Kinase inhibitor rescues the liver

The healthy liver has a remarkable capacity to regenerate. However, injury — due to disease or extended hepatectomy — diminishes this regenerative capacity, and hepatocytes may be unable to sufficiently regenerate to maintain a critical functional liver mass. Writing in Cell, Zwirner et al. now report the development of a first-in-class MKK4 inhibitor, which boosts liver regeneration and prevents liver failure in mouse and pig hepatectomy models. In a phase I trial in healthy volunteers, the small molecule demonstrated excellent safety and pharmacokinetics.

The dual specificity protein kinase MKK4 is a component of the stress-activated protein kinase (SAPK)/mitogen-activated protein kinase (MAPK)-signalling networks, which is activated upon exposure of a cell to stress stimuli. Previously, the authors reported MKK4 to be a master regulator of hepatocyte regeneration. In mice, short hairpin RNA (shRNA)-mediated silencing of MKK4 unlocked the endogenous regenerative capacity of hepatocytes in injured livers. As there are currently no therapies to restore hepatocyte regeneration, Zwirner et al. set out to assess the potential of therapeutically inhibiting MKK4.

First, to mimic the systemic activity of MKK4 inhibitors and assess potential safety, Zwirner et al. engineered MKK4 shRNA transgenic mice. When subjected to partial hepatectomy, these mice exhibited enhanced liver regeneration compared to control mice. Importantly, MKK4 silencing did not affect body weight and there were no signs of toxicity or increased risk of liver tumorigenesis.

The authors therefore proceeded with a drug discovery programme. MKK4 represents a relatively unexplored therapeutic target, with little information available regarding the conformational structure of the protein and insufficiently resolved crystal structures of the kinase domain. Zwirner et al. therefore began with a literature search to investigate reported off-target profiles of known protein kinase inhibitors. This interrogation revealed that the clinically approved BRAF^V600E inhibitor vemurafenib exerted potential MKK4 inhibitory activity.

A series of modifications to the chemical structure of vemurafenib, in conjunction with nuclear magnetic resonance spectroscopy studies, ultimately resulted in the identification of a potent, selective MKK4 inhibitor (MKK4i), HRX215.

In mice, HRX215 displayed favourable pharmacokinetic properties after oral administration. HRX215 treatment prior to partial hepatectomy increased the number of proliferating hepatocytes; HRX215 had no effect on hepatocyte proliferation in mice that had not undergone surgery. Mechanistically, when MKK4i-dosed mice were subjected to partial hepatectomy, SAPK signalling was rerouted via MKK7 and JNK1, which subsequently activated an ATF2- and ELK1-dependent pro-regenerative programme, as previously reported in mice subjected to shRNA-mediated MKK4 silencing.

HRX215 also enhanced hepatocyte proliferation upon partial hepatectomy in fibrotic mouse livers and attenuated apoptosis in a mouse model of CCL₄-induced liver damage. Furthermore, the MKK4i exhibited antisteatotic and antifibrotic efficacy in mouse models of alcoholic steatohepatitis and chronic CCl₄ treatment-induced liver fibrosis.

In addition, HRX215 induced liver regeneration in pig hepatectomy models. Strikingly, in a lethal 85% hepatectomy model, intravenous treatment of pigs with HRX215 either 12 hours before or 12 hours after surgery prevented post-hepatectomy liver failure, enabling survival of 4 out of 6, and 5 out of 6 animals, respectively. By contrast, 5 of the 6 carrier-treated animals fell into coma, necessitating euthanization.

Subsequent toxicity studies in rats and dogs demonstrated no effect of HRX215 on the cardiovascular, neurological or respiratory systems and no drug-related toxicity.

Based on the encouraging preclinical data, a double-blind placebo-controlled phase I clinical trial was performed in 48 healthy male volunteers. HRX215 administered orally in a capsule formulation demonstrated favourable pharmacokinetics, safety and tolerability.

Together, these findings support further clinical investigation of HRX215 as a therapeutic option to prevent or treat liver failure in various settings, such as following oncological liver resections or after graft transplantation.