Pharmacologic activity and pharmacokinetics of metabolites of regorafenib in preclinical models

Dieter Zopf, Iduna Fichtner, Ajay Bhargava, Wolfram Steinke, Karl-Heinz Thierauch, Konstanze Diefenbach, Scott Wilhelm, Frank-Thorsten Hafner, Michael Gerisch, Dieter Zopf, Iduna Fichtner, Ajay Bhargava, Wolfram Steinke, Karl-Heinz Thierauch, Konstanze Diefenbach, Scott Wilhelm, Frank-Thorsten Hafner, Michael Gerisch

Abstract

Regorafenib is an orally administered inhibitor of protein kinases involved in tumor angiogenesis, oncogenesis, and maintenance of the tumor microenvironment. Phase III studies showed that regorafenib has efficacy in patients with advanced gastrointestinal stromal tumors or treatment-refractory metastatic colorectal cancer. In clinical studies, steady-state exposure to the M-2 and M-5 metabolites of regorafenib was similar to that of the parent drug; however, the contribution of these metabolites to the overall observed clinical activity of regorafenib cannot be investigated in clinical trials. Therefore, we assessed the pharmacokinetics and pharmacodynamics of regorafenib, M-2, and M-5 in vitro and in murine xenograft models. M-2 and M-5 showed similar kinase inhibition profiles and comparable potency to regorafenib in a competitive binding assay. Inhibition of key target kinases by all three compounds was confirmed in cell-based assays. In murine xenograft models, oral regorafenib, M-2, and M-5 significantly inhibited tumor growth versus controls. Total peak plasma drug concentrations and exposure to M-2 and M-5 in mice after repeated oral dosing with regorafenib 10 mg/kg/day were comparable to those in humans. In vitro studies showed high binding of regorafenib, M-2, and M-5 to plasma proteins, with unbound fractions of ~0.6%, ~0.9%, and ~0.4%, respectively, in murine plasma and ~0.5%, ~0.2%, and ~0.05%, respectively, in human plasma. Estimated free plasma concentrations of regorafenib and M-2, but not M-5, exceeded the IC50 at human and murine VEGFR2, suggesting that regorafenib and M-2 are the primary contributors to the pharmacologic activity of regorafenib in vivo.

Keywords: Antitumor activity; metabolite; multikinase inhibitor; pharmacology; regorafenib.

© 2016 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.

Figures

Figure 1
Figure 1
Biochemical kinase selectivity profiles of regorafenib, M‐2, and M‐5. Only kinases with Kd values ≤100 nmol/L are displayed (Table S1 for Kd values of the studied kinase panel). Image generated using the TREEspot™ Software Tool and reprinted with permission from KINOMEscan®, a division of DiscoveRx Corporation, © DiscoverRx Corporation 2010.
Figure 2
Figure 2
Inhibitory effects of regorafenib, M‐2, and M‐5 in cell‐based mechanistic assays. (A) VEGFR2 autophosphorylation in HuVECs. (B) VEGFR3 autophosphorylation in HuLECs and effects on potential intracellular signaling kinases ERK1/2 and AKT. *Indicates unspecific signals. (C) Inhibition of cell migration, analyzed by scratch assay in HuLECs. Black lines demarcate the borders of the confluent cell layer. (D) Inhibition of FGFR2 autophosphorylation in SNU‐16 tumor cells. Regorafenib data in A, B, and C were taken from Schmieder et al. 14.
Figure 3
Figure 3
Effects of regorafenib, M‐2, and M‐5 on the growth of human xenografts in mice. Data show relative tumor volume in mice bearing xenografts of (A) human breast cancer cell line MDA‐MB‐231 (KRASG13D, BRAFG464V) and (B) human CRC cell line HT‐29 (BRAFV600E) following oral administration of 10 mg/kg/day of regorafenib, M‐2, or M‐5 for 27 days, starting at Day 13 or Day 11, respectively, after tumor inoculation (palpable tumor size; n = 8; *P < 0.05 for regorafenib versus vehicle; †P < 0.05 for M‐2 versus vehicle; ‡P < 0.05 for M‐5 versus vehicle)

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