Soluble epoxide hydrolase: a novel therapeutic target in stroke

Abstract

The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke.

Figure 1

Soluble epoxide hydrolase (sEH) expression in brain. Western blot analysis of brain vascular and non-vascular compartments shows that sEH is predominantly expressed in brain’s parenchymal, and to a lesser degree in cerebral vessels. Vascular smooth muscle α-actin is restricted to the vessel fraction, suggesting effective separation of both compartments. Western blot is representative of three blots.

Figure 2

Localization of soluble epoxide hydrolase (sEH) immunoreactivity in mouse brain neuronal cell bodies and processes. Immunoreactivity for sEH in the cerebral cortex (A to C) and striatum (D) is localized to neuronal cell bodies (dotted arrows) and processes (solid arrows). Three immunohistochemistry runs were performed on n = 3 mice.

Figure 3

Pharmacokinetic profile of sEH inhibitor AUDA-BE and its metabolites in plasma. AUDA-BE was administered to mice as a single intraperitoneal injection of either 40 mg/kg (A) or 10 mg/kg (B). 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester and the metabolites AUDA and AUBA were analyzed at 1, 3, 6, and 24 h after injection. Control injections of sesame oil without AUDA-BE led to concentrations of parent drug or metabolites below the detection limits (0.96, 1.10, and 5.78 nmol/L for AUDA, AUDA-BE, and AUBA, respectively). Control values were subtracted from corresponding values at each time point. AUBA is a biologically inactive indicator metabolite. The mean value at each time point represents two animals.

Figure 4

12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester inhibits soluble epoxide hydrolase enzymatic activity in mouse brain tissue after systemic administration. Activity was measured at 1, 3, 6, and 24 h after single AUDA-BE administration (10 mg/kg intraperitoneally) using sEH-specific surrogate substrate [³H]-trans-1,3-diphenylpropene oxide (tDPPO). Average activity over 24 h was reduced from 1.92 ± 0.06nmol/mg in vehicle-treated mice to 1.56 ± 0.13 nmol/mg protein in AUDA-BE-treated mice (n = 2) at each of four time points: 1, 3, 6, and 24 h after injection, P < 0.05.

Figure 5

12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester decreases infarct size after middle cerebral artery occlusion (MCAO) in mice. Infarct size was reduced from 33 ± 2% (n = 5) in vehicle-treated mice to 20±5% (n = 10) and 16±6% (n = 5) when AUDA-BE was administered (10 mg/kg intraperitoneally) 1 h before (pre) or at the time of reperfusion (post) after 2-h MCAO. *Different from vehicle (P<0.05).

Figure 6

P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargylloxyphenyl) hexanamide (MS-PPOH) eliminates protection by AUDA-BE. Infract size was reduced by AUDA-BE alone (10 mg/kg, a single intraperitoneal injection at reperfusion) from 33±2% to 16±6% (n = 5 per group). However, when combined with MS-PPOH (0.5 mg/200 µL over 24 h before MCAO, via subcutaneously implanted osmotic mini-pumps), AUDA-BE loses it protective effect (infarct size 34±7%, n = 5, not different from vehicle). *Different from vehicle (P<0.05).

Figure 7

Blood flow rates distribution in mouse brain during MCAO. Flow rates were quantified by [¹⁴C]-iodoantipyrine (IAP) autoradiography at the end of 2-h MCAO in mice treated with vehicle (sesame oil, n = 5) or AUDA-BE (10 mg/kg intraperitoneally, 30 mins before MCAO, n = 5). (A) Color-coded distribution of regional CBF rates. (B) Blood flow distribution in the ipsilateral hemisphere. No differences were observed in the amount of tissue (mm³) perfused with any given flow rate (mL/100 g per min) between vehicle- and AUDABE-treated mice (n = 5 per group).