Allosteric MEK1/2 inhibitor refametinib (BAY 86-9766) in combination with sorafenib exhibits antitumor activity in preclinical murine and rat models of hepatocellular carcinoma

Abstract

Objective: The objectives of the study were to evaluate the allosteric mitogen-activated protein kinase kinase (MEK) inhibitor BAY 86-9766 in monotherapy and in combination with sorafenib in orthotopic and subcutaneous hepatocellular carcinoma (HCC) models with different underlying etiologies in two species.

Design: Antiproliferative potential of BAY 86-9766 and synergistic effects with sorafenib were studied in several HCC cell lines. Relevant pathway signaling was studied in MH3924a cells. For in vivo testing, the HCC cells were implanted subcutaneously or orthotopically. Survival and mode of action (MoA) were analyzed.

Results: BAY 86-9766 exhibited potent antiproliferative activity in HCC cell lines with half-maximal inhibitory concentration values ranging from 33 to 762 nM. BAY 86-9766 was strongly synergistic with sorafenib in suppressing tumor cell proliferation and inhibiting phosphorylation of the extracellular signal-regulated kinase (ERK). BAY 86-9766 prolonged survival in Hep3B xenografts, murine Hepa129 allografts, and MH3924A rat allografts. Additionally, tumor growth, ascites formation, and serum alpha-fetoprotein levels were reduced. Synergistic effects in combination with sorafenib were shown in Huh-7, Hep3B xenografts, and MH3924A allografts. On the signaling pathway level, the combination of BAY 86-9766 and sorafenib led to inhibition of the upregulatory feedback loop toward MEK phosphorylation observed after BAY 86-9766 monotreatment. With regard to the underlying MoA, inhibition of ERK phosphorylation, tumor cell proliferation, and microvessel density was observed in vivo.

Conclusion: BAY 86-9766 shows potent single-agent antitumor activity and acts synergistically in combination with sorafenib in preclinical HCC models. These results support the ongoing clinical development of BAY 86-9766 and sorafenib in advanced HCC.

Figure 1

Synergistic antiproliferative activity of BAY 86-9766 and sorafenib in HCC cell lines. Isobologram analysis of the interaction between BAY 86-9766 and sorafenib in human Hep3B cells and rat MH3924A cells is shown in A. Points below the straight diagonal line indicate a synergistic interaction. The combination index analysis for the interaction between the two drugs in these HCC lines is also shown in A (D1, sorafenib; D2, BAY 86-9766). Effects of BAY 86-9766 and sorafenib, as single agents and in combination, in MH3924A cells are shown in B. Phosphorylation of ERK was more potently blocked due to combination treatment in comparison to BAY 86-9766 and sorafenib monotreatment after 12 hours of compound incubation. Additionally, compensatory up-regulation of phosphorylated MEK was diminished in the combination-treated cells.

Figure 2

Effect of BAY 86-9766 and sorafenib, as single agents and in combination, in the orthotopic HBV-driven human Hep3B xenograft model. Kaplan-Meier survival curves and results are presented in A; comparison of survival with BAY 86-9766 monotherapy versus vehicle in the orthotopic murine Hepa129 HCC allograft model is presented in B.

Figure 3

Effect of BAY 86-9766 and sorafenib, as single agents and in combination, on tumor growth, metastatic spread, and alkaline phosphatase levels in the orthotopic rat MH3924A allograft model. The primary tumor, which has been cut out and measured on day 21 following orthotopic implantation, is depicted in A. Significance is indicated by asterisk (P < .05). Representative photos of the primary tumor from rats treated with vehicle control or combination therapy are shown in B. The percentage of animals with metastases is presented in C. Mean (+SD) serum alkaline phosphatase levels are shown in D.

Figure 4

Effect of BAY 86-9766 and sorafenib, as single agents and in combination, in MH3924A cells and MH3924A allografts. Inhibitory effect of BAY 86-9766 on phosphorylation of ERK: pERK staining of liver tumor paraffin sections of animals (A) from (i) vehicle group, (ii) BAY 86-9766 group, (iii) sorafenib group, and (iv) combination group. The proportions of cells staining positive for Ki-67 are shown in B. Inhibitory effect of BAY 86-9766 on angiogenesis with staining with von Willebrand factor is shown in C. Significance is indicated by asterisk (P < .05). Kaplan-Meier survival curves and median survival data are shown in D.

Figure 4

Effect of BAY 86-9766 and sorafenib, as single agents and in combination, in MH3924A cells and MH3924A allografts. Inhibitory effect of BAY 86-9766 on phosphorylation of ERK: pERK staining of liver tumor paraffin sections of animals (A) from (i) vehicle group, (ii) BAY 86-9766 group, (iii) sorafenib group, and (iv) combination group. The proportions of cells staining positive for Ki-67 are shown in B. Inhibitory effect of BAY 86-9766 on angiogenesis with staining with von Willebrand factor is shown in C. Significance is indicated by asterisk (P < .05). Kaplan-Meier survival curves and median survival data are shown in D.

Figure 5

Pharmacokinetics of BAY 86-9766 and sorafenib in rats with orthotopic MH3924A allografts. Unbound plasma concentration-time curves after last dose of combined treatment with 3 mg/kg qd BAY 86-9766 and 6 mg/kg qd sorafenib (given 5.75 hours later) are shown in A, with the horizontal lines depicting the IC₅₀ values of the respective drugs in the MH3924A cells in vitro. Pharmacokinetic parameters for each drug individually and in combination are shown in B.