repurposed compounds np-135 and np-160 reduce fibrosis in … · 2019. 12. 16. · • cenicriviroc...

Repurposed Compounds NP-135 and NP-160 Reduce Fibrosis in a NASH Pre-clinical Mouse Model Mark Williams, PhD, MBA CSO, Algernon Pharmaceuticals Vancouver, Canada Email: [email protected] Overview Methods Results: Experiment 1 Results: Experiment 2 Conclusions & Future Work Acknowledgements References Induction of NASH 1 • Male C57BL/6 mice received 200 μg streptozotocin SC 2 days post-birth (causing mild islet inflammation and islet destruction) • High-fat diet ad libitum from 4 weeks of age (57% kcal fat) (causing fatty changes to the liver, NASH and fibrosis) Treatment • From weeks 6-9, ten groups (n = 8/group) received a NASH test compound QD PO • One group (n = 8) received vehicle control (0.5% CMC) • One group (n = 8) received telmisartan as a positive control • Individual body weight measured daily during treatment period • Survival, clinical signs and behaviour monitored Study endpoints • Animals were sacrificed at 9 weeks • Liver weights and liver to body-weight ratio • Plasma and serum biochemistry • Histological analyses of liver sections (NAFLD score and fibrosis) Figure 1.3 ALT levels Figure 1.1 NAFLD activity scores 0 50 100 150 200 250 750 800 Vehicle NP-222 NP-251 NP-120 NP-160 NP-101 NP-221 NP-321 NP-204 NP-287 NP-391 telmisartan Plasma ALT U/L Vehicle NP-222 NP-251 NP-120 NP-160 NP-101 NP-221 NP-321 NP-204 NP-287 NP-391 telmisartan 0 NAFLD activity score 1 2 3 4 5 6 7 * * *** *; p<0.05, **; p<0.01, ***; p<0.001 (vs. vehicle) Vehicle NP-160 NP-287 telmisartan Figure 1.5 Sirius red-stained liver sections Figure 2.3 Reduction in fibrosis Parameter (mean ± SD) Vehicle (n = 8) NP-160 (n = 7) NP-287 (n = 7) Telmisartan (n = 8) Plasma Biochemistry Plasma ALT (U/L) 62 ± 18 43 ± 11 # 47 ± 17 # 50 ± 15 Plasma AST (U/L) 110 ± 39 83 ± 19* 81 ± 19* 148 ± 77 Plasma glucose (mg/dL) 768 ± 117 853 ± 80* 695 ± 93 1095 ± 187** Plasma total cholesterol (mg/dL) 161 ± 13 167 ± 41 154 ± 10 186 ± 50* Plasma triglyeride (mg/dL) 738 ± 268 865 ± 206 561 ± 331 523 ± 130* Serum Biochemistry Serum total cholesterol (mg/dL) 145 ± 7 138 ± 24 138 ± 14 167 ± 44* Serum HDL-cholesterol (mg/dL) 102 ± 11 90 ± 17* 100 ± 11 123 ± 28* Serum LDL-cholesterol (mg/dL) 12 ± 1 11 ± 4 13 ± 3 22 ± 11** Serum VLDL-cholesterol (mg/dL) 25 ± 10 28 ± 21 20 ± 10 18 ± 8* Serum triglyceride (mg/dL) 430 ± 94 467 ± 244 287 ± 185 * 212 ± 84* Liver biochemistry Liver triglyceride (mg/g liver) 30.9 ± 10.5 27.3 ± 5.6 20.5 ± 4.8** 12.4 ± 3.0** Liver hydroxyproline (μg/g total protein) 0.58 ± 0.09 0.53 ± 0.12 0.76 ± 0.12** 0.92 ± 0.16** MDA/protein (nmol/mg) 0.49 ± 0.40 0.31 ± 0.12 0.36 ± 0.18 0.66 ± 0.47 Table 1.1 Biochemistry Bonferroni multiple comparison test. *; p<0.1, **; p<0.05 (vs. Vehicle), #; p=n.d. Bonferroni multiple comparison test. *; p<0.05, (vs. Vehicle) Figure 1.2 HE-stained liver sections NP-287 Vehicle NP-160 telmisartan Figure 2.1 NAFLD activity scores 0 1 2 3 4 5 6 7 NAFLD activity score Normal NP-160 NP-135 cenicriviroc telmisartan Vehicle Figure 2.2 HE-stained liver sections • Eleven repurposed drugs were tested in the STAM TM mouse model of NASH • Three compounds significantly reduced both NAFLD scores and liver fibrosis • One compound (NP-135) showed superior reduction in fibrosis to cenicriviroc Introduction • Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) advance, if untreated, to liver cirrhosis, fibrosis, hepatocellular carcinoma, liver failure and liver-related death. In the United States, NASH affects approximately 2- 5% of the population, and an additional 10-30% have NAFLD. • Algernon Pharmaceuticals pursued a drug repurposing strategy, wherein drugs approved outside the USA and Europe, whose long-term safety data was known, were tested for efficacy in animal models of disease • Here, we describe the results of two experiments investigating a number of repurposed compounds in the STAM TM murine model of liver disease 1 NP-160 (20 mg/kg) and NP-287 (8 mg/kg) reduce NAFLD activity scores NP-160 (20 mg/kg) and NP-287 (8 mg/kg) reduce signs of liver damage NP-160 (20 mg/kg) and NP-287 (8 mg/kg) significantly reduce liver fibrosis Vehicle Normal NP-135 cenicriviroc ****; p<0.0001 (vs. Normal) #; p<0.05, ##; p<0.01, ###; p<0.001 (vs. Vehicle) x200 x200 x200 x200 x200 x200 x200 x200 NP-160 (40 mg/kg) and NP-135 (200 mg/kg) reduce NAFLD activity scores • HE-stained liver sections in mice treated with test compounds showed less steatosis, less lobular inflammation and less degeneration of liver cells and nuclei (ballooning) than the Vehicle • NP-160 (20 mg/kg) and NP-287 (8 mg/kg) reduced the NAFLD score by 42% and 56%, respectively (1.9 and 2.5 points) • NP-160 (20 mg/kg) and NP-287 (8 mg/kg) trended towards a reduction in liver damage as measured by plasma ALT and AST levels (ALT by 31% and 23%, AST by 25% and 26%, respectively) • NP-287 (8 mg/kg) significantly reduced liver triglycerides (by 26%) and increased liver hydroxyproline (by 43%) • Sirius red-stained liver sections from the Vehicle group showed increased collagen deposition in the pericentral region of the liver lobule • NP-160 and NP-287 reduced fibrosis by 42% and 39%, respectively, compared to Vehicle • In the same model, telmisartan reduced fibrosis by 26% • HE-stained liver sections in mice treated with Vehicle exhibited micro- and macrovesicular fat deposition, inflammatory cell infiltration and hepatocellular ballooning compared to Normal mice • NP-160 (40 mg/kg) and NP-135 (200 mg/kg) reduced the NAFLD score by 29% and 26%, respectively (1.25 and 1.1 points) • Cenicriviroc (40 mg/kg) – a dual-CCR2/CCR5 antagonist in clinical trials for NASH 2 – reduced the NAFLD score by 34% (1.5 points) 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% NP-160 NP-135 Cenicriviroc Telmisartan Reduction in fibrosis (vs. Vehicle) Figure 2.4 Sirius red-stained liver sections NP-160 (40 mg/kg) and NP-135 (200 mg/kg) significantly reduce liver fibrosis Vehicle Normal NP-135 cenicriviroc • Sirius red-stained liver sections from the Vehicle group showed increased collagen deposition in the pericentral region of the liver lobule • NP-160 (40 mg/kg) and (200 mg/kg) reduced fibrosis by 60% and 84%, respectively, compared to Vehicle • In the same model, cenicriviroc (40 mg/kg) reduced fibrosis by 54% We thank Yuichiro Shibazaki, Taishi Hashiguchi and Yuka Shirakata of SMC Laboratories for experimental contributions. 1. Fujii M et. al. A murine model for non-alcoholic steatohepatitis showing evidence of association between diabetes and hepatocellular carcinoma. Med. Mol. Morphol. 2013;46:141-152. 2. Friedman S, Sanyal A, Goodman Z, Lefebvre E, Gottwald M, Fischer L, Ratziu V. Efficacy and safety study of cenicriviroc for the treatment of non-alcoholic steatohepatitis in adult subjects with liver fibrosis: CENTAUR Phase 2b study design. Contemp Clin Trials 2016;47:356-65. • Several repurposed compounds were screened in the STAM TM model of NASH, and a number of these compounds including NP-160, NP-135 and NP-287 showed significant reductions in both NAFLD score and liver fibrosis compared to Vehicle • NP-160 and NP-135 compared favourably to cenicriviroc, a dual-CCR2/CCR5 antagonist currently in clinical trials for NASH • Early results suggest that the mechanism involves an anti-hypoxic effect • A phase IIa clinical trial is currently being planned for 2019 to assess the efficacy of the lead compound(s) ****; p<0.0001 (vs. vehicle) Figure 1.4 Reduction in fibrosis 0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100% NP-160 NP-287 Telmisartan Reduction in fibrosis (vs. Vehicle) *; p<0.05, **; p<0.01 (vs. vehicle) * ** ** 0 20 40 60 80 100 Hypoxia (%) • NP-135 reduced hypoxia by 51.5% and 64% at concentrations of 5 μM and 10 μM, respectively NP-135 reduces hypoxia in vitro Figure 2.5 Reduction in hypoxia • The effect of NP-135 on hypoxia was studied using a WB experiment in HEK-293 cell lines • 2000 cells/well were seeded • Hypoxia was induced with 10 μM PHT • The treatment time was 24h • For band quantifications, samples were normalized by β-Actin **** **** **** **** x200 x200 x200 x200 x200 x200 x200 x200 **** # ### ##

repurposed compounds np-135 and np-160 reduce fibrosis in … · 2019. 12. 16. · • cenicriviroc...

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