Substituted phenyl groups improve the pharmacokinetic profile and anti-inflammatory effect of urea-based soluble epoxide hydrolase inhibitors in murine models

Abstract

Soluble epoxide hydrolase inhibitors (sEHIs) are anti-inflammatory, analgesic, anti-hypertensive, cardio- and renal-protective in multiple animal models. However, the earlier adamantyl-containing urea-based inhibitors are rapidly metabolized. Therefore, new potent inhibitors with the adamantyl group replaced by a substituted phenyl group were synthesized to presumptively offer better pharmacokinetic (PK) properties. Here we describe the improved PK profile of these inhibitors and the anti-inflammatory effect of the most promising one in a murine model. The PK profiles of inhibitors were determined following p.o. administration and serial bleeding in mice. The anti-inflammatory effect of 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU), the most promising inhibitor among the five sEHIs tested, was investigated in a lipopolysaccharide (LPS)-challenged murine model. The earlier broadly-used adamantyl-containing sEHI, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), was used for comparison. Compared with the earlier adamantyl-containing urea-based inhibitors, substituted phenyl-containing urea-based inhibitors afford more favorable PK properties, such as higher C_maxs, larger AUCs and longer t_1/2s, which, as expected, show more stable metabolic stability. Moreover, oral administration of TPPU dramatically reversed the shifts caused by LPS-challenge in plasma levels of inflammatory cytokines, epoxides and corresponding diols, which is more potent than t-AUCB. The substituted phenyl-containing sEHIs are more metabolically stable than those with adamantyl group, resulting in more potent efficacy in vivo. This indicates a new strategy for development of sEHIs for further study toward clinical trials.

Introduction

Soluble epoxide hydrolase (sEH) is a ubiquitous enzyme that catalyzes the conversion of epoxides into the corresponding vicinal diols by the addition of water (Hammock et al., 1980, Morisseau and Hammock, 2005, Newman et al., 2005). For example, sEH catalyzes the metabolism of anti-inflammatory and vasodilatory epoxyeicosatrienoic acids (EETs) into the more polar and pro-inflammatory dihydroxyeicosatrieneoic acid (DHETs) (Chacos et al., 1983, Morisseau and Hammock, 2005, Newman et al., 2005). Pharmacological inhibition of sEH by sEH inhibitors (sEHIs) has been demonstrated to be an effective approach to reduce infammation, pain, and hypertension among others (Ingraham et al., 2011, Qiu et al., 2011). Significant progress has been made in recent years in the development of the amide-, urea- and carbamate-based compounds as potent sEHIs (Morisseau et al., 1999). N,N′-Dicyclohexylurea (DCU) was the first sEHI tested in spontaneously hypertensive rats (SHRs), and it was effective in lowering blood pressure (Yu et al., 2000). Another sEHI 1-cyclohexyl-3-dodecyl urea (CDU) was reported not only to attenuate human vascular smooth muscle cell proliferation dose-dependently in vitro (Davis et al., 2002), but also to be anti-hypertensive and renal protective in a rodent model of angiotensin II-induced hypertension (Imig et al., 2002, Zhao et al., 2004). However, these inhibitors have high melting points and poor solubility in either water or oil, which limits their pharmacological use. Therefore a new series of N,N′-substituted urea was then designed to desirably improve the water solubility and melting point from the early inhibitors (Kim et al., 2004, Morisseau et al., 2002). Among such series, 1-adamantanyl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea (AEPU), N-adamantyl-N′-dodecylurea (ADU), 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and its butyl ester (AUDA-BE) have been widely tested in animal models for anti-inflammation (Schmelzer et al., 2005, Smith et al., 2005), analgesic (Inceoglu et al., 2006, Schmelzer et al., 2006), anti-hypertension (Imig et al., 2005, Loch et al., 2007, Revermann et al., 2009), renal-protection (Huang et al., 2007, Parrish et al., 2009), and cardio-protection (Dorrance et al., 2005, Xu et al., 2006). These compounds have improved water solubility and melting points in comparison to the earlier sEHIs such as DCU and CDU. However, they were rapidly metabolized and excreted due to the facile oxidation of their flexible alkyl chain, which limits their use in vivo. Therefore, conformationally-restricted sEHIs such as 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU) and trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) were synthesized based on the hypothesis that conformationally-restricted compounds will eliminate beta oxidation, resulting in improved metabolic stability (Hwang et al., 2007, Jones et al., 2006). This hypothesis was supported by subsequent pharmacokinetics (PK) studies in murine and canine models (Liu et al., 2009, Tsai et al., 2010).

In several biological studies both the cis- and trans-isomers of AUCB demonstrated anti-inflammatory (Liu et al., 2009, Manhiani et al., 2009, Rose et al., 2010), anti-hypertensive (Revermann et al., 2009), cardio-protective (Ai et al., 2009, Chaudhary et al., 2010, Li et al., 2009) and renal-protective effects (Manhiani et al., 2009). However, the adamantyl group has been shown in pharmacology to be metabolically unstable (Lamoureux and Artavia, 2010). For example, the adamantyl group at t-AUCB gave an attractive PK profile for once a day oral dosing in dogs, but in other species it required higher and more frequent doses for efficacy. Interestingly, we found in previous studies that TPAU (containing 4-trifluoro-methoxy-phenyl group) is more metabolically stable than APAU (containing adamantyl group) (Liu et al., 2009, Tsai et al., 2010), suggesting a possibility that replacement of an adamantyl group by a substituted phenyl group may improve the metabolic stability of urea-based sEHIs. To test this hypothesis, a series of substituted phenyl urea-based compounds were synthesized. The PK profiles of five such inhibitors that afford favorable IC₅₀s in vitro (Table 1) were then tested in a murine model at four different doses with single oral administration. Here we present the PK profiles of these compounds and the anti-inflammatory effect of 1-(4-trifluoro-methoxy-phenyl)-3-(1-propionylpiperidin-4-yl)urea (TPPU), the most promising compound among the five tested compounds in murine models.