Inhibitor Report for: URB597

Early life stress (ELS) may increase predisposition to depression. Despite extensive research, there is still a lack of knowledge of how to optimally treat depression. We aimed to establish a role for the endocannabinoid (ECB) system within the hippocampal-nucleus accumbens (NAc) network as a possible effective target in combating the pathophysiological development of depression-like behavior and neuronal alterations that are precipitated by ELS. Male and female rats were exposed to ELS during post-natal days (P) 7-14, injected with the fatty acid amide hydrolase (FAAH) inhibitor URB597 or the monoacylglycerol lipase (MAGL) inhibitor JZL184 for 2 weeks during late-adolescence (P45-60). Rats were tested starting at P90 for depressive- and anxiety-like behaviors as well as social preference and recognition; alterations in FAAH and MAGL activity; the expression of brain-derived neurotrophic factor (BDNF); and plasticity in the hippocampal-NAc pathway. FAAH and MAGL inhibitors during late-adolescence prevented: (i) the long-term effects of ELS on depression- and anxiety-like behavior and the impairment in social behavior and neuronal plasticity in males and females; (ii) ELS-induced alterations in MAGL activity in males' hippocampus and females' hippocampus and NAc; and (iii) ELS-induced alterations in BDNF in males' hippocampus and NAc and females' hippocampus. Significant correlations were observed between alterations in MAGL and BDNF levels and the behavioral phenotype. The findings suggest that alterations in MAGL activity and BDNF expression in the hippocampal-NAc network contribute to the depressive-like behavioral phenotype in ELS males and females. Moreover, the study suggests FAAH and MAGL inhibitors as potential intervention drugs for depression.

Pressure overload, which is typical of hypertension, is known to evoke alterations not only in the morphology of the heart but also in the preference of myocardial energetic substrates usage. Nowadays, the endocannabinoid system (ECS) serves as a potential therapeutic target for cardiovascular disorders and, simultaneously, affects whole body metabolism homeostasis. Therefore, an open question is whether ECS, apart from decreasing blood pressure, also affects cardiac muscle metabolism in hypertensive conditions. All experiments were conducted on a genetic model of primary hypertension i.e. spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) served as a normotensive control. ECS was chronically activated by 2-weeks intraperitoneal injections of fatty acid amide hydrolase (FAAH) inhibitor - URB597. Lipid analyses in the left ventricle and serum were based on ex vivo heart perfusion in Langendorff perfusion system, thin layer chromatography, and gas liquid chromatography. The total expression of selected proteins was determined using Western blot as well as immunohistochemical techniques. As expected, URB597 markedly reduced systolic as well as mean blood pressures in SHRs. Moreover, prolonged FAAH inhibition resulted in stimulation of (3)H-palmitate uptake and incorporation into different lipid fractions in cardiomyocytes in the hypertensive as well as normotensive conditions. An increase in fatty acid oxidation caused by URB597 treatment was observed only in WKY rats, but not SHRs, and was accompanied by an elevation in peroxisome proliferator-activated receptor alpha (PPARalpha) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) expressions. Chronic activation of ECS significantly upregulates palmitate uptake and its esterification but not oxidation in the SHR's myocardium.

The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10microM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3mgkg(-1)) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

Early life stress (ELS) may increase predisposition to depression. Despite extensive research, there is still a lack of knowledge of how to optimally treat depression. We aimed to establish a role for the endocannabinoid (ECB) system within the hippocampal-nucleus accumbens (NAc) network as a possible effective target in combating the pathophysiological development of depression-like behavior and neuronal alterations that are precipitated by ELS. Male and female rats were exposed to ELS during post-natal days (P) 7-14, injected with the fatty acid amide hydrolase (FAAH) inhibitor URB597 or the monoacylglycerol lipase (MAGL) inhibitor JZL184 for 2 weeks during late-adolescence (P45-60). Rats were tested starting at P90 for depressive- and anxiety-like behaviors as well as social preference and recognition; alterations in FAAH and MAGL activity; the expression of brain-derived neurotrophic factor (BDNF); and plasticity in the hippocampal-NAc pathway. FAAH and MAGL inhibitors during late-adolescence prevented: (i) the long-term effects of ELS on depression- and anxiety-like behavior and the impairment in social behavior and neuronal plasticity in males and females; (ii) ELS-induced alterations in MAGL activity in males' hippocampus and females' hippocampus and NAc; and (iii) ELS-induced alterations in BDNF in males' hippocampus and NAc and females' hippocampus. Significant correlations were observed between alterations in MAGL and BDNF levels and the behavioral phenotype. The findings suggest that alterations in MAGL activity and BDNF expression in the hippocampal-NAc network contribute to the depressive-like behavioral phenotype in ELS males and females. Moreover, the study suggests FAAH and MAGL inhibitors as potential intervention drugs for depression.

Pressure overload, which is typical of hypertension, is known to evoke alterations not only in the morphology of the heart but also in the preference of myocardial energetic substrates usage. Nowadays, the endocannabinoid system (ECS) serves as a potential therapeutic target for cardiovascular disorders and, simultaneously, affects whole body metabolism homeostasis. Therefore, an open question is whether ECS, apart from decreasing blood pressure, also affects cardiac muscle metabolism in hypertensive conditions. All experiments were conducted on a genetic model of primary hypertension i.e. spontaneously hypertensive rats (SHRs) and Wistar Kyoto rats (WKY) served as a normotensive control. ECS was chronically activated by 2-weeks intraperitoneal injections of fatty acid amide hydrolase (FAAH) inhibitor - URB597. Lipid analyses in the left ventricle and serum were based on ex vivo heart perfusion in Langendorff perfusion system, thin layer chromatography, and gas liquid chromatography. The total expression of selected proteins was determined using Western blot as well as immunohistochemical techniques. As expected, URB597 markedly reduced systolic as well as mean blood pressures in SHRs. Moreover, prolonged FAAH inhibition resulted in stimulation of (3)H-palmitate uptake and incorporation into different lipid fractions in cardiomyocytes in the hypertensive as well as normotensive conditions. An increase in fatty acid oxidation caused by URB597 treatment was observed only in WKY rats, but not SHRs, and was accompanied by an elevation in peroxisome proliferator-activated receptor alpha (PPARalpha) and beta-hydroxyacyl-CoA dehydrogenase (beta-HAD) expressions. Chronic activation of ECS significantly upregulates palmitate uptake and its esterification but not oxidation in the SHR's myocardium.

Chemotherapy-induced neuropathic pain is a distressing and commonly occurring side effect of many commonly used chemotherapeutic agents, which in some cases may prevent cancer patients from being able to complete their treatment. Cannabinoid based therapies have the potential to manage or even prevent pain associated with this syndrome. Pre-clinical animal studies that investigate the modulation of the endocannabinoid system (endogenous cannabinoid pathway) are being conducted to better understand the mechanisms behind this phenomenon. Five recent pre-clinical studies identified from Medline published between 2013 and 2016 were selected for review. All studies evaluated the effect of small-molecule agonists or antagonists on components of the endocannabinoid system in rats or mice, using cisplatin or paclitax-el-induced allodynia as a model of chemotherapy-induced neuropathic pain. Activation of the cannabinoid receptor-2 (CB-2) receptor by AM1710 blocked paclitaxel-induced mechanical and cold allodynia in one study. Four studies investigating the activation of both cannabinoid receptor-1 (CB-1) and CB-2 receptors by dual-agonists (WIN55,21 and CP55,940), or by the introduction of inhibitors of endocannabinoid metabolisers (URB597, URB937, JZL184, and SA-57) showed reduction of chemotherapy-induced al-lodynia. In addition, their results suggest that anti-allodynic effects may also be mediated by additional receptors, including TRPV1 and 5-hydroxytryptamine (5-HT1A). Pre-clinical studies demon-strate that the activation of endocannabinoid CB-1 or CB-2 receptors produces physiological effects in animal models, namely the reduction of chemotherapy-induced allodynia. These studies also provide in-sight into the biological mechanism behind the therapeutic utility of cannabis compounds in managing chemotherapy-induced neuropathic pain, and provide a basis for the conduct of future clinical studies in patients of this population.

The present study explored the protective effects of cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase inhibitor URB597 (URB) against neuroinflammation in rats with chronic cerebral hypoperfusion (CCH). Activated microglia, astrocytes, and nuclear factor kappa B (NF-kappaB) p65-positive cells were measured by immunofluorescence. Reactive oxygen species (ROS) was assessed by dihydroethidium staining. The protein levels of cluster of differentiation molecule 11b (OX-42), glial fibrillary acidic protein (GFAP), NF-kappaB p65, inhibitor of kappa B alpha (IkappaB-a), IkappaB kinase a/beta (IKK a/beta), phosphorylated IKK a/beta (p-IKK a/beta), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), tumor necrosis factor (TNF)-alpha, and interleukin-1beta (IL-1beta) were examined by western blotting or enzyme-linked immunosorbent assay. All the protein levels of OX-42, GFAP, TNF-a, IL-1beta, COX-2, and iNOS are increased in CCH rats. WIN and URB downregulated the levels of OX-42, GFAP, TNF-alpha, IL-1beta, COX-2 and iNOS and inhibited CCH-induced ROS accumulation in CCH rats, indicating that WIN and URB might exert their neuroprotective effects by inhibiting the neuroinflammatory response. In addition, the NF-kappaB signaling pathway was activated by CCH in frontal cortex and hippocampus, while the aforementioned changes were reversed by WIN and URB treatment. These findings suggest that WIN and URB treatment ameliorated CCH-induced neuroinflammation through inhibition of the classical pathway of NF-kappaB activation, resulting in mitigation of chronic ischemic injury.

The present study further investigated the protective effects of cannabinoid receptor agonist WIN55,212-2 (WIN) and fatty acid amide hydrolase (FAAH) inhibitor URB597 (URB) on chronic cerebral hypoperfusion (CCH)-induced cognitive impairment in rats. Spatial learning and memory were assessed with the Morris water maze and by measuring Long-term potentiation. The expression of microtubule-associated protein-2 (MAP)-2, growth-associated protein-43 (GAP)-43, synaptophysin, cannabinoid receptor 1 (CB1), brain-derived neurotrophic factor (BDNF), FAAH, N-acylphosphatidylethanolamine phospholipase D(NAPE-PLD) and monoacyl glycerol lipase (MGL) as well as phosphoinositide 3-kinase (PI3K)/AKT signaling pathway molecules and downstream targets including AKT, phosphorylated (p-)AKT, cyclic AMP response element- binding protein (CREB), p-CREB, Bcl-2-associated death protein (BAD), p-BAD, glycogen synthase kinase (GSK)-3beta, p-GSK-3beta, forkhead box protein (FOXO) 3A and p-FOXO3A was determined by western blotting. WIN and URB treatment improved learning and memory performance, effects that were abolished by co-administration of the PI3K/AKT inhibitor LY294002. Moreover, WIN and URB reversed the decreases in MAP-2 and synaptophysin expression resulting from CCH, and stimulated BDNF and CB1 expression as well as CREB, FOXO3A, GSK-3beta, and BAD phosphorylation, confirming that WIN and URB mediate neuroprotection by preventing neuronal apoptosis and improving cognition via PI3K/AKT signaling. These findings suggest that WIN and URB are promising agents for therapeutic management of CCH.

Carboxylesterase type 1 (CES1) and CES2 are serine hydrolases located in the liver and small intestine. CES1 and CES2 actively participate in the metabolism of several pharmaceuticals. Recently, carbamate compounds were developed to inhibit members of the serine hydrolase family via covalent modification of the active site serine. URB597 and JZL184 inhibit fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively; however, carboxylesterases in liver have been identified as a major off-target. We report the kinetic rate constants for inhibition of human recombinant CES1 and CES2 by URB597 and JZL184. Bimolecular rate constants (k(inact)/K(i)) for inhibition of CES1 by JZL184 and URB597 were similar [3.9 (+/-0.2) x 10(3) M(-1) s(-1) and 4.5 (+/-1.3) x 10(3) M(-1) s(-1), respectively]. However, k(inact)/K(i) for inhibition of CES2 by JZL184 and URB597 were significantly different [2.3 (+/-1.3) x 10(2) M(-1) s(-1) and 3.9 (+/-1.0) x 10(3) M(-1) s(-1), respectively]. Rates of inhibition of CES1 and CES2 by URB597 were similar; however, CES1 and MAGL were more potently inhibited by JZL184 than CES2. We also determined kinetic constants for spontaneous reactivation of CES1 carbamoylated by either JZL184 or URB597 and CES1 diethylphosphorylated by paraoxon. The reactivation rate was significantly slower (4.5x) for CES1 inhibited by JZL184 than CES1 inhibited by URB597. Half-life of reactivation for CES1 carbamoylated by JZL184 was 49 +/- 15 h, which is faster than carboxylesterase turnover in HepG2 cells. Together, the results define the kinetics of inhibition for a class of drugs that target hydrolytic enzymes involved in drug and lipid metabolism.

Tetrahymena thermophila is a model organism for molecular and cellular biology. Previous studies from our group showed that Tetrahymena contains major components of the endocannabinoid system, such as various endocannabinoids and FAAH. In mammalian cells the endocannabinoid 2-arachidonoylglycerol is inactivated mainly by MAGL. In this study we showed that 2-arachidonoylglycerol and 2-oleoylglycerol are hydrolyzed by the combined actions of MAGL and FAAH. MAGL-like activity was examined in the presence of FAAH specific inhibitors, URB597 or AM374 and showed optimum pH of 8-9, apparent K(M) of 14.1muM and V(max) of 5.8nmol/minxmg. The enzyme was present in membrane bound and cytosolic isoforms; molecular mass was determined at approximately 45 and approximately 40kDa. MAGL and FAAH could also inactivate endogenous signaling lipids, which might play an important role in Tetrahymena as suggested in mammals. Tetrahymena could be used as a model system for testing drugs targeting enzymes of the endocannabinoid system.

The O-arylcarbamate URB597 (cyclohexylcarbamic acid 3'-carbamoylbiphenyl-3-yl ester; also referred to as KDS-4103) is a potent inhibitor of fatty-acid amide hydrolase (FAAH), an intracellular serine hydrolase responsible for the inactivation of the endogenous cannabinoid anandamide. URB597 demonstrates a remarkable degree of selectivity for FAAH over other serine hydrolases (e.g. cholinesterases) or other components of the endocannabinoid system (e.g. cannabinoid receptors). However, in a proteomic-based selectivity screen based on the displacement of fluorophosphonate-rhodamine (FPR) from mouse brain proteins, it was recently shown that URB597 prevents FPR binding to triacylglycerol hydrolase (TGH) with a median inhibitory concentration of 192nM. To determine whether this effect correlates with inhibition of TGH activity, we investigated the ability of URB597 to inhibit triolein hydrolysis in rat liver and heart tissues, which are rich in TGH, as well as white adipose tissue (WAT), which is rich in adipose triacylglycerol lipase (TGL) and hormone-sensitive lipase. The results show that URB597 does not affect triolein hydrolysis in any of these tissues at concentrations as high as 10microM, whereas it inhibits FAAH activity at low nanomolar concentrations. Moreover, intraperitoneal (i.p.) administration of URB597 at doses that maximally inhibit FAAH in vivo (0.3-3mgkg(-1)) exerts no effect on triolein hydrolysis and tissue triacylglycerol (TAG) levels in rat liver, heart or WAT. The results indicate that URB597, while potent at inhibiting FAAH, does not affect TGH and TGL activities in rat tissues.

In the present article, we review the pharmacological properties of KDS-4103 (URB597), a highly potent and selective inhibitor of the enzyme fatty-acid amide hydrolase (FAAH), which catalyzes the intracellular hydrolysis of the endocannabinoid anandamide. In vitro, KDS-4103 inhibits FAAH activity with median inhibitory concentrations (IC(50)) of 5 nM in rat brain membranes and 3 nM in human liver microsomes. In vivo, KDS-4103 inhibits rat brain FAAH activity after intraperitoneal (i.p.) administration with a median inhibitory dose (ID(50)) of 0.15 mg/kg. The compound does not significantly interact with other cannabinoid-related targets, including cannabinoid receptors and anandamide transport, or with a broad panel of receptors, ion channels, transporters and enzymes. By i.p. administration to rats and mice KDS-4103 elicits significant, anxiolytic-like, antidepressant-like and analgesic effects, which are prevented by treatment with CB1 receptor antagonists. By contrast, at doses that significantly inhibit FAAH activity and substantially raise brain anandamide levels, KDS-4103 does not evoke classical cannabinoid-like effects (e.g., catalepsy, hypothermia, hyperphagia), does not cause place preference, and does not produce generalization to the discriminative effects of the active ingredient of cannabis, Delta9-tetrahydrocannabinol (Delta9-THC). These findings suggest that KDS-4103 acts by enhancing the tonic actions of anandamide on a subset of CB(1) receptors, which may normally be engaged in controlling emotions and pain. KDS-4103 is orally available in rats and cynomolgus monkeys. Sub-chronic repeated dose studies (1,500 mg/kg, per os) in these two species have not demonstrated systemic toxicity. Likewise, no toxicity was noted in bacterial cytotoxicity tests in vitro and in the Ames test. Furthermore, no deficits were observed in rats on the rotarod test after acute i.p. treatment with KDS-4103 at doses up to 5 mg/kg or in a functional observation battery after oral doses up to 1,500 mg/kg. The results suggest that KDS-4103 will offer a novel approach with a favorable therapeutic window for the treatment of anxiety, depression and pain.