Effect of a Soluble Epoxide Hydrolase Inhibitor , UC 1728 , on LPS-Induced Uveitis in the Rabbit Abbreviations

Cytochrome P450 epoxygenase isozymes convert free arachidonic acid into eicosanoids named epoxyeicosatrienoic acids (EETs) that have roles in regulating inflammation. EETs are rapidly converted to dihydroxyeicosatrienoic acids (DiHETs) by soluble epoxide hydrolase (sEH). Little is known about the potential role of these metabolites in uveitis, but conversion of EETs to DiHETs could contribute to the inflammation. We tested a potent and orally available inhibitor of sEH for its ability to reduce ocular inflammation in a rabbit LPS-induced model of uveitis. Rabbits were treated by subcutaneous injection with the sEH inhibitor (UC1728, 3 mg/kg), or the vehicle control (PEG400) and uveitis was assessed at 6, 24 and 48 h post-intracameral LPS injection using a modified Hackett-McDonald scoring system. Eyes treated by intra-cameral injection of PBS, or by aseptic preparation served as further controls. Signs of inflammation in this model were mild and transient. Treatment with UC1728 did not significantly reduce inflammation compared to animals treated with the PEG400 vehicle. Blood levels of UC1728 were a thousand fold higher than the in vitro determined inhibitory potency (IC50) of the compound suggesting a significant degree of inhibition of sEH in the rabbit. The lack of efficacy suggests that sEH or its substrates the EETs may not be involved in mediating inflammation in this model of uveitis. Citation: Gillian J, Aktas MZ, Hennes-Beean E, Aaron W, Inna KV, et al. Effect of a Soluble Epoxide Hydrolase Inhibitor, UC1728, on LPSInduced Uveitis in the Rabbit. J Ocular Biol. 2016;4(1): 7. J Ocular Biol 3(2): 7 (2016) Page 02 ISSN: 2334-2838 Soluble epoxide hydrolase has been a target for drug development for some time and a large number of compounds have been tested in various models [3,7,24-28]. DiHETs have largely been found inactive or less active than EETs in assays that used these molecules barring few exceptions [29]. The finding that linoleic metabolites, DiHOMEs (dihydroxy octadecamoenoate esters), are more toxic to the lung than their corresponding EpOME (monoepoxide derivatives of linoleic acid) precursors suggest that they may be pro-inflammatory [30]. Early sEH inhibitors suffered from poor solubility and less than ideal pharmacokinetics. More recently, orally available inhibitors with improved potency have been synthesized [31]. One compound, trans-4-{4-[3-(4-trifluormethoxy-phenyyl)-ureido]-cyclohexyloxy}benzoic acid (UC1728) is a potential inhibitor of rabbit sEH with an IC50 of 2.0 nM. UC1728 also has a favorable pharmacokinetic profile with peak serum levels (320 nM) in mice given a dose of 1 mg/kg, occurring between 5 and 7 h post-administration with a halflife of 15 to 20 h. It was successful in reducing the severe pain and inflammation associated with laminitis in horses [32]. Thus UC1728 is an excellent “probe” to understand the roles of sEH and EETs and a potential candidate for further drug development. Uveitis encompasses a number of ocular inflammatory conditions and accounts for 5-20% of blindness in the USA and Europe. In developing countries uveitis can account for as much as 25% of legal blindness [33,34]. Corticosteroids are commonly used to treat uveitis but not all patients respond. Immunosuppressive drugs such as cyclosporine, azathioprine, mycophenolate mofetil, and methotrexate are used as second line drugs. More recently, inhibitors that target TNF-α, such as infliximab, etanercept, and adalimumab, have been effective in patients that are refractory to other treatments [35]. Several of these therapeutic agents have unfavorable side effects or toxicity and carry the potential for serious adverse effects, thus additional therapeutic agents are needed to effectively treat uveitis. Three basic types of animal models have been used to test therapeutic agents for uveitis. One acute model involves the intra-vitreal or intracameral injection of bacterial endotoxin [36,37]. Another acute model involves immunizing animals and then injecting the antigen into the anterior chamber [38]. A chronic model involves immunizing animals with interphotoreceptor retinoid-binding protein (IRBP) or visual arrestin [39]. The chronic models mimic an autoimmunemediated mechanism with T-cell involvement [35,40]. Inhibitors of the sEH have been shown to be anti-inflammatory in numerous models of acute inflammation, most prominently LPS induced systemic inflammation [41,42]. In this model inhibition of sEH increases survival at 24 h, reverses hypotension, decreases pain and decreases the levels of pro-inflammatory AA metabolites. Therefore the goal of this study was to test the hypothesis that UC1728 will be efficacious in reducing the acute inflammation induced by endotoxin in the rabbit anterior chamber model of uveitis. We found that UC1728 did not significantly reduce the severity of aqueous cell and flare or iris congestion. Mean blood level of UC1728 was 2070±196 nM 48 h after injection of UC1728 which is about a thousand fold above the in vitro IC50 on rabbit sEH arguing the lack of effect is not due to low drug levels. These results suggest that sEH, and by inference EETs and DiHETs do not play a significant role in the acute inflammation induced by endotoxin injection into the anterior chamber of rabbit eyes.