In vitro and in vivo metabolism of a novel cannabinoid-1 receptor inverse agonist, taranabant, in rats and monkeys.

The metabolism and excretion of taranabant (MK-0364, N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2{[5-(trifluoromethyl)pyridine-2-yl]oxy}propanamide), a potent cannabinoid-1 receptor inverse agonist, were evaluated in rats and rhesus monkeys. Following administration of [¹4C]taranabant, the majority of the radioactivity was excreted within 72 h. In both rats and rhesus monkeys, taranabant was eliminated primarily via oxidative metabolism, followed by excretion of metabolites into bile. Major pathways of metabolism that were common to rats and rhesus monkeys included hydroxylation at the benzylic carbon adjacent to the cyanophenyl ring to form a biologically active circulating metabolite M1, and oxidation of one of the two geminal methyl groups of taranabant or M1 to the corresponding diastereomeric carboxylic acids. Oxidation of the cyanophenyl ring, followed by conjugation with glutathione or glucuronic acid, was a major pathway of metabolism only in the rat and was not detected in the rhesus monkey. Metabolism profiles of taranabant in liver microsomes in vitro were qualitatively similar in rats, rhesus monkeys and humans and included formation of M1 and oxidation of taranabant or M1 to the corresponding carboxylic acids via oxidation of a geminal methyl group. In human liver microsomes, metabolism of taranabant was mediated primarily by CYP3A4.

Importance of mechanistic drug metabolism studies in support of drug discovery: A case study with an N -sulfonylated dipeptide VLA-4 antagonist in rats.

N-(1-(3,5-dichlorobenzenesulfonyl)-2S-methyl-azetidine-2-carbonyl)-L-4-(2',6'-dimethoxyphenyl)phenylalanine (1) is a potent antagonist of the very late activating (VLA) antigen-4. During initial screening, 1 exhibited an apparent plasma clearance (CL) of 227 ml min(-1) kg(-1) in Sprague-Dawley rats following an intravenous bolus dose formulated in an aqueous solution containing 40% polyethylene glycol. Such a high CL value led to speculation that the elimination of compound 1 involved extra-hepatic tissues. However, the apparent plasma CL was reduced to 97 ml in(-1) kg(-1) when a 2-min time point was added to sample collections, and further decreased to 48 ml min(-1) kg(-1) after the dose was formulated in rat plasma. The lung extraction of 1 in rats was negligible whereas the hepatic extraction was > or =90%, based on comparison of area under the curve (AUC) values derived from intra-artery, intravenous, and portal vein administration. In rats dosed intravenously with [(14)C]-1, approximately 91% of the radioactivity was recovered in bile over 48 h, with 85% accounted for in the first 4-h samples. The biliary radioactivity profile consisted of approximately 30% intact parent compound, 20% 1-glucuronide, and 50% oxidation products resulting from O-demethylation or hydroxylation reactions. When incubated with rat liver microsomes, oxidative metabolism of 1 was inhibited completely by 1-aminobenzotriazole (ABT), whereas the oxidation and glucuronidation reactions were little affected in the presence of cyclosporin A (CsA). In contrast, the hepatic extraction of 1 in rats was unperturbed in animals pre-dosed with ABT, but was reduced approximately 60% following treatment with CsA. In vitro, 1 was a substrate of the rat organic anion transporter Oatp1b2, and its cellular uptake was inhibited by CsA. In addition, the hepatic extraction of 1 was approximately 30% lower in Eisai hyperbilirubinaemic rats which lack functional multidrug resistant protein-2 (MRP2). Collectively, these data suggest that the clearance of 1 in rats likely is a result of the combined processes of hepatic oxidation, glucuronidation and biliary excretion, all of which are facilitated by active hepatic uptake of parent compound and subsequent active efflux of both unchanged parent and its metabolites into bile. It was concluded, therefore, that multiple mechanisms contribute to the clearance of 1 in rats, and suggest that appropriate pharmacokinetic properties might be difficult to achieve for this class of compounds. This case study demonstrates that an integrated strategy, incorporating both rapid screening and mechanistic investigations, is of particular value in supporting drug discovery efforts and decision-making processes.

Substituted 2-aminopyridines as inhibitors of nitric oxide synthases.

A series of substituted 2-aminopyridines was prepared and evaluated as inhibitors of human nitric oxide synthases (NOS). 4,6-Disubstitution enhanced both potency and specificity for the inducible NOS with the most potent compound having an IC50 of 28 nM.

Inhibition of stromelysin-1 (MMP-3) by P1'-biphenylylethyl carboxyalkyl dipeptides.

Carboxyalkyl peptides containing a biphenylylethyl group at the P1' position were found to be potent inhibitors of stromelysin-1 (MMP-3) and gelatinase A (MMP-2), in the range of 10-50 nM, but poor inhibitors of collagenase (MMP-1). Combination of a biphenylylethyl moiety at P1', a tert-butyl group at P2', and a methyl group at P3' produced orally bioavailable inhibitors as measured by an in vivo model of MMP-3 degradation of radiolabeled transferrin in the mouse pleural cavity. The X-ray structure of a complex of a P1-biphenyl inhibitor and the catalytic domain of MMP-3 is described. Inhibitors that contained halogenated biphenylylethyl residues at P1' proved to be superior in terms of enzyme potency and oral activity with 2(R)-[2-(4'-fluoro-4-biphenylyl)ethyl]-4(S)-n-butyl-1,5-pentane dioic acid 1-(alpha(S)-tert-butylglycine methylamide) amide (L-758,354, 26) having a Ki of 10 nM against MMP-3 and an ED50 of 11 mg/kg po in the mouse pleural cavity assay. This compound was evaluated in acute (MMP-3 and IL-1 beta injection in the rabbit) and chronic (rat adjuvant-induced arthritis and mouse collagen-induced arthritis) models of cartilage destruction but showed activity only in the MMP-3 injection model (ED50 = 6 mg/kg iv).

Inhibition of matrix metalloproteinases by N-carboxyalkyl peptides.

An extensive study of the requirements for effective binding of N-carboxyalkyl peptides to human stromelysin, collagenase, and to a lesser extent, gelatinase A has been investigated. These efforts afforded inhibitors generally in the 100-400 nM range for these matrix metalloproteinases. The most significant increase in potency was obtained with the introduction of a beta-phenylethyl group at the P1' position, suggesting a small hydrophobic channel into the S1' subsite of stromelysin. One particular compound, N-[1(R)-carboxyethyl]-alpha(S)-(2-phenylethyl)glycyl-L-leucine,N- phenylamide (79a), is relatively selective for rabbit stromelysin with a K(i) = 6.5 nM and may prove useful for elucidating the role of endogenously-produced stromelysin in lapine models of tissue degradation.

Substituted 4,6-diaminoquinolines as inhibitors of C5a receptor binding.

The anaphylatoxin C5a is implicated in a number of inflammatory diseases. It is a highly cationic protein with 13 of 74 amino acids being either arginine or lysine. A search focusing on positively charged molecules, particularly amine-containing functionalities, led to the discovery of substituted 4,6-diaminoquinolines 1 [N,N'-bis(4-amino-2-methyl-6-quinolyl)urea] and 7 [6-N-(2-chlorocinnamoyl)-4,6-diamino-2-methylquinoline] as inhibitors of C5a receptor binding. These two compounds inhibited the binding of radiolabeled C5a to its receptor isolated from human neutrophils with IC50's = 3.3 and 12 micrograms/mL, respectively. Our efforts to enhance their potencies by chemical modification revealed a narrow profile of potency for effective C5a receptor binding inhibition.