Sorafenib has soluble epoxide hydrolase inhibitory activity, which contributes to its effect profile in vivo

Determination of the K_I of sorafenib with the human soluble epoxide hydrolase (1 nmol/L) using [³H]-t-DPPO as substrate. For each substrate concentration (3.6–30.0 μmol/L), the velocity is plotted as a function of soluble epoxide hydrolase inhibitor concentration (0–15 nmol/L), allowing the determination of an apparent inhibition constant (K_Iapp). K_Iapps are plotted as a function of the substrate concentration (inset). For [S] = 0, a K_I value of 17 nmol/L was found. Similar plots were obtained with four other inhibitors and the human soluble epoxide hydrolase (see Table 1).

Docking sorafenib with the human soluble epoxide hydrolase enzyme. Sorafenib was manually docked into the active site of human soluble epoxide hydrolase. For this, we used the published X-ray crystal structure of human soluble epoxide hydrolase complex with a urea-based soluble epoxide hydrolase inhibitor (Protein Data Bank accession number 1ZD3) from http://www.rcsb.org. Between two plausible binding modes for sorafenib, the orientation given herein is the one with the lower calculated enthalpy and thus is the most probable. The opposite binding mode resulted in steric clashes with the residues of the binding site, such as Met418. Sorafenib was bound by making H-bonding interactions between the urea group and the residues Tyr381, Tyr465, and Asp333 at the active site of the human soluble epoxide hydrolase enzyme. These amino acids are shown as ball-and-stick structures on the backbone ribbon diagram.

Conventional soluble epoxide hydrolase inhibitors of varying potency do not cause significant apoptosis or attenuate phosphorylation of ERK or VEGF receptor. A, ACHN and A498 cells were serum starved for 18 h and treated with vehicle (QM lane is serum-free quiescence media alone; vehicle is DMSO at1 μL/ml), platelet-derived growth factor, PD98059 (10 μmol/L), sorafenib (10 μmol/L), and soluble epoxide hydrolase inhibitors (10 μmol/L) for 1 h. After incubation, all cells except quiescence media control were stimulated with platelet-derived growth factor (10 ng/mL) for 15 min. Cells were harvested and immunoblotted with phospho-ERK (202Tyr204Thr) or nonphosphorylated ERK, as indicated. β-Actin is a gel loading control. B, ACHN cells were treated as in A, except that the soluble epoxide hydrolase inhibitors were treated at three different doses, as indicated. C, ACHN cells were treated in 10% serum-containing complete media with vehicle (DMSO at 1 μL/mL), PD98059 (10 μmol/L), sorafenib (10 μmol/L), and soluble epoxide hydrolase inhibitors (10 μmol/L) for 24 and 48 h. After incubation, cells were harvested and immunoblotted with phospho-VEGF receptor, VEGF receptor, and PARP. PARP activation is indicated by the appearance of the lower–molecular weight cleavage product. β-Actin is a gel loading control.

Conventional soluble epoxide hydrolase inhibitors of varying potency do not alter cell growth or apoptosis. ACHN and A498 cells were incubated with serum-free quiescence media for 18 h and then treated with 10% serum-containing media containing vehicle (DMSO; 1 μL/mL), sorafenib (10 μmol/L), and indicated soluble epoxide hydrolase inhibitors (10 μmol/L) for an additional 48 h. A, an MTT assay was done as described in Materials and Methods. The visible absorbance of each well was quantified using a microplate reader. B, an assay of caspase-1 and caspase-3 activity was assessed as described in Materials and Methods. The visible absorbance of each well was quantified using a microplate reader.

Neither sorafenib nor sunitinib synergizes with soluble epoxide hydrolase inhibitors on cell survival. A, ACHN and A498 cells were treated as described in Fig. 5 with the soluble epoxide hydrolase inhibitors AUBA and t-AUCB (50 μmol/L) in the presence of vehicle (DMSO), sorafenib (5 μmol/L), or sunitinib (0.5 μmol/L). B, ACHN cells were treated as in A. Epoxyeicosatrienic acids or DMSO vehicle was added to the wells at 10 μmol/L. After 48 h, the MTT assay was done as described in Materials and Methods. The visible absorbance of each well was quantified using a microplate reader.

Blood concentration-time course of sorafenib with oral administration to mice. Sorafenib (as the tosylate at 5 mg/kg) was administered to male Swiss-Webster mice as described in Materials and Methods. Each data point is the mean ± SD of three mice plotted in a log/linear scale. Blood for sorafenib analysis was collected from the tail vein of mice at different time after oral gavage. The data of t-AUCB is from ref. 30. Dotted lines A and B represent the inhibitory IC₅₀s of sorafenib and t-AUCB against the murine soluble epoxide hydrolases, respectively.