"Introduction: Cyclophilins are peptidyl-prolyl isomerases that regulate several biological processes. Importantly, cyclophilin-A promotes the remodelling of extracellular matrix (ECM) proteins by activating matrix metalloproteinases and is implicated in the correct folding of collagen fibres. Cyclophilin inhibition has shown beneficial therapeutic effects at various stages of liver disease, including steatosis, fibrosis, and hepatocellular carcinoma. As mortality caused by liver disease and its complications is on the rise, the possibility of targeting cyclophilins represents a strategy that is worth investigating. CRV431 is a pan-cyclophilin inhibitor (non-immunosuppressant cyclosporin derivative) that is currently in clinical development for non-alcoholic liver disease (Phase 2A).
Here, we developed a pipeline for evaluating the effects of CRV431 in two patient-derived models of liver disease, and in particular its role in ECM remodelling. The objectives were:
i) to study the ECM scaffold remodelling by primary human hepatic stellate cells (HSCs - major ECM-producing cells in liver fibrosis) upon fibrosis induction and treatment with CRV431;
ii) to investigate the effects of hepatotoxic insults and CRV431 treatment in a 3D-organotypic and multicellular model (patient-matched precision-cut liver slices - PCLS).
Methodology: Patient-matched PCLS and primary HSCs were prepared from human liver specimens (n=4). PCLS were exposed to hepatotoxic insults (ethanol 250mM, fatty acids 0.1mM, LPS 10µg/ml) with or without 5µM CRV431, for up to 5 days. Quiescent HSCs were activated using TGFβ1, for up to 10 days in the presence/absence of 5µM CRV431. PCLS tissue functionality was evaluated by histology, cytokeratin-18 release and mitochondrial assays. In both PCLS and HSCs, fibrosis/HSC activation status was assessed by gene expression, immunofluorescence and secretion of fibrotic markers. Pro-inflammatory cytokines in the culture media of both PCLS and HSC were quantified by Luminex. ECM remodeling was assessed via atomic force microscopy (AFM) in PCLS, while in HSC cultures, deposited ECM was decellularised and characterized via proteomics and confocal imaging. Fibre thicknesses and alignment was analysed via a Fiji-ImaJ plug-in software fit for the porpoise.
Results: Upon administration of hepatotoxic compounds in PCLS, liver damage was induced, shown by apoptosis makers and pro-fibrogenic/pro-inflammatory cytokines increase. CRV431 reduced the expression and secretion of damage-associated markers and restored a balanced cytokine profile in organotypic liver slices.
In activated HSCs, CRV431 reduced aSMA expression and collagen and fibronectin deposition when added simultaneously or after TGF‐β1 activation. In addition, CRV431 treatment induced modification of ECM fibre alignment, showing that specific ECM protein remodeling is a mechanism of action of CRV431. Importantly, both in 2D and 3D models, CRV431 was not hepatotoxic and did not induce cell death.
Conclusion: Our results confirm the role of cyclophilins in liver fibrosis, including HSC activation, and ECM fibre deposition and alignment. These data reveal for the first time the potential for cyclophilin inhibitor CRV431 to reduce liver fibrosis and propose a mechanism of action of the drug using two patient-derived models. The data support the possibility of using this drug as a potentially safe and effective therapy in liver disease patients."