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Upregulation of the RAS-RAF-MEK-ERK-MAPK pathway is involved in the development of several human tumors, aortic aneurysms, atherosclerosis, and cardiomyopathy. Refametinib, a highly selective MEK-inhibitor, has already shown antineoplastic activity in phase II trials. Furthermore, it showed potency to attenuate aortic root growth in murine models. Current formulations of this drug however necessitate oral gavage as a delivery method for long-term studies, which is labor-intensive and induces stress and occasional injury, potentially confounding results. Therefore, we developed a novel oral administration method for refametinib. A 2-hydroxypropyl-beta-cyclodextrin (HPBCD) based drinking water preparation of refametinib was formulated, for which a selective, analytical UHPLC-UV method was developed to assess the in-use stability. Next, 16 week old male wild-type C57Bl/6J mice received either a daily dose of 50 or 75 mg/kg/day refametinib or were given regular drinking water during 7 days. In both dosage groups the refametinib plasma levels were measured (n = 10 or 7, respectively). Furthermore, pERK/total ERK protein levels were calculated in the myocardial and aortic tissue of mice receiving a daily dose of 50 mg/kg/day refametinib and untreated mice (n = 4/group). After 7 days no significant degradation of refametinib was observed when dissolved in drinking water provided that drinking bottles were protected from UV/visible light. Furthermore, a dose-dependent increase in refametinib plasma levels was found whereby active plasma levels (> 1.2 µg/mL) were obtained even in the lowest dose-group of 50 mg/kg/day. A significant reduction of pERK/total ERK protein levels compared to untreated mice was observed in aortic and myocardial tissue of mice receiving a daily dose of 50 mg/kg/day refametinib. Importantly, a relatively high mortality rate was noted in the highest dose group (n = 5). This approach provides a valid alternative oral administration method for refametinib with a reduced risk of complications due to animal manipulation and without loss of functionality, which can be implemented in future research regarding the malignant upregulation of the RAS-RAF-MEK-ERK-MAPK pathway. However, care must be taken not to exceed the toxic dose.

The continuous selective hydrogenation of the iodo-nitroaromatic refametinib active pharmaceutical ingredient (API) intermediate (2,3-difluoro-5-methoxy-6-nitro-phenyl)-(2-fluoro-4-iodo-phenyl)-amine (DIM-NA) to the corresponding iodo-aniline 3,4-difluoro-N2-(2-fluoro-4-iodo-phenyl)-6-methoxy-benzene-1,2-diamine (DIM-DAB) was investigated using a conventional Raney cobalt catalyst in a cocurrent trickle-bed reactor. Suitable reaction conditions targeting full conversion and high selectivity, that is, low degrees of hydro-dehalogenation of the labile iodine substituent, were evaluated in investigations employing a short 4 mL screening reactor. The process was then transferred to a longer 40 mL reactor. Raney cobalt was found to be a highly selective catalyst, yielding only very small amounts of the desiodinated aniline desiodo-DIM-DAB or other side products. Productivities up to 144 g/h crude DIM-DAB were achieved operating the 40 mL reactor with a 0.2 M reactant solution in the presence of 3.4 equiv of H2...

Purpose: Refametinib, an oral MEK inhibitor, has demonstrated antitumor activity in combination with sorafenib in patients with RAS-mutated hepatocellular carcinoma (HCC). Two phase II studies evaluated the efficacy of refametinib monotherapy and refametinib plus sorafenib in patients with RAS-mutant unresectable or metastatic HCC. Patients and Methods: Eligible patients with RAS mutations of cell-free circulating tumor DNA (ctDNA) determined by beads, emulsion, amplification, and magnetics technology received twice-daily refametinib 50 mg ± sorafenib 400 mg. Potential biomarkers were assessed in ctDNA via next-generation sequencing (NGS). Results: Of 1,318 patients screened, 59 (4.4%) had a RAS mutation, of whom 16 received refametinib and 16 received refametinib plus sorafenib. With refametinib monotherapy, the objective response rate (ORR) was 0%, the disease control rate (DCR) was 56.3%, overall survival (OS) was 5.8 months, and progression-free survival (PFS) was 1.9 months. With refametinib plus sorafenib, the ORR was 6.3%, the DCR was 43.8%, OS was 12.7 months, and PFS was 1.5 months. In both studies, time to progression was 2.8 months. Treatment-emergent toxicities included fatigue, hypertension, and acneiform rash. Twenty-seven patients had ctDNA samples available for NGS. The most frequently detected mutations were in TERT (63.0%), TP53 (48.1%), and β-catenin (CTNNB1; 37.0%). Conclusions: Prospective testing for RAS family mutations using ctDNA was a feasible, noninvasive approach for large-scale mutational testing in patients with HCC. A median OS of 12.7 months with refametinib plus sorafenib in this small population of RAS-mutant patients may indicate a synergistic effect between sorafenib and refametinib—this preliminary finding should be further explored. Clin Cancer Res; 24(19); 4650–61. ©2018 AACR.

Although MEK blockade has been highlighted as a promising antitumor drug, it has poor clinical efficacy in KRAS mutant colorectal cancer (CRC). Several feedback systems have been described in which inhibition of one intracellular pathway leads to activation of a parallel signaling pathway, thereby decreasing the effectiveness of single-MEK targeted therapies. Here, we investigated a bypass mechanism of resistance to MEK inhibition in KRAS CRC. We found that KRAS mutant CRC cells with refametinib, MEK inhibitor, induced MIF secretion and resulted in activation of STAT3 and MAPK. MIF knockdown by siRNA restored sensitivity to refametinib in KRAS mutant cells. In addition, combination with refametinib and 4-IPP, a MIF inhibitor, effectively reduced the activity of STAT3 and MAPK, more than single-agent treatment. As a result, combined therapy was found to exhibit a synergistic growth inhibitory effect against refametinib-resistant cells by inhibition of MIF activation. These results reveal that MIF-induced STAT3 and MAPK activation evoked an intrinsic resistance to refametinib. Our results provide the basis for a rational combination strategy against KRAS mutant colorectal cancers, predicated on the understanding of cross talk between the MEK and MIF pathways.

Sorafenib is the only approved systemic treatment for advanced hepatocellular carcinoma patients and all the recently published randomized controlled trials on new systemic drugs have been unsuccessful. This is likely due to a lack of understanding of tumor progression, molecular drivers, and liver toxicity, as well as flaws in trial design. An important signaling pathway in hepatocarcinogenesis is the MEK cascade involved in various cellular responses, including adaptation and survival. A key role in this cascade is played by MEK, of which MEK 1/2 represent the prototypes and an interesting target for new oncological drugs. This review analyzes recent developments and future perspectives on the role of MEK inhibitors in hepatocellular carcinoma treatment.

Background: Activating KRAS mutations are reported in up to 90% of pancreatic cancers. Refametinib potently inhibits MEK1/2, part of the MAPK signaling pathway. This phase I/II study evaluated the safety and efficacy of refametinib plus gemcitabine in patients with advanced pancreatic cancer. Methods: Phase I comprised dose escalation, followed by phase II expansion. Refametinib and gemcitabine plasma levels were analyzed for pharmacokinetics. KRAS mutational status was determined from circulating tumor DNA. Results: Ninety patients overall received treatment. The maximum tolerated dose was refametinib 50mg twice daily plus standard gemcitabine (1000mg/m2 weekly). The combination was well tolerated, with no pharmacokinetic interaction. Treatment-emergent toxicities included thrombocytopenia, fatigue, anemia, and edema. The objective response rate was 23% and the disease control rate was 73%. Overall response rate, disease control rate, progression-free survival, and overall survival were higher in patients without detectable KRAS mutations (48% vs. 28%, 81% vs. 69%, 8.8 vs. 5.3months, and 18.2 vs. 6.6months, respectively). Conclusion: Refametinib plus gemcitabine was well tolerated, with a promising objective response rate, and had an acceptable safety profile and no pharmacokinetic interaction. There was a trend towards improved outcomes in patients without detectable KRAS mutations that deserves future investigation. [Figure not available: see fulltext.]

Background: Mutations affecting the Wnt/β-catenin pathway have been found in 26-40% of HCC samples. Aberrant constitutive activation of this pathway leads to uncontrolled cell proliferation growth and survival of tumor cells. Thus, identification of Wnt/β-catenin pathway inhibitors might lead to a new treatment option for a large subset of HCC patients. In the present study, we aim to investigate the effects of Sorafenib plus the allosteric MEK1/2 inhibitor Refametinib (Sorafenib/Refametinib) on Wnt/β-catenin signaling in vitro and in PDX models of HCC. Methods: We treated 15 patient-derived HCC xenograft models with i) 10 mg/kg Sorafenib QD, ii) 15 mg/kg Refametinib QD, and iii) Sorafenib/Refametinib. Western blotting was employed to determine pharmacodynamic changes in biomarkers relevant Wnt/β-catenin signaling. Apoptosis, cell proliferation and localization of β-catenin were analyzed by immunohistochemistry. In-vitro explant culture from 3 patient-derived HCC lines was also tested. Results: In both wild-type and activated-mutated β-catenin HCC primary cells, Refametinib and Sorafenib/Refametinib potently inhibited cell proliferation and caused cell death, which were correlated with downregulation of Dvl3 and β-catenin target genes (Axin2, c-myc, cyclin D1 and survivin). Inhibition of p-LRP6 at Ser1490 and p-β-catenin at Tyr142 and elevation of E-cadherin were also observed. These changes were not observed in Sorafenib-treated samples. In addition, the Sorafenib/Refametinib combination also suppressed Wnt3a-induced p-LRP6 at Ser1490 and β-catenin accumulation in cells. In vivo, significant reductions in p-LRP6 at Ser1490 and p-β-catenin at Tyr142 were observed in tumor samples from the Sorafenib/Refametinib treatment group and to a lesser extent, samples from the Refametinib. Although the overall protein levels of β-catenin were not significantly changed, non-phosphorylated (active) β-catenin Ser33/37/Thr41 and WNT/β-catenin target genes in Refametinib- and Sorafenib/Refametinib-treated tumors were significantly reduced. While vehicle-treated β-catenin-mutated tumors showed strong cytoplasmatic and nuclear β-catenin localization, most of β-catenin in Sorafenib/Refametinib- and, to a lesser extent, Refametinib-treated tumors was accumulated at the membrane. Conclusion: Sorafenib/Refametinib and, to a lesser extent, Refametinib inhibit the growth of patient derived explant cultures and PDX models. They also inactivate β-catenin signaling. Since activating mutations in the Wnt/β-catenin pathway are detected in a high proportion of HCC, combination of Sorafenib and Refametinib may represent an alternative treatment for β-catenin-dependent HCC. Citation Format: Huynh T. Hung, Richard Ong, Florian Puehler, Arne Scholz, Oliver Politz, Dieter Zopf, Dominik Mumberg, Karl Ziegelbauer. Sorafenib/Refametinib potently inhibits Wnt/β-catenin in vitro and patient-derived xenograft models of human hepatocellular carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2276.

2588 Background: Dual inhibition of the PI3K and MAPK signaling pathways is a promising strategy for optimal anticancer therapy. In this phase 1b trial we determined the maximum tolerated dose (MTD...

4129 Background: Refametinib is a potent oral allosteric MEK 1/2 inhibitor with both single-agent and synergistic activity in combination with gemcitabine in preclinical models of pancreas cancer (...

4025 Background: Refametinib is a potent oral allosteric MEK 1/2 inhibitor with single-agent and synergistic activities in combination with gemcitabine in preclinical pancreatic cancer (PC) models....