PHARMACEUTICAL USE OF AN EXTENDED-RELEASE COMPOSITION CONTAINING PIRFENIDONE FOR THE TREATMENT AND REVERSAL OF HUMAN STEATOHEPATITIS (NAFLD/NASH)

Abstract

The present invention relates to the use of a pharmaceutical composition in the form of extended-release tablets containing Pirfenidone for treating NAFLD/NASH and advanced liver fibrosis by decreased serum cholesterol and triglycerides as well as reducing the content of hepatic fat accumulation, both in the form of macrosteatosis and microsteatosis. Additionally, its use as an agonist for PPARgamma (peroxisome proliferation receptor activated gamma), PPARalpha (peroxisome proliferation receptor activated alpha), LXR and CPT1, key molecules in the metabolism of fatty degradation and inflammation of the liver. In addition, another use is the induction of decreased expression of NFkB master gene, transcriptional inducer of hepatic inflammatory process factor. All of these events results in the reversal of NAFLD/NASH and advanced liver fibrosis.

Claims

1. A method of treating or reversing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH), the method comprising administering to the subject a pharmaceutical composition comprising between 100 mg and 600 mg of pirfenidone.

2. The method of claim 1, wherein the method leads to regression of hepatic fibrosis.

3. The method of claim 1, wherein the method decreases serum cholesterol and triglycerides.

4. The method of claim 1, wherein the method decreases hepatic fat accumulation.

5. The method of claim 1, wherein the method induces the elimination of excess liver fat.

6. The method of claim 1, wherein the method decreases the expression of NFkB.

7. The method of claim 1, wherein the method decreases hepatic inflammation.

8. The method of claim 1, wherein the method decreases serum levels of IL-17A.

9. The method of claim 1, wherein the method decreases serum levels of IL-6.

10. The method of claim 1, wherein the method decreases serum levels of IL-1β.

11. The method of claim 1, wherein the method increases serum levels of IL-10.

12. The method of claim 1, wherein the method decreases serum levels of IFN-γ.

13. The method of claim 1, wherein the method decreases serum levels of TNF-α.

14. The method of claim 1, wherein the method decreases expression of TGF-β1.

15. The method of claim 1, wherein the method increases expression of SREBP1.

16. The method of claim 1, wherein the method increases expression of CPT1A.

17. The method of claim 1, wherein the method increases expression of PPAR gamma.

18. The method of claim 1, wherein the extended-release tablet comprises 100 mg, 200 mg, 300 mg, 400 mg, or 600 mg of pirfenidone.

19. The method of claim 1, wherein the method is a method of treating or reversing non-alcoholic steatohepatitis (NASH).

20. The method of claim 1, wherein a daily dosage comprises an equivalent of at least approximately 100 mg/kg of pirfenidone per day.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0029] Other features and advantages of the invention will be clear from the following detailed description of the objectives and preferred embodiments of the appended claims and of the accompanying figures, wherein:

[0030] FIGS. 1(A-D): Shows the body weights, the glucose levels and the levels of the ALT and AST liver enzymes of the mice.

[0031] FIG. 2: Histological examination of the tissues of the livers of the mice included in the control, HF and those treated with PFD-LP.

[0032] FIGS. 3(A-F): Shows the analysis of proinflammatory cytokines in the serum of the mice included in each of the indicated groups.

[0033] FIG. 4: Shows the expression of profibrogenic and proinflammatory marker genes.

[0034] FIG. 5: Shows the western blots with the protein expression of the mediators of fat metabolism in NAFLD/NASH, the key metabolic transcription factors LXR and PPARalpha were evaluated in the liver tissue.

[0035] FIG. 6: Shows the western blots with the protein expression of the key transcriptional factors in the regulation of the inflammatory process in the liver, such as PPARgamma and NFkB.

DETAILED DESCRIPTION OF THE INVENTION

Animals Used in the Experiments

[0036] Mice of six to eight weeks of age, male C57BL/6NHsd (Harlan, Mexico City) were housed in an atmosphere of 22±2° C. in 12-hour light/dark cycles. 5-7 mice were randomly assigned to the standard Chow diet (control) or high-fat/high carbohydrate (HF) diet for 16 weeks. The control group received diet Harlan TM-2018 (18% of calories from fat) and had free access to pure water, while the HF group received Harlan diet TD-06414 (60% of calories from lipids) and had free access to water with high fructose enriched at a concentration of 42 g/L (proportions in 55% fructose and 45% sucrose). The group of PFD mice (HF+PFD) received the HF diet for 8 weeks, followed by the HF diet for 8 weeks and 100 mg/kg/day of PFD extended release formulation. All regimens received 0.1 ml of vehicle. After a night of fasting, blood samples were analyzed. The weights of the mice and glucose were recorded weekly from start to sacrifice.

Body Weight, Glucemia, Cholesterol, Triglycerides, VLDL Cholesterol and Aminotransferases.

[0037] The characteristics of mice with steatohepatitis (NASH/NAFLD) induced by high-fat diet (FH) are shown in FIG. 1A-1D. Significant differences were found between the groups, being the mice of the HF group those that gained the most weight (up to 37% against the control) and the HF+PFD-LP group having 11% less weight than the HF group (FIG. 1A). Regarding glycemia, higher serum glucose levels were observed in the HF group compared to the control group, with a significant difference from week 9 to 13. However, in the last five weeks of treatment, there was no difference significant between HF+PFD-LP vs. control group (FIG. 1B). We also found higher serum levels of AST (FIG. 1C), ALT (FIG. 1D), and cholesterol, triglycerides and VLDL-cholesterol (Table 1) in the HF group compared to HF+PFD or control group.

TABLE-US-00001 TABLE 1 Determinations in serum of cholesterol, triglycerides and VLDL-cholesterol Control HF HF + PFD Cholesterol  78 ± 22 (10) 153 ± 27 (10).sup.a 120 ± 24 (8) (mg/dl) Triglycerides 128 ± 28 (10) 212 ± 44 (10).sup.a .sup. 127 ± 38 (8).sup.b (mg/dl) VLDL (mg/dl) 30 ± 6 (10) 34 ± 7 (10)  25 ± 8 (8) The values are the averages ± SD. The numbers of the mice are indicated in parentheses. .sup.aP < 0.05 compared with the control. .sup.bP < 0.05 compared with HF.

Histology

[0038] Histological examination of liver tissues of the HF group showed substantial microvesicular steatosis and macrovesicular steatosis with inflammatory changes (FIG. 2). Steatosis in the HF group was predominantly macrovesicular and was severe in acinar zone 1 with severe microvesicular steatosis and macrovesicular steatosis in acinar zone 2. Evidence of severe microvesicular steatosis was found in acinar zone 3 and moderate balloon degeneration in hepatocytes in that area; the inflammation was predominantly periportal with neutrophils and mononuclear cells, which also shows periportal fibrosis with a score of 1 (0-4), such histological changes are compatible with stage 1C of hepatic fibrosis and NAFLD in class 3. PFD-LP induced a dramatic decrease in liver fat content. These results correlate with gross hepatic observations. Regarding central and peripheral fat, HF+PFD-LP showed a lower level of fat compared to the HF group.

Analysis of Cytokines in Serum

[0039] In order to correlate the histological results with systemic markers, the pro-inflammatory cytokines of serum IL-17A, IL-6, IL-1β, IFN-γ and TNF-α were analyzed. Serum IL-6 levels (FIG. 3A) were significantly reduced in the HF+PFD group (145.8±15.0 ng/ml) compared to the HF group (211±30 ng/ml). The serum levels of IL-1β (FIG. 3B) significantly shows the reduction in the HF+PFD group (69.3±13 ng/ml) compared to the HF group (177.4±20.6 ng/ml). IFN-γ is shown in FIG. 3C and was significantly lower in the PFD-LP group (18.2±8 ng/ml) compared to the HF group (221.3±50 ng/ml). Similarly, TNF-α (FIG. 3D) shows a significant reduction in serum levels in the HF+PFD group (32.9±21.1 ng/ml) compared to the HF group (254.6±70 ng/ml). Worthy of mention, the level of IL-17A (FIG. 3F), showed a dramatic reduction in mice treated with PFD-LP (271.1±149.6) compared to the HF group, which showed a significant increase in serum levels of IL-17A (1983.5±400 ng/ml). All the cytokines analyzed in the HF group showed a highly significant increase compared to the control group. Finally, it was observed that the serum level of the anti-inflammatory cytokine IL-10 (FIG. 3E) was significantly higher in the HF+PFD group (37.8±10.4 ng/nil) compared to the HF group (15.4±9.1 ng/ml).

Expression of Profibrogenic and Proinflamatory Marker Genes

[0040] The level of liver messenger RNA showed a decrease in the expression of TGF-β1, and a significant down-regulation of COL1A1 and TNF-α in the PFD-LP+HF group. In addition, a significant reduction in the expression of genes CD11 b and MCP1 was observed in comparison with the HF group (FIG. 4).

PFD-LP Modulates the Mediators of the Metabolism of the Hepatic Fats.

[0041] To analyze the effect of PFD on the modulation of fat metabolism mediators in NAFLD/NASH, the key metabolic transcription factors LXR and PPARalpha were evaluated in liver tissue. As shown in FIG. 5, the protein levels of LXR (8082±847) and PPARalpha (11506±1167) increased significantly in the PFD+HF group compared to the control (2,634±1,042 and 3,890±1,130, respectively) and the HF group (3375±898 and 7308±1117 respectively). To understand better the effect of PFD-LP on key proteins such as SREBP1 and CPT1A that are targeted by LXR and PPAR respectively, they were also evaluated. As shown in FIG. 5, PFD-LP induced an increase in the precursor protein SREBP1 (6057±847) compared to the control (785±396) and the HF group (2,622±1,161). However, SREBP1 in its cleaved form (active form) showed a tendency to decrease, but no significant differences were found between the three groups (4567±1620), HF (1776±893) and PFD+HF (1638±303). In addition, a significant increase was observed in CPT1A expression (enzyme responsible for internalizing hepatic fats in the mitochondria to be “burned”) in the PFD+HF group (9303±809), compared to the control (4303±820) and the HF group (6.172±1.356).

[0042] Finally, FIG. 6 shows us that key transcriptional factors in the regulation of the inflammatory process in the liver such as PPARgamma and NFkB are specifically modulated in a way that results in the decrease of hepatic inflammation.

BIBLIOGRAPHY

[0043] 1 Marra F, Lotersztajn S. Pathophysiology of NASH: perspectives for a targeted treatment. Curr Pharm Des 2013:19(29):5250-5269. [0044] 2. Ferramosca A, Vincenzo Zara. Modulation of hepatic steatosis by dietary fatty acids. World J Gastroenterol 2014; 20(7):1746-1755 [0045] 3. Paschos P, Paletas K. Non alcoholic fatty liver disease and metabolic syndrome. Hippokratia 2009; 13 (1):9-19. [0046] 4. Van Rooyen D M, Larter C Z, Haigh W G, Yeh M M, Ioannou G, Kuver R, et. al. Hepatic free cholesterol accumulates in obese, diabetic mice and causes nonalcoholic steatohepatitis. Gastroenterology 2011; 141:1393-403. [0047] 5. Kang H, Greenson J K, Omo J T, Guillot C, Peterman D, Anderson L, et. al. Metabolic syndrome is associated with greater histologic severity, higher carbohydrate, and lower fat diet in patients with NAFLD. Am J Gastroenterol 2006; 101:2247-53. [0048] 6. Lim J S, Mietus-Snyder M, Valente A, Schwarz J M, Lustig R H. The role of fructose in the pathogenesis of NAFLD and the metabolic syndrome. Nat Rev Gastroenterol Hepatol 2010; 7:251-64. [0049] 7. Harley I T, Stankiewicz T E, Giles D A, Softic S, Flick L M, Cappelletti M, et al. IL-17 signaling accelerates the progression of nonalcoholic fatty liver disease in mice. Hepatology. 2014; 59(5):1830-9. [0050] 8. Zúñiga L A, Shen W-J, Joyce-Shaikh B, Pyatnova E A, Richards A G, Thom C, et al. IL-17 Regulates Adipogenesis, Glucose Homeostasis, and Obesity. J Immunol. 2010 Dec. 1; 185(11):6947-59. Lafdil F, Miller A M, Ki S H, Gao B. Th17 cells and their associated cytokines in liver diseases. Cell Mol Immunol 2010; 7(4):250-254. [0051] 9. Weaver C T, Hatton R D. Interplay between the TH17 and Treg cell lineages: a co-evolutionary perspective. Nat Rev Immunol 2009; 9:883-9. [0052] 10. Korn T, Bettelli E, Oukka M, Kuchroo V K. IL-17 and Th17 Cells. Annu Rev Immunol 2009; 27:485-517. [0053] 11. Tang Y, Bian Z, Zhao L, Liu Y, Liang S, Wang 0, et. al. Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease. Clin Exp Immunol 2011; 166(2):281-90. [0054] 12. Wang Y-X. PPARs: diverse regulators in energy metabolism and metabolic diseases. Cell Res. 2010; 20(2):124-37. [0055] 13. Yan Xing, Tingting Zhao, Xiaoyan Gao, Yuzhang Wu. Liver X receptor a is essential for the capillarization of liver sinusoidal endothelial cells in liver injury. [0056] 14. Yoshikawa T, Ide T, Shimano H, Yahagi N, Amemiya-Kudo M, Matsuzaka T, et al. Cross-Talk between Peroxisome Proliferator-Activated Receptor (PPAR) a and Liver X Receptor (LXR) in Nutritional Regulation of Fatty Acid Metabolism. I. PPARs Suppress Sterol Regulatory Element Binding Protein-1c Promoter through Inhibition of LXR Signaling. Mol Endocrinol. 2003; 17(7):1240-54. [0057] 15. Cui G, Qin X, Wu L, Zhang Y, Sheng X, Yu Q, Sheng H, Xi B, Zhang J Z, Zang Y Q. Liver X receptor (LXR) mediates negative regulation of mouse and human Th17 differentiation. J Clin Invest. 2011; 121(2):658-670. [0058] 16. Ducheix S, Montagner A, Polizzi A, Lasserre F, Marmugi A, Bertrand-Michel J, et al. Essential fatty acids deficiency promotes lipogenic gene expression and hepatic steatosis through the liver X receptor. J Hepatol. 2013; 58(5):984-92.