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Evaluation of NV556, a Novel Cyclophilin Inhibitor, as a Potential Antifibrotic Compound for Liver Fibrosis

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Evaluation of NV556, a Novel Cyclophilin Inhibitor, as a Potential Antifibrotic Compound for Liver Fibrosis

Sonia Simón Serrano et al. Cells.

Abstract

Hepatic fibrosis can result as a pathological response to nonalcoholic steatohepatitis (NASH). Cirrhosis, the late stage of fibrosis, has been linked to poor survival and an increased risk of developing hepatocellular carcinoma, with limited treatment options available. Therefore, there is an unmet need for novel effective antifibrotic compounds. Cyclophilins are peptidyl-prolyl cis-trans isomerases that facilitate protein folding and conformational changes affecting the function of the targeted proteins. Due to their activity, cyclophilins have been presented as key factors in several stages of the fibrotic process. In this study, we investigated the antifibrotic effects of NV556, a novel potent sanglifehrin-based cyclophilin inhibitor, in vitro and in vivo. NV556 potential antifibrotic effect was evaluated in two well-established animal models of NASH, STAM, and methionine-choline-deficient (MCD) mice, as well as in an in vitro 3D human liver ECM culture of LX2 cells, a human hepatic stellate cell line. We demonstrate that NV556 decreased liver fibrosis in both STAM and MCD in vivo models and decreased collagen production in TGFβ1-activated hepatic stellate cells in vitro. Taken together, these results present NV556 as a potential candidate for the treatment of liver fibrosis.

Keywords: 3D in vitro model; NV556; STAM; cyclophilin; decellularized human liver; hepatic stellate cells (HSC); liver fibrosis; methionine-choline-deficient (MCD) diet; nonalcoholic steatohepatitis.

Conflict of interest statement

S.S.S., A.G., E.E., and M.J.H are employees of NeuroVive Pharmaceutical AB, which develops NV556. S.S.S., A.G., E.E., and M.J.H. own shares in NeuroVive Pharmaceutical AB. L.L. and G.M. are employees of Engitix Ltd., which develops proprietary 3D extracellular matrix technology. K.R. and M.P. receive consultancies from Engitix Ltd., and G.M., K.R., L.L., and M.P. own shares in Engitix Ltd. M.G. and S.M. are employees of and own shares in Isomerase Therapeutics Ltd. J.K., P.G., and R.M. have nothing to disclose.

Figures

Figure 1
Figure 1
Effect of NV556 treatment in methionine-choline-deficient (MCD)-diet-fed mice. (A) Experimental timeline and parameters measured: (B) body weight, (C) AST, and ALT, (D) liver/body weight ratio (%), (E) liver total cholesterol, liver triglycerides, and liver fatty acids; and (F) NAFLD scoring–inflammation, ballooning, steatosis score, and (G) fibrosis stained by Sirius red. Representative photomicrographs of Sirius red staining (Scale bars indicate 100 µm). Data are represented as mean ± SD and statistically analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test for C, D, and E. F and G were statistically analyzed by Kruskal Wallis, followed by Dunn’s multiple comparison test, *p < 0.05, **p < 0.01, ***p < 0.001, n = 10 mice per treatment.
Figure 2
Figure 2
Effect of NV556 treatment in the STAM model of nonalcoholic steatohepatitis (A) Experimental timeline and parameters measured: (B) body weight and (C) liver/body-weight ratio, (D) plasma triglyceride, whole blood glucose, plasma insulin, (E) liver triglyceride, and (F) NAFLD scoring: inflammation, ballooning, and steatosis score (G) fibrosis area: representative photomicrographs at 200× and values of Sirius-red positive areas. Data are represented as mean ± SD and statistically analyzed by one-way ANOVA, followed by Dunnett’s multiple comparison test for C, D, E, and G. F was statistically analyzed by Kruskal–Wallis, followed by Dunn’s multiple comparison test *p < 0.05, **p < 0.01, ***p < 0.001, n = 8 mice per treatment.
Figure 3
Figure 3
NV556 effect on gene expression (A,B) and procollagen secretion (C) in a 3D human liver model reseeded with LX2 cells. Data in A and B are represented as mean of relative expression over nontreated control cells ± SD, and data in AC are statistically analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01 and ***p < 0.001, n = 4 scaffolds per condition investigated.
Figure 4
Figure 4
NV556 effect on gene expression (A,B) and procollagen secretion (C) in a 3D human liver model reseeded with LX2 cells. Data in A and B are represented as mean of relative expression over nontreated control cells ± SD and Data in AC are statistically analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01 and ***p < 0.001, n = 4 scaffolds per condition investigated.
Figure 5
Figure 5
NV556 dose-response effect on gene expression in 3D human liver model reseeded with LX2 cells. Data are represented as mean of relative expression to inactive control ± SD for LOX (A), COL1A1 (B), COL3A1 (C) and COL4A1 (D) expression and statistically analyzed by one-way ANOVA, followed by Tukey’s multiple comparison test. *p < 0.05, **p < 0.01, and ***p < 0.001. #p <0.05 for TGF-β1 versus nontreated Control cells, n = 4 scaffolds per condition investigated.

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