve the above-described alteration and slow progression of the above-described symptoms. The therapeutically effective amount of the GHRH molecule may vary depending on factors such as the disease state, age, sex, and weight of the subject, and the ability of the compound to elicit a desired response in the subject. The dosage regimen may be adjusted to provide the optimal therapeutic response. A therapeutically effective amount also refers to an amount wherein any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, e.g., to prevent or inhibit the rate of onset or progression of the above-mentioned symptoms. A prophylactically effective amount can be determined as described above for a therapeutically effective amount. The specific dosage regimen for any particular subject may be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions.

According to another aspect of the present disclosure, a therapeutic composition of the present disclosure comprising a GH secretagogue may be provided in a container, kit or package (e.g., a commercial package) further comprising instructions for its use for the prevention or treatment of a condition described herein.

Accordingly, the present disclosure further provides a kit or package comprising a GHRH molecule or the above composition, optionally together with instructions for preventing or treating the symptoms described herein.

As used herein, the term "subject" or "patient" refers to a warm-blooded animal, such as a mammal, e.g., a cat, dog, mouse, guinea pig, horse, cow, sheep, or human. In one embodiment, the subject is a mammal. In another embodiment, the subject is a human.

Examples of the invention

The present disclosure is further illustrated by the following non-limiting examples.

Example 1: materials and methods

Participant selection

Participants were recruited at massachusetts general hospital (MGH, boston, massachusetts) and the national institutes of health (NIH, besida, maryland). Potentially eligible participants were identified by local physician recommendations, notification to participants participating in the volunteer program of the MGH subject, and NIH's HIV and NAFLD patient population. 61 HIV-infected males and females met eligibility criteria and participated in baseline assessments.

Participants were eligible for participation in the study if they were between 18-70 years of age and had been diagnosed with HIV infection and hepatic steatosis and demonstrated a liver fat fraction of > 5% as measured by Magnetic Resonance Spectroscopy (MRS). Participants in heavy drinking (female daily)>20g or male daily>30g) Excluded are participants with hepatitis B, active hepatitis C, alpha-1 antitrypsin deficiency, Wilson's disease, hemochromatosis or autoimmune hepatitis. Those patients with a history of hepatitis c qualified only after successful treatment, i.e. hepatitis c resolved at least one year prior to study entry, and no HCV viral load was detected at the screening visit. Participants with known liver cirrhosis, biopsies showing stage 4 fibrosis or other severe chronic diseases were also excluded. If the hemoglobin A1c is less than or equal to 7 percent, the antidiabetic drug maintains stable time for more than or equal to 6 months, and insulin or thiazole diketone drugs are not used, the mild diabetes participants also meet the conditions. Participants also need a stable antiviral treatment regimen of 3 months or more, with stable use of any antihypertensive or lipid lowering drug for 3 months or more, and, if applicable, stable use of vitamin E for 6 months or more before study entry. Use of Chronic OnchidiumParticipants with systemic corticosteroids, methotrexate, amiodarone, tamoxifen or GH were excluded, as were participants with any active malignancy. Women 50 years or older were asked to have a negative breast X-ray examination within 1 year after the baseline visit and men with a history of prostate cancer were excluded. Participants with a history of hypopituitarism or other diseases known to affect the GH axis are also ineligible. For safety reasons, other exclusion experimental conditions were as follows: hemoglobin<11g/dL；CD4⁺Counting<100 cells/mm³(ii) a HIV viral load>400 copies/mL; prostate specific antigen<5ng/mL。

Research planning

The study included a 12-month double-blind treatment phase during which the participants were treated at a rate of 1: a ratio of 1 was randomized to receive either 2mg daily or equivalent placebo treatment with temsirolimus followed by a 6 month open period during which all participants received temsirolimus treatment. A pre-specified preliminary analysis comparing the change in temsirolimus versus placebo treatment over 12 months is reported here. Randomness was graded by research institutes (NIH and MGH) and vitamin E usage, defined as sustained daily usage ≥ 400 international units. A random list is prepared by the study statistician using the permuted block algorithm in each rank, where the block sizes vary randomly. Temsirolimus is administered subcutaneously daily at a dose of 2mg approved by the FDA; participants received training for drug configuration and self-injection at baseline visit and injections were performed at home, returning used vials to assess compliance. The fraction Z of IGF-1 was monitored throughout the study by an independent endocrinologist of MGH, otherwise considered irrelevant to the study. The pre-specified IGF-1 score, Z ≧ 3 threshold, was included in the regimen as a trigger to reduce the temorelin dose to 1mg and virtual dose reduction in the placebo group, but not required by any patient during the double-blind phase of the study.

All subjects received nutritional counseling from clinical research dieticians at baseline, 6 months and 12 months. Visit is carried out in a fasting stateIn (1). Screening visits included medical history and physical examination, qualification laboratory examination, MRS, and Magnetic Resonance Imaging (MRI), the latter used to assess the cross-sectional area of Visceral Adipose Tissue (VAT) at the L4 vertebra. Screening visit MRI/MRS, HbA1c, CD4⁺Counts and HIV viral load were used as baseline measurements. Baseline assessments included liver biopsies; whole body dual energy x-ray absorptiometry (DXA); fasting assessment of liver function test, blood lipids, serum inflammatory markers, IGF-1 and HbA1 c; and bionutrient assessments, including 4-day food records, variable activity questionnaires (Kriska AM et al, Diabetes Care (Diabetes Care) 1990; 13:401-11), and anthropometric measurements taken in triplicate. Baseline assessments were repeated at 12 months as well as assessments of MRI/MRS, HbA1c and immunological parameters. Metaphase MRI/MRS was performed in a 6 month visit.

Measurement of results

The main result is the use¹HFF for H MRS measurements, performed in the morning after an 8 hour fast. Fat fraction was calculated by dividing the area under the lipid peak of the spectrum by the total area under the water and lipid peaks. Image acquisition follows standard protocols of MGH and NIH. Liver fat content was quantified using the jMRUI semi-automated software from NIH and the automated LC-Model software from MGH. The MGH rereads the NIH scans using an LC-Model with a correlation coefficient between measurements of 0.97, corresponding to the correlation between two scans performed on the same machine before and after repositioning, and uses the LC-Model for analysis (Breedella MA et al, J computer aided tomography (J Comput Assist Tomogr) 2010; 34: 372-6). The diagnostic accuracy of MRS for hepatic steatosis compared to the assessment of liver biopsies by an experienced pathologist was 0.94 (95% CI 0.88-1.0) area under the subject's working curve (Georgoff P et al, J. Radiology of America (AJR Am J Roentgenol) 2012; 199: 2-7). If HFF ≧ 5%, the subject is eligible.

All liver biopsy samples were histologically scored by an blinded central pathologist (DEK) using the NAS CRN scoring system (Kleiner DE et al, hepatology 2005; 41: 1313-21). The sum of the grades of steatosis (grade 0-3), hepatocyte swelling (grade 0-2) and lobular inflammation (grade 0-3) constitutes NAS with fibrosis graded independently between 0-4 (Kleiner DE et al, hepatology 2005; 41: 1313-21). Whether steatohepatitis is present or not is determined by histological examination (DEK). The progression of fibrosis is considered to be any increase in the stage of fibrosis between baseline and 12 months. An abdominal cross-sectional MRI of the L4 vertebra was read centrally and used to quantify VAT and Subcutaneous Adipose Tissue (SAT) regions.

Participants in MGH also underwent normoglycemic hyperinsulinemic clamp surgery to assess insulin sensitivity. As mentioned before, after a 14-hour fast, a low dose (20 mU/m insulin) was administered²Min) for 2 hours, followed by high dose (insulin 80 mU/m)²Min) for 2 hours (Braun LR et al, "effect of pitavastatin on insulin sensitivity and liver fat: a Randomized Clinical Trial (Effects of Pitavastatin on Insulin Sensitivity and Liver Fat: A Randomized Clinical Trial 2018 journal of Clinical endocrine metabolism (J Clin endocrine Metab); 103:4176-86). Within the last 20 minutes of the low and high dose clamps, insulin stimulated glucose disposal (M) was calculated using the DeFronzo method (DeFronzo RA, Tobin JD, Andres R, journal of physiology in the United states (Am J Physiol) 1979; 237: E214-23) as a primary indicator of liver and systemic insulin sensitivity, respectively.

Laboratory analysis was performed using standard methods. Clinical experiments were measured at the NIH clinical laboratory and MGH was measured at LabCorp and Quest. IGF-1 was measured centrally in the Quest laboratory. C-reactive protein was measured using electrochemiluminescence (Meso Scale Discovery, rockville, ma) and adiponectin was measured using ELISA (R & D Systems, minneapolis, minnesota).

Statistical analysis

The pre-assigned primary study endpoint was change in HFF between baseline and 12 months. Secondary study endpoints included changes in liver pathology, ALT, blood lipids, glucose metabolism, and systemic inflammatory markers. 60 sample sizes were selected for 80% efficacy to detect treatment differences with liver fat fraction changes of 0.85 standard deviation or more over 12 months, assuming a drug withdrawal rate of 25% (i.e. 45 evaluable patients were withdrawn) and bilateral alpha of 0.05. IRB was licensed to recruit 61 st participant after one patient at MGH center discontinued his drug at the end of baseline visit.

According to a pre-specified analysis plan, the variation of HFF is evaluated by a random intercept mixed effects model that uses a limited maximum likelihood to evaluate the effect estimates of time x randomized interactions. All available data is used in the analysis, which is based on the intended treatment. The same analysis was used for other study endpoints measured at multiple time points during double-blinding, including VAT, SAT, ALT, BMI, and glucose. For secondary study endpoints measured only at baseline and 12 months, a paired t-test was performed using all available data. These data are expressed as mean ± standard deviation, or for categorical variables, as numbers and percentages. The student's t-test for continuous variables and the pearson chi-square statistics for categorical variables were used to evaluate the comparisons between groups at baseline. The pearson correlation coefficient is used to evaluate the relationship between successive variables. Two data points, a baseline ALT value and a baseline CRP value, were excluded due to more than 5 standard deviations above the sample mean. A two-sided α of 0.05 is a predefined threshold for statistical significance. Research data was collected and managed using a research electronic data Capture (REDCap) tool hosted by a cooperative medical company (Harris PA et al, J Biomed Inform 2009; 42: 377-81). The sensitivity analysis was performed in 100 iterations (10 prior to discarding) using multiple interpolation of missing data. All data analyses were supervised by the study statistician (HL).

Example 2: results

Patient characteristics, compliance

Of the 143 participants screened, 61 entered the randomized treatment portion of the trial. NIH recruited 15 participants and MGH recruited 46 participants. The reasons for participant flow and patient exclusion are shown in figure 1. As shown in figure 1,4 participants in the temorelin group and 2 participants in the placebo group were discontinued prior to any follow-up images. 5 participants in the temorelin group and 2 participants in the placebo group were discontinued after obtaining follow-up images for preliminary analysis. The total drug withdrawal rate among the groups was not significantly different, and P was 0.12.

Clinical features (table 1) and measurements of body composition and metabolism (fig. 2) were similar between groups at baseline (fig. 2). At baseline, 33% of the population had a histological diagnosis of NASH. 43% of the population suffer from stage 1 or higher fibrosis; no one had stage 4 fibrosis at baseline according to the protocol. These ratios were similar in the treatment group (table 1). The ART protocol was similar between groups (table 1).

Table 1: baseline demographic and clinical characteristics

For any of the variables shown above, there were no statistically significant differences between groups at baseline.

Continuous variables are expressed as mean ± standard deviation.

Current smokers also belong to the category "ex-smokers".

Vitamin E usage is defined as regular daily intake of > 400 International units of vitamin E.

Abbreviations: g, g; HIV, human immunodeficiency virus; kcal, kcal; NASH, not

Alcoholic steatohepatitis; NRTI, nucleoside reverse transcriptase inhibitors; PI, protease inhibitors;

NNRTIs, non-nucleoside reverse transcriptase inhibitors.

The compliance to daily injections by returned empty vial counts was similar between treatment groups: placebo 87 ± 16% and temorelin 80 ± 15% (P ═ 0.11). As shown in FIG. 2, the change in IGF-1 values demonstrated the expected effect of temorelin to increase IGF-1 in an amount of 117ng/mL (95% CI [76, 157], P < 0.0001). None of the subjects had an IGF-1 score Z exceeding a pre-specified dose reduction threshold (Z score >3), but one subject had received a dose reduction to 1mg due to symptoms that may be associated with growth hormone. The subject exited the study soon after dose reduction.

Markers of hepatic fat fraction, liver pathology and inflammation

Table 2A shows the Hepatic Fat Fraction (HFF) at baseline and at the end of 1 year of treatment for temsirolimus and placebo treated subjects completing the study. Table 2B shows the NASH score, steatosis score, hepatocyte expansion score, and lobular inflammation score at baseline and at the end of 1 year of treatment for temozerin and placebo treated subjects completing the study. Table 2C shows the fibrosis phase at baseline and at the end of 1 year of treatment for temsirolimus and placebo treated subjects completing the study.

TABLE 2A

TABLE 2B

TABLE 2C

Compared to placebo, temorelin significantly reduced HFF (effect amount-4.1% (95% CI-7.5, -0.7), P ═ 0.02). As shown in fig. 3A, the change between baseline and 12 months corresponds to a relative change of-37% (95% CI-67, -7) of liver fat (fig. 3B). In the temorelin group, 35% of the individuals had a reduction in HFF to < 5%, whereas in the placebo group 4% of the individuals experienced this (fig. 3C, P ═ 0.007 for comparison). Furthermore, temorelin is effective in reducing HFF in the most afflicted subjects (i.e., subjects with the greatest magnitude of baseline HFF elevation, e.g., > 15%), with some patients showing an absolute reduction in HFF of more than 10%: patient 7 decreased from 16.97 to 2.87; patient 24 decreased from 33.19 to 17.4; patient number 26 decreased from 15.03 to 2.14; patient No. 55 decreased from 33.3 to 21 (table 2A).

Throughout the population, temorelin prevented the progression of fibrosis during treatment, with 2 in the temorelin group showing progression and 9 in the placebo group (P ═ 0.04) (fig. 4A). Changes in fibrosis during the study were positively correlated with changes in NAS score (p-value of analysis of variance 0.0003, fig. 4B). Throughout the population, temorelin did not significantly change the NAS score relative to placebo (effect dose-0.3, 95% CI-1.0, 0.5). However, in randomly dosed temorelin, patients with higher baseline NAS scores had a greater decrease in score during treatment (r-0.48, P-0.04), while no similar relationship was observed in the placebo group (r-0.14, P-0.52). Notably, NAS score of patient 55 receiving temorelin treatment decreased from 7 to 4 points, which was associated with improvement in fibrosis (from stage 2 to stage 1A) (tables 2B and 2C). Similarly, in the tesamorelin treated population, the change in NAS score during treatment was positively correlated with the change in HFF (r-0.51, P-0.03), whereas no similar relationship was observed in placebo-treated patients (r-0.14, P-0.52).

Compared to placebo, temorelin did not significantly reduce either ALT or GGT during treatment, although both effect levels showed a modest reduction (figure 2). By limiting the population to patients with elevated ALT (>30U/L) at baseline, temorelin did significantly reduce ALT after 12 months relative to placebo (effect amount-29U/L [ 95% CI-3, -55], P ═ 0.03, fig. 3D). Temorelin reduced CRP relative to placebo (figure 2), but had no effect on adiponectin.

Lipid and glucose metabolism and body composition

No effect of temorelin on low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) or triglycerides (FIG. 2) was observed in this study. Temorelin had no significant effect on fasting plasma glucose or hemoglobin A1c during treatment (FIG. 2). In a subset of patients receiving normoglycemic hyperinsulinemic clamp, temorelin did not affect the therapeutic effect of the desired glucose infusion rate during the low dose clamp (low dose M0.0 mg/kg/min [ 95% CI-1.1, 1.1], P ═ 0.996) nor the insulin-stimulated glucose uptake during the high dose clamp (high dose M-0.9mg/kg/min [ 95% CI-2.4, 0.7).

Table 3 shows visceral adiposity status at baseline and at the end of 1 year of treatment in temozerin and placebo-treated subjects completing the study.

TABLE 3

Temorelin significantly reduced VAT area relative to placebo, with no effect on SAT area (figure 2). Temorelin moderately increased the lean body mass by DXA with no significant effect on the systemic fat mass (figure 2). Body mass index and waist circumference were unchanged.

Nourishing and physical activity

According to the assessment of the modifiable activity questionnaire, the daily caloric and macronutrient intake recorded for the four day food, the self-reported number of drinking weekly and the hours of activity per week did not vary significantly from baseline to 12 months.

Sensitivity analysis

Sensitivity analysis of the primary study endpoint was performed using multiple interpolations of missing data. This data confirms the main result, an estimated effect of HFF reduction of-3.8% (95% coverage-5.4, -2.2).

Adverse effects

Adverse events for each study group are shown in table 4. The number of Severe Adverse Events (SAE) observed was limited, with no difference between treatment groups. None was judged to be related to study drug. In the placebo group, 2 people developed SAE and were hospitalized: one was hospitalized with suicide attempts and the other was haematoma after liver biopsy. In the temorelin group, 4 people developed SAE and were hospitalized: 1 has a history of apoplexy and temporary hemiplegia, and the reason is unknown; 1, the user has suicidal ideation; 1 human uremia; pneumonia was caused only by influenza in 1 person.

Events occurring in 1 in the placebo group and 4 in the temorelin group met the prior regimen criteria for investigator drug withdrawal (P ═ 0.17, including two hyperglycemic withdrawal in the temorelin group (week 2 and month 6 visits). 1 person was known to have diabetes at baseline.

Table 4: adverse events

The numbers refer to the number of patients who have had an event.

P values for event numbers were compared by pearson's chi-square test per group. The study failed to detect a difference in adverse events and only displays the p-value of the summary event.

Example 3: gene Set Enrichment Analysis (GSEA) of samples from the temozorelin group and placebo group

RNAseq data from the temorelin and placebo groups were generated using Hallmark and select gene sets for Gene Set Enrichment Analysis (GSEA) (Subramanian, Tamayo et al, 2005, Proc. Natl. Acad. Sci. USA (PNAS) 102,15545 & 15550; Mootha, Lindgren et al, 2003, Nature genetics (Nat Genet) 34,267 & 273) to determine whether temorelin administration was associated with overexpression and/or downregulation of a particular gene set or pathway. In using RNAlater^TMAfter preparation, paired liver biopsy specimens from this study (temorelin, n-19; placebo, n-24) were used. Changes in gene expression were successfully assessed in 18 patients in the temorelin group and 21 patients in the placebo group. Illumina TruSeq^TMAutomated variants of the standard mRNA sample preparation kit were used to provide 50M reads aligned in pairs. Each run was one 101bp double-ended, reading one eight base index barcode. Data was analyzed using Broad Picard Pipeline, including demultiplexing and data aggregation. Alignment is accomplished using the STAR alignment algorithm. RNA sequencing of the Tru-Seq strand-specific large insert includes plating, poly-A selection and strand-specific cDNA synthesis, library preparation (450 and 550bp insert size), sequencing (101bp paired reads), and sample identification QC check (when sample identification of matching DNA samples is selected). The product uses chain-specific Illumina TruSeq^TMThe protocol provides for library construction, with sequence coverage up to 50M paired reads or 50M total reads. Standardized gene set enrichment methods were used to compare the changes over time for a particular gene set in each treatment group. Data were analyzed using heat maps, enrichment maps and dot maps of individual genes. The genes at the leading edge were examined in a related pathway and were examined for relationship to changes in liver biopsy phenotype, including stage of fibrosis.

The results of these studies are summarized below:

cell metabolism

Upregulating oxidative phosphorylation with temorelin therapy;

the frontal genes correspond to the major coding genes:

component of the electron transport chain;

omicron chaperone proteins required for assembly of the electron transport chain;

enhancement of mitochondrial activity can reduce Reactive Oxygen Species (ROS) levels, preventing liver inflammation and fibrosis.

Inflammation(s)

Tesamorelin leads to a large downregulation of multiple inflammatory pathways. (e.g., TNF- α, IL-6, and IL-2);

inflammation is an important process of NAFLD progression (e.g. NASH);

gene analysis appears to be more sensitive to detect inflammatory changes that are not openly reflected in histopathological analysis;

the frontal genes correspond to the major coding genes:

component of innate and adaptive immune cells;

omicron cytokine.

Tissue repair

Tesamorelin leads to down-regulation of genes involved in tissue repair;

fibrosis is a manifestation of chronic/deregulated wound healing responses. Notably, in participants receiving treatment with tesamorelin, down-regulation of a gene pathway involved in tissue repair was found to correspond to a reduction in genes associated with the fibrosis stage;

down-regulation of the apoptotic gene in a participant in treatment with tesamorelin;

downregulation of the uv response gene of the temorelin treatment participant;

down-regulation of Epithelial Mesenchymal Transition (EMT) genes in a participant in treatment with temorelin;

transforming growth factor-beta (TGF- β) is a key mediator of fibrosis, EMT and tumorigenesis, and is down-regulated in participants receiving temorelin therapy.

Cell renewal

Tesamorelin leads to down-regulation of genes involved in cell turnover;

the earliest event of NAFLD, even before steatosis, has been shown to be liver proliferation; and is

Has been considered an important step in liver damage;

the repeated cycles of hepatocyte apoptosis and proliferation increase the risk of abnormal repair in certain individuals, ultimately leading to tumorigenesis.

Cancer prognosis gene set

Temeprelin leads to an up-regulation of a good prognosis of HCC and a down-regulation of a poor prognosis gene set of HCC; down-regulation of the Yap Taz pathway gene set involved in fibrosis and tumorigenesis;

consistent with its down-regulation of inflammatory, fibrotic and cell renewal pathways, these pathways may ultimately reduce the risk of malignancy.

Tesamorelin appears to reduce the likelihood of liver malignancy, even while stimulating the GH pathway and increasing the systemic IGF-1 pathway.

Relationship of specific pathways to fibrotic genetic alterations (in the Temorelin group)

The set of genes involved in oxidative phosphorylation is inversely correlated with changes in fibrotic genes;

the gene set involved in inflammation, tissue repair, and cell turnover is positively correlated with changes in fibrotic genes.

Although the present disclosure has been described hereinabove by way of specific embodiments thereof, it can be modified without departing from the spirit and nature of the present disclosure as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

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GHRH or analogues thereof for the treatment of liver disease

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