Metopimazine is primarily metabolized by a liver amidase in humans.

Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first-pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin-monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP.

Discovery of APD371: Identification of a Highly Potent and Selective CB2 Agonist for the Treatment of Chronic Pain.

The discovery of a novel, selective and fully efficacious CB2 agonist with satisfactory pharmacokinetic and pharmaceutical properties is described. Compound 6 was efficacious in a rat model of osteoarthritis pain following oral administration and, in contrast to morphine, maintained its analgesic effect throughout a 5-day subchronic treatment paradigm. These data were consistent with our hypothesis that full agonist efficacy is required for efficient internalization and recycling of the CB2 receptor to avoid tachyphylaxis. Based on its overall favorable preclinical profile, 6 (APD371) was selected for further development for the treatment of pain.

Discovery of 2-(((1r,4r)-4-(((4-Chlorophenyl)(phenyl)carbamoyl)oxy)methyl)cyclohexyl)methoxy)acetate (Ralinepag): An Orally Active Prostacyclin Receptor Agonist for the Treatment of Pulmonary Arterial Hypertension.

The design and synthesis of a new series of potent non-prostanoid IP receptor agonists that showed oral efficacy in the rat monocrotaline model of pulmonary arterial hypertension (PAH) are described. Detailed profiling of a number of analogues resulted in the identification of 5c (ralinepag) that has good selectivity in both binding and functional assays with respect to most members of the prostanoid receptor family and a more modest 30- to 50-fold selectivity over the EP3 receptor. In our hands, its potency and efficacy are comparable or superior to MRE269 (the active metabolite of the clinical compound NS-304) with respect to in vitro IP receptor dependent cAMP accumulation assays. 5c had an excellent PK profile across species. Enterohepatic recirculation most probably contributes to a concentration-time profile after oral administration in the cynomolgus monkey that showed a very low peak-to-trough ratio. Following the identification of an acceptable solid form, 5c was selected for further development for the treatment of PAH.

Identification of Human Sulfotransferases Involved in Lorcaserin N-Sulfamate Formation.

Lorcaserin [(R)-8-chloro-1-methyl-2,3,4,5-tetrahydro-1H-3-benzazepine] hydrochloride hemihydrate, a selective serotonin 5-hydroxytryptamine (5-HT) 5-HT(2C) receptor agonist, is approved by the U.S. Food and Drug Administration for chronic weight management. Lorcaserin is primarily cleared by metabolism, which involves multiple enzyme systems with various metabolic pathways in humans. The major circulating metabolite is lorcaserin N-sulfamate. Both human liver and renal cytosols catalyze the formation of lorcaserin N-sulfamate, where the liver cytosol showed a higher catalytic efficiency than renal cytosol. Human sulfotransferases (SULTs) SULT1A1, SULT1A2, SULT1E1, and SULT2A1 are involved in the formation of lorcaserin N-sulfamate. The catalytic efficiency of these SULTs for lorcaserin N-sulfamate formation is widely variable, and among the SULT isoforms SULT1A1 was the most efficient. The order of intrinsic clearance for lorcaserin N-sulfamate is SULT1A1 > SULT2A1 > SULT1A2 > SULT1E1. Inhibitory effects of lorcaserin N-sulfamate on major human cytochrome P450 (P450) enzymes were not observed or minimal. Lorcaserin N-sulfamate binds to human plasma protein with high affinity (i.e., >99%). Thus, despite being the major circulating metabolite, the level of free lorcaserin N-sulfamate would be minimal at a lorcaserin therapeutic dose and unlikely be sufficient to cause drug-drug interactions. Considering its formation kinetic parameters, high plasma protein binding affinity, minimal P450 inhibition or induction potential, and stability, the potential for metabolic drug-drug interaction or toxicological effects of lorcaserin N-sulfamate is remote in a normal patient population.

Discovery of a novel trans-1,4-dioxycyclohexane GPR119 agonist series.

The design and optimization of a novel trans-1,4-dioxycyclohexane GPR119 agonist series is described. A lead compound 21 was found to be a potent and efficacious GPR119 agonist across species, and possessed overall favorable pharmaceutical properties. Compound 21 demonstrated robust acute and chronic regulatory effects on glycemic parameters in the diabetic or non-diabetic rodent models.

Discovery of 1a,2,5,5a-tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalen-4-carboxamides as potent and selective CB2 receptor agonists.

The design and synthesis of novel 1a,2,5,5a-tetrahydro-1H-2,3-diaza-cyclopropa[a]pentalen-4-carboxamide CB2 selective ligands for the potential treatment of pain is described. Compound (R,R)-25 has good balance between CB2 agonist potency and selectivity over CB1, and possesses overall favorable pharmaceutical properties. It also demonstrated robust in vivo efficacy mediated via CB2 activation in the rodent models of inflammatory and osteoarthritis pain after oral administration.

Discovery of APD334: Design of a Clinical Stage Functional Antagonist of the Sphingosine-1-phosphate-1 Receptor.

APD334 was discovered as part of our internal effort to identify potent, centrally available, functional antagonists of the S1P1 receptor for use as next generation therapeutics for treating multiple sclerosis (MS) and other autoimmune diseases. APD334 is a potent functional antagonist of S1P1 and has a favorable PK/PD profile, producing robust lymphocyte lowering at relatively low plasma concentrations in several preclinical species. This new agent was efficacious in a mouse experimental autoimmune encephalomyelitis (EAE) model of MS and a rat collagen induced arthritis (CIA) model and was found to have appreciable central exposure.

(7-Benzyloxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetic Acids as S1P1 Functional Antagonists.

S1P1 is a validated target for treatment of autoimmune disease, and functional antagonists with superior safety and pharmacokinetic properties are being sought as second generation therapeutics. We describe the discovery and optimization of (7-benzyloxy-2,3-dihydro-1H-pyrrolo[1,2-a]indol-1-yl)acetic acids as potent, centrally available, direct acting S1P1 functional antagonists, with favorable pharmacokinetic and safety properties.

Discovery and optimization of 5-fluoro-4,6-dialkoxypyrimidine GPR119 agonists.

A series of 5-fluoro-4,6-dialkoxypyrimidine GPR119 modulators were discovered and optimized for in vitro agonist activity. A lead molecule was identified that has improved agonist efficacy relative to our clinical compound (APD597) and possesses reduced CYP2C9 inhibitory potential. This optimized lead was found to be efficacious in rodent models of glucose control both alone and in combination with a Dipeptidyl peptidase-4 (DPP-4) inhibitor.

Biopharmaceutics permeability classification of lorcaserin, a selective 5-hydroxytryptamine 2C agonist: method suitability and permeability class membership.

The objectives of the study were (1) to demonstrate that a Caco-2 cell-based permeability assay, developed in our laboratory, is suitable to identify the permeability classification according to the US Food and Drug Administration Biopharmaceutics Classification System guidance, and (2) to use the validated Caco-2 method to determine permeability class membership of lorcaserin. Lorcaserin, marketed in United States as Belviq, is a selective human 5-hydroxytryptamine 2C agonist used for weight management. First, the permeability of twenty commercially available drugs was determined in the apical-to-basolateral direction at a final concentration of 10 µM, with the pH of transporter buffer in the apical and basolateral compartments being 6.8 and 7.4, respectively. A rank-order relationship between in vitro permeability results and the extent of human intestinal absorption for the drugs tested was observed. Second, the apparent permeability coefficient values of lorcaserin at 2, 20, and 200 µM and apical pH values of 6.8 and 7.4 in the apical-to-basolateral direction were determined using the validated method and found to be comparable to those of the high-permeability internal standard metoprolol. Lorcaserin permeability across Caco-2 cell monolayers was not dependent on the variation of apical pH. Furthermore, lorcaserin was not a substrate for efflux transporters such as P-glycoprotein. In conclusion, using the validated Caco-2 permeability assay, it was shown that lorcaserin is a highly permeable compound.

Kinetics of 5-HT2B receptor signaling: profound agonist-dependent effects on signaling onset and duration.

The kinetics of drug-receptor interactions can profoundly influence in vivo and in vitro pharmacology. In vitro, the potencies of slowly associating agonists may be underestimated in assays capturing transient signaling events. When divergent receptor-mediated signaling pathways are evaluated using combinations of equilibrium and transient assays, potency differences driven by kinetics may be erroneously interpreted as biased signaling. In vivo, drugs with slow dissociation rates may display prolonged physiologic effects inconsistent with their pharmacokinetic profiles. We evaluated a panel of 5-hydroxytryptamine2B (5-HT2B) receptor agonists in kinetic radioligand binding assays and in transient, calcium flux assays, and inositol phosphate accumulation assays; two functional readouts emanating from Gαq-mediated activation of phospholipase C. In binding studies, ergot derivatives demonstrated slow receptor association and dissociation rates, resulting in significantly reduced potency in calcium assays relative to inositol phosphate accumulation assays. Ergot potencies for activation of extracellular signal-regulated kinases 1 and 2 were also highly time-dependent. A number of ergots produced wash-resistant 5-HT2B signaling that persisted for many hours without appreciable loss of potency, which was not explained simply by slow receptor-dissociation kinetics. Mechanistic studies indicated that persistent signaling originated from internalized or sequestered receptors. This study provides a mechanistic basis for the long durations of action in vivo and wash-resistant effects in ex vivo tissue models often observed for ergots. The 5-HT2B agonist activity of a number of ergot-derived therapeutics has been implicated in development of cardiac valvulopathy in man. The novel, sustained nature of ergot signaling reported here may represent an additional mechanism contributing to the valvulopathic potential of these compounds.

Fused tricyclic indoles as S1P1 agonists with robust efficacy in animal models of autoimmune disease.

Two series of fused tricyclic indoles were identified as potent and selective S1P(1) agonists. In vivo these agonists produced a significant reduction in circulating lymphocytes which translated into robust efficacy in several rodent models of autoimmune disease. Importantly, these agonists were devoid of any activity at the S1P(3) receptor in vitro, and correspondingly did not produce S1P(3) mediated bradycardia in telemeterized rat.

Identification of human UDP-glucuronosyltransferases involved in N-carbamoyl glucuronidation of lorcaserin.

Lorcaserin, a selective serotonin 5-HT(2C) receptor agonist, is a weight management agent in clinical development. Lorcaserin N-carbamoyl glucuronidation governs the predominant excretory pathway of lorcaserin in humans. Human UDP-glucuronosyltransferases (UGTs) responsible for lorcaserin N-carbamoyl glucuronidation are identified herein. Lorcaserin N-carbamoyl glucuronide formation was characterized by the following approaches: metabolic screening using human tissues (liver, kidney, intestine, and lung) and recombinant enzymes, kinetic analyses, and inhibition studies. Whereas microsomes from all human tissues studied herein were found to be catalytically active for lorcaserin N-carbamoyl glucuronidation, liver microsomes were the most efficient. With recombinant UGT enzymes, lorcaserin N-carbamoyl glucuronidation was predominantly catalyzed by three UGT2Bs (UGT2B7, UGT2B15, and UGT2B17), whereas two UGT1As (UGT1A6 and UGT1A9) played a minor role. UGT2B15 was most efficient, with an apparent K(m) value of 51.6 ± 1.9 µM and V(max) value of 237.4 ± 2.8 pmol/mg protein/min. The rank order of catalytic efficiency of human UGT enzymes for lorcaserin N-carbamoyl glucuronidation was UGT2B15 > UGT2B7 > UGT2B17 > UGT1A9 > UGT1A6. Inhibition of lorcaserin N-carbamoyl glucuronidation activities of UGT2B7, UGT2B15, and UGT2B17 in human liver microsomes by mefenamic acid, bisphenol A, and eugenol further substantiated the involvement of these UGT2B isoforms. In conclusion, multiple human UGT enzymes catalyze N-carbamoyl glucuronidation of lorcaserin; therefore, it is unlikely that inhibition of any one of these UGT activities will lead to significant inhibition of the lorcaserin N-carbamoyl glucuronidation pathway. Thus, the potential for drug-drug interaction by concomitant administration of a drug(s) that is metabolized by any of these UGTs is remote.

Identification of human cytochrome P450 and flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel selective human 5-hydroxytryptamine 2C agonist.

Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 µM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 µM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 µM), 7-hydroxylorcaserin (IC(50) = 0.213 µM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 µM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze the formation of four primary oxidative metabolites of lorcaserin, suggesting that lorcaserin has a low probability of drug-drug interactions by concomitant medications.

Discovery and structure-activity relationship of 3-methoxy-N-(3-(1-methyl-1H-pyrazol-5-yl)-4-(2-morpholinoethoxy)phenyl)benzamide (APD791): a highly selective 5-hydroxytryptamine2A receptor inverse agonist for the treatment of arterial thrombosis.

Serotonin, which is stored in platelets and is released during thrombosis, activates platelets via the 5-HT(2A) receptor. 5-HT(2A) receptor inverse agonists thus represent a potential new class of antithrombotic agents. Our medicinal program began with known compounds that displayed binding affinity for the recombinant 5-HT(2A) receptor, but which had poor activity when tested in human plasma platelet inhibition assays. We herein describe a series of phenyl pyrazole inverse agonists optimized for selectivity, aqueous solubility, antiplatelet activity, low hERG activity, and good pharmacokinetic properties, resulting in the discovery of 10k (APD791). 10k inhibited serotonin-amplified human platelet aggregation with an IC(50) = 8.7 nM and had negligible binding affinity for the closely related 5-HT(2B) and 5-HT(2C) receptors. 10k was orally bioavailable in rats, dogs, and monkeys and had an acceptable safety profile. As a result, 10k was selected further evaluation and advanced into clinical development as a potential treatment for arterial thrombosis.