Pharmacokinetic screening of soluble epoxide hydrolase inhibitors in dogs

Abstract

Epoxyeicosatrienoic acids that have anti-hypertensive and anti-inflammatory properties are mainly metabolized by soluble epoxide hydrolase (sEH, EC 3.3.2.3). Therefore, sEH has emerged as a therapeutic target for treating various cardiovascular diseases and inflammatory pain. N,N′-Disubstituted ureas are potent sEH inhibitors in vitro. However, in vivo usage of early sEH inhibitors has been limited by their low bioavailability and poor physiochemical properties. Therefore, a group of highly potent compounds with more drug-like physiochemical properties were evaluated by monitoring their plasma profiles in dogs treated orally with sEH inhibitors. Urea compounds with an adamantyl or a 4-trifluoromethoxyphenyl group on one side and a piperidyl or a cyclohexyl ether group on the other side of the urea function showed pharmacokinetic profiles with high plasma concentrations and long half lives. In particular, the inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB) not only is very potent with good physiochemical properties, but also shows high oral bioavailability for doses ranging from 0.01 to 1 mg/kg. This compound is also very potent against the sEH of several mammals, suggesting that t-AUCB will be an excellent tool to evaluate the biology of sEH in multiple animal models. Such compounds may also be a valuable lead for the development of veterinary therapeutics.

Introduction

Epoxyeicosatrienoic acids (EETs) are endogenous signalling molecules produced by cytochrome P450 from arachidonic acid (Spector, 2008). The EETs act as vasodilators by activating Ca²⁺-activated K⁺ channels in vascular smooth muscle cells (Fleming, 2007). EETs also possess anti-inflammatory properties in endothelial cells (Node et al., 1999). EETs and related epoxy fatty acids are mainly metabolized by the soluble epoxide hydrolase (sEH; EC 3.3.2.3) (Liu et al., 2005, Newman et al., 2005), which hydrolyzes the EETs into dihydroxyeicosatrienoic acids (DHETs), resulting in partial or complete loss of their initial biological activities. Inhibition of sEH stabilizes endogenous EETs and thus reduces hypertension in multiple rodent models (Imig et al., 2002, Imig et al., 2005, Jung et al., 2005, Chiamvimonvat et al., 2007, Huang et al., 2007, Loch et al., 2007). The inhibition of sEH also reduces the production of nitric oxide, cytokines and pro-inflammatory lipid mediators, in addition to significantly improving survival in lipopolysaccharide (LPS)-induced systemic inflammation in mice (Liu et al., 2005, Schmelzer et al., 2005, Smith et al., 2005). The sEH is also a potential target for the treatment of ischemic stroke (Dorrance et al., 2005, Zhang et al., 2007a) and cardiac hypertrophy (Xu et al., 2006). Furthermore, sEH inhibition reduces pain in several rodent models (Inceoglu et al., 2006). Thus, sEH is an emerging target for pharmacological treatment of cardiovascular diseases (Monti et al., 2008), inflammation, and possibly other diseases (Gauthier et al., 2007, Harris et al., 2008).

N,N′-Disubstituted ureas are potent sEHIs (sEH inhibitors) with K_is in the low nanomolar range (Morisseau et al., 1999b). Early urea-based inhibitors, such as 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), 12-(3-adamantane-1-yl-ureido)-dodecanoic acid n-butyl ester (AUDA-nBE) and 1-adamantan-1-yl-3-{5-[2-(2-ethoxyethoxy)ethoxy]pentyl}urea (AEPU) (Imig et al., 2002, Kim et al., 2004), were used to investigate the biology associated with the inhibition of sEH in animal models (Dorrance et al., 2005, Imig et al., 2005, Jung et al., 2005, Schmelzer et al., 2005, Sellers et al., 2005, Smith et al., 2005, Inceoglu et al., 2006, Xu et al., 2006, Ai et al., 2007, Huang et al., 2007, Loch et al., 2007, Zhang et al., 2007a, Li et al., 2008, Olearczyk et al., 2009, Parrish et al., 2009). However, their in vivo usage is limited due to poor physiochemical properties, rapid metabolism and/or poor bioavailability. Tedious formation such as the use of nanocrystals from cryomilling with careful selection of salts and solvents are needed to formulate such materials for efficient delivery in a small capsule (Ghosh et al., 2008). As an alternative to such formulation approaches, a medicinal chemistry approach was taken to develop new potent inhibitors that are more water soluble and more metabolically stable by methodically modifying their structures (Zhao et al., 2004, Hwang et al., 2006, Jones et al., 2006, Li et al., 2006, Morisseau et al., 2006, Hwang et al., 2007, Kim et al., 2007a, Kim et al., 2007b, Kasagami et al., 2009, Shen et al., 2009).

While simple, rapid and efficient methods have been developed to estimate the inhibitory potency of new compounds (Jones et al., 2005, Wolf et al., 2006) as well as their solubility and other physiochemical properties, it is more difficult and costly to evaluate their pharmacokinetic properties. It is believed that compounds with favorable pharmacokinetics are more likely to be efficacious and safe (Dingemanse and Appel-Dingemanse, 2007). We previously developed a rapid pharmacokinetic screening method using cassette dosing and measuring compounds with minuscule serial bleedings in mice (Watanabe et al., 2006). This method was efficient in classifying compounds on their relative bioavailability. However, because of the small size of the animal used, it is difficult to extrapolate to larger animals or humans. Although we routinely monitor blood levels with 5 μl of blood for these compounds (Watanabe et al., 2006), the small volume of blood in a mouse limits our ability to monitor multiple blood biomarkers and, in particular, oxylipins which are promising indicators of the efficacy of sEHIs in vivo. Herein we report the use of dogs (black Labrador retrievers) for rapid pharmacokinetics screen of sEHIs. Due to the relatively large body size of the dogs (∼20 kg each), this method allows for easier administration of drugs and collection of plasma with larger sample size. Moreover, dogs are much closer phylogenetically and in size to humans than rodents, making extrapolation to humans more reliable (Tibbitts, 2003). Finally, there is a need for novel anti-inflammatory and pain therapies in dogs and cats (Lascelles et al., 2005). For example, the non-steroid anti-inflammatory drugs (NSAIDs) are potent canine analgesic and anti-inflammatory drugs, but their use is limited by a variety of adverse effects such as causing gastrointestinal disorders. Thus, the canine data obtained herein will not only be valuable in refining the structure of sEHI for clinical use in humans and for use in canine models of human diseases, but also these data could lead to the development of novel veterinary therapeutics either used alone or as synergists with existing drugs such as NSAIDs (Schmelzer et al., 2006).