Inhibitor Report for: Ar9281

Glycemic regulation improves myocardial function in diabetic patients, but finding optimal therapeutic strategies remains challenging. Recent data have shown that pharmacological inhibition of soluble epoxide hydrolase (sEH), an enzyme that decreases the endogenous levels of protective epoxyeicosatrienoic acids (EETs), improves glucose homeostasis in insulin-resistant mice. Here, we tested whether the administration of sEH inhibitors preserves cardiac myocyte structure and function in hyperglycemic rats. University of California-Davis-type 2 diabetes mellitus (UCD-T2DM) rats with nonfasting blood glucose levels in the range of 150-200 mg/dl were treated with the sEH inhibitor 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU) for 6 wk. Administration of APAU attenuated the progressive increase of blood glucose concentration and preserved mitochondrial structure and myofibril morphology in cardiac myocytes, as revealed by electron microscopy imaging. Fluorescence microscopy with Ca(2+) indicators also showed a 40% improvement of cardiac Ca(2+) transients in treated rats. Sarcoplasmic reticulum Ca(2+) content was decreased in both treated and untreated rats compared with control rats. However, treatment limited this reduction by 30%, suggesting that APAU may protect the intracellular Ca(2+) effector system. Using Western blot analysis on cardiac myocyte lysates, we found less downregulation of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), the main route of Ca(2+) reuptake in the sarcoplasmic reticulum, and lower expression of hypertrophic markers in treated versus untreated UCD-T2DM rats. In conclusion, APAU enhances the therapeutic effects of EETs, resulting in slower progression of hyperglycemia, efficient protection of myocyte structure, and reduced Ca(2+) dysregulation and SERCA remodeling in hyperglycemic rats. The results suggest that sEH/EETs may be an effective therapeutic target for cardioprotection in insulin resistance and diabetes.

BACKGROUND AND AIMS: This study evaluated the responses to soluble epoxide hydrolase (s-EH) inhibition, an essential enzyme in the metabolism of arachidonic acid, on food intake, body weight and metabolic parameters in mice fed a high fat-high fructose diet (HFD) for 10 weeks. METHODS AND RESULTS: After 5 weeks of HFD, mice were divided into two groups: 1) s-EH inhibitor (AR9281, 200mg/kg/day by gavage twice daily), and 2) vehicle (0.3ml per gavage). Food intake, body weight, oxygen consumption (VO(2)), carbon dioxide production (VCO(2)), respiratory quotient (RQ), and motor activity were measured weekly for more 5 weeks. HFD increased body weight (37+/-1 vs. 26+/-1g), and plasma of glucose (316+/-8 vs. 188+/-27mg/dl), insulin (62.1+/-8.1 vs. 15.5+/-5.0muU/ml), and leptin levels (39.4+/-3.6 vs. 7.5+/-0.1ng/ml) while reducing VO(2), VCO(2) and motor activity. s-EH inhibition for 5 weeks decreased caloric intake by ~32% and increased VO(2) by ~17% (42.8+/-1.4 vs. 50.2+/-1.5ml/kg/min) leading to significant weight loss. Inhibition of s-EHi also caused significant reductions in plasma leptin levels and visceral fat content. Uncoupling protein 1 (UCP1) content in brown adipose tissue was also elevated by ~50% during s-EH inhibition compared to vehicle treatment. CONCLUSION: These results suggest that s-EH inhibition with AR9281 promotes weight loss by reducing appetite and increasing metabolic rate, and that increased UCP1 content may contribute to the increase in energy expenditure.

Epoxyeicosanoids, including the epoxyeicosatrienoic acids are signaling molecules which appear to help ameliorate the effects of a wide variety of pathological conditions. The enzyme soluble epoxide hydrolase (sEH) metabolizes these molecules by converting them to their corresponding vicinal diols. Inhibition of sEH either by knockout or chemical inhibitors increases epoxyeicosanoid levels in vivo and provides significant organ protection in models of brain, cardiac, and renal injury. sEH also appears to be involved in modulating inflammation, pain pathways, pulmonary function, hypertension, and diabetes. Potent sEH inhibitors have been developed in academic, pharmaceutical, and biotech laboratories and described in the patent and scientific literature. Most of the inhibitor scaffolds employ a urea or amide which functions as an active-site transition state mimic. Arete Therapeutics compound AR9281 successfully completed phase Ia and 1b studies. A phase IIa proof of concept trial for treatment of impaired glucose tolerance has been completed, but the results are not yet reported.

Multi-target inhibitors have become increasing popular as a means to leverage the advantages of poly-pharmacology while simplifying drug delivery. Here, we describe dual inhibitors for soluble epoxide hydrolase (sEH) and fatty acid amide hydrolase (FAAH), two targets known to synergize when treating inflammatory and neuropathic pain. The structure activity relationship (SAR) study described herein initially started with t-TUCB (trans-4-[4-(3-trifluoromethoxyphenyl-l-ureido)-cyclohexyloxy]-benzoic acid), a potent sEH inhibitor that was previously shown to weakly inhibit FAAH. Inhibitors with a 6-fold increase of FAAH potency while maintaining high sEH potency were developed by optimization. Interestingly, compared to most FAAH inhibitors that inhibit through time-dependent covalent modification, t-TUCB and related compounds appear to inhibit FAAH through a time-independent, competitive mechanism. These inhibitors are selective for FAAH over other serine hydrolases. In addition, FAAH inhibition by t-TUCB appears to be higher in human FAAH over other species; however, the new dual sEH/FAAH inhibitors have improved cross-species potency. These dual inhibitors may be useful for future studies in understanding the therapeutic application of dual sEH/FAAH inhibition.

AR9281, a potent and selective inhibitor of soluble epoxide hydrolase (s-EH), is in clinical development targeting hypertension and type 2 diabetes. The safety, pharmacokinetics, and pharmacodynamics of AR9281 were evaluated in double-blind, randomized, placebo-controlled, ascending, single oral dose (10-1000 mg) and multiple dose (100-400 mg every 8 hours for 7 days) studies in healthy subjects. AR9281 was well tolerated, and no dose-related adverse events were observed during either study. The drug was rapidly absorbed with a mean terminal half-life ranging from 3 to 5 hours. The area under the plasma concentration-time curve increased in an approximately dose-proportional manner up to the 500-mg dose and exhibited a greater than dose linearity at higher doses. AR9281 directly and dose-dependently inhibited blood s-EH activity with 90% inhibition or greater over an 8-hour period at the 250-mg dose and over a 12-hour period at the 500-mg dose. Multiple doses of AR9281 ranging from 100 to 400 mg every 8 hours resulted in a sustained inhibition of s-EH activity at 90% or greater during the trough. The current studies provide proof of safety and target inhibition of AR9281 in healthy subjects. AR9281 pharmacokinetic and pharmacodynamic characteristics support a twice-daily or thrice-daily dosing regimen in patients.

Glycemic regulation improves myocardial function in diabetic patients, but finding optimal therapeutic strategies remains challenging. Recent data have shown that pharmacological inhibition of soluble epoxide hydrolase (sEH), an enzyme that decreases the endogenous levels of protective epoxyeicosatrienoic acids (EETs), improves glucose homeostasis in insulin-resistant mice. Here, we tested whether the administration of sEH inhibitors preserves cardiac myocyte structure and function in hyperglycemic rats. University of California-Davis-type 2 diabetes mellitus (UCD-T2DM) rats with nonfasting blood glucose levels in the range of 150-200 mg/dl were treated with the sEH inhibitor 1-(1-acetypiperidin-4-yl)-3-adamantanylurea (APAU) for 6 wk. Administration of APAU attenuated the progressive increase of blood glucose concentration and preserved mitochondrial structure and myofibril morphology in cardiac myocytes, as revealed by electron microscopy imaging. Fluorescence microscopy with Ca(2+) indicators also showed a 40% improvement of cardiac Ca(2+) transients in treated rats. Sarcoplasmic reticulum Ca(2+) content was decreased in both treated and untreated rats compared with control rats. However, treatment limited this reduction by 30%, suggesting that APAU may protect the intracellular Ca(2+) effector system. Using Western blot analysis on cardiac myocyte lysates, we found less downregulation of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), the main route of Ca(2+) reuptake in the sarcoplasmic reticulum, and lower expression of hypertrophic markers in treated versus untreated UCD-T2DM rats. In conclusion, APAU enhances the therapeutic effects of EETs, resulting in slower progression of hyperglycemia, efficient protection of myocyte structure, and reduced Ca(2+) dysregulation and SERCA remodeling in hyperglycemic rats. The results suggest that sEH/EETs may be an effective therapeutic target for cardioprotection in insulin resistance and diabetes.

BACKGROUND AND AIMS: This study evaluated the responses to soluble epoxide hydrolase (s-EH) inhibition, an essential enzyme in the metabolism of arachidonic acid, on food intake, body weight and metabolic parameters in mice fed a high fat-high fructose diet (HFD) for 10 weeks. METHODS AND RESULTS: After 5 weeks of HFD, mice were divided into two groups: 1) s-EH inhibitor (AR9281, 200mg/kg/day by gavage twice daily), and 2) vehicle (0.3ml per gavage). Food intake, body weight, oxygen consumption (VO(2)), carbon dioxide production (VCO(2)), respiratory quotient (RQ), and motor activity were measured weekly for more 5 weeks. HFD increased body weight (37+/-1 vs. 26+/-1g), and plasma of glucose (316+/-8 vs. 188+/-27mg/dl), insulin (62.1+/-8.1 vs. 15.5+/-5.0muU/ml), and leptin levels (39.4+/-3.6 vs. 7.5+/-0.1ng/ml) while reducing VO(2), VCO(2) and motor activity. s-EH inhibition for 5 weeks decreased caloric intake by ~32% and increased VO(2) by ~17% (42.8+/-1.4 vs. 50.2+/-1.5ml/kg/min) leading to significant weight loss. Inhibition of s-EHi also caused significant reductions in plasma leptin levels and visceral fat content. Uncoupling protein 1 (UCP1) content in brown adipose tissue was also elevated by ~50% during s-EH inhibition compared to vehicle treatment. CONCLUSION: These results suggest that s-EH inhibition with AR9281 promotes weight loss by reducing appetite and increasing metabolic rate, and that increased UCP1 content may contribute to the increase in energy expenditure.

1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea 14a (AR9281), a potent and selective soluble epoxide hydrolase inhibitor, was recently tested in a phase 2a clinical setting for its effectiveness in reducing blood pressure and improving insulin resistance in pre-diabetic patients. In a mouse model of diet induced obesity, AR9281 attenuated the enhanced glucose excursion following an intraperitoneal glucose tolerance test. AR9281 also attenuated the increase in blood pressure in angiotensin-II-induced hypertension in rats. These effects were dose-dependent and well correlated with inhibition of the sEH activity in whole blood, consistent with a role of sEH in the observed pharmacology in rodents.

Epoxyeicosanoids, including the epoxyeicosatrienoic acids are signaling molecules which appear to help ameliorate the effects of a wide variety of pathological conditions. The enzyme soluble epoxide hydrolase (sEH) metabolizes these molecules by converting them to their corresponding vicinal diols. Inhibition of sEH either by knockout or chemical inhibitors increases epoxyeicosanoid levels in vivo and provides significant organ protection in models of brain, cardiac, and renal injury. sEH also appears to be involved in modulating inflammation, pain pathways, pulmonary function, hypertension, and diabetes. Potent sEH inhibitors have been developed in academic, pharmaceutical, and biotech laboratories and described in the patent and scientific literature. Most of the inhibitor scaffolds employ a urea or amide which functions as an active-site transition state mimic. Arete Therapeutics compound AR9281 successfully completed phase Ia and 1b studies. A phase IIa proof of concept trial for treatment of impaired glucose tolerance has been completed, but the results are not yet reported.

Endothelial dysfunction is a hallmark of, and plays a pivotal role in the pathogenesis of cardiometabolic diseases, including type II diabetes, obesity, and hypertension. It has been well established that epoxyeicosatrienoic acids (EETs) act as an endothelial derived hyperpolarization factor (EDHF). Soluble epoxide hydrolase (s-EH) rapidly hydrolyses certain epoxylipids (e.g. EETs) to less bioactive diols (DHETs), thereby attenuating the evoked vasodilator effects. The aim of the present study was to examine if inhibition of s-EH can restore impaired endothelial function in three animal models of cardiometabolic diseases. Isolated vessel rings of the aorta and/or mesenteric artery from mice or rats were pre-contracted using phenylephrine or U46619. Endothelium-dependent and independent vasorelaxation to acetylcholine and sodium nitroprusside (SNP) were measured using wire myography in vessels isolated from db/db or diet-induced obesity (DIO) mice, and angiotensin II-induced hypertensive rats treated chronically with s-EH inhibitors AR9281 or AR9276 or with vehicle. Vasorelaxation to acetylcholine, but not to SNP was severely impaired in all three animal models. Oral administration of AR9281 or AR9276 abolished whole blood s-EH activity, elevated epoxy/diol lipid ratio, and abrogated endothelial dysfunction in all three models. Incubating the mesenteric artery of db/db mice with L-NAME and indomethacin to block nitric oxide (NO) and prostacyclin formation did not affect AR9821-induced improvement of endothelial function. These data indicate that inhibition of s-EH ameliorates endothelial dysfunction and that effects in the db/db model are independent of the presence of NO and cyclooxygenase derived prostanoids. Thus, preserving vasodilator EETs by inhibition of s-EH may be of therapeutic benefit by improving endothelial function in cardiometabolic diseases.