MEK1/2 Inhibition in Murine Heart and Aorta After Oral Administration of Refametinib Supplemented Drinking Water

Abstract

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.

cSTAR analysis identifies endothelial cell cycle as a key regulator of flow-dependent artery remodeling

“…This finding is consistent with reports that HIF1 contributes to inflammatory processes during atherogenesis 19 . Interestingly, the available literature data also suggest that atherogenesis is suppressed by inhibitors of MEK 20 , PAK 21 , and BET bromodomain 22 proteins. For ITK inhibitors, the situation is less clear.…”

An Overview of Investigational and Experimental Drug Treatment Strategies for Marfan Syndrome

“… 182 Recent improvements in the formulation of refametinib, with confirmed inhibition of ERK1/2 activity in mouse aortic and myocardial tissue, will facilitate future experiments with this inhibitor. 184 …”

“…182 Recent improvements in the formulation of refametinib, with confirmed inhibition of ERK1/2 activity in mouse aortic and myocardial tissue, will facilitate future experiments with this inhibitor. 184 Via an alternative noncanonical pathway, the TGF-β receptor complex signals through TGF-β-activated kinase 1 (TAK1) to activate the MAPK kinase (MKK) 4/7 and MKK3/6, which are upstream of JNK and p38 kinase, respectively. 185 Treatment of Fbn1 C1039G/+ mice with the SP600125 JNK inhibitor led to a significant reduction of aortic root growth without affecting ERK1/2 signaling activity.…”