Abu Sadeque

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 (IC50 = 1.914 μM), 7-hydroxylorcaserin (IC50 = 0.213 μM), and 1-hydroxylorcaserin formation (IC50 = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high Km components in HLM and 7-hydroxylorcaserin showed lower Km 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.

Identification of Human UDP-Glucuronosyltransferases Involved in N-Carbamoyl Glucuronidation of Lorcaserin

Lorcaserin, a selective serotonin 5-HT2C 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 Km value of 51.6 ± 1.9 μM and Vmax 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.

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.

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.

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.

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.

(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 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 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.

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.