Bindhu V. Karanam

In vitro metabolism of FK-506 in rat, rabbit, and human liver microsomes: identification of a major metabolite and of cytochrome P450 3A as the major enzymes responsible for its metabolism.

The metabolism of the immunosuppressant FK-506 was shown to be catalyzed primarily by cytochrome P450 isozymes of the P450 3A subfamily. Antibodies against rat P450 3A inhibited FK-506 metabolism by 82% in rat liver microsomes and by 35-56% in liver microsomes from humans, dexamethasone-induced rats, and erythromycin-induced rabbits. Poor species cross-reactivity of the antibodies, metabolic switching, and/or some metabolism by P450 isozymes other than P450 3A may be responsible for the incomplete inhibition observed. Besides anti-rat P450 3A, antibodies against rat P450 1A also appeared to have some inhibitory effect implicating these particular cytochrome P450 isozymes as having a minor role in FK-506 metabolism. The formation of 13-desmethyl FK-506, identified here as a major metabolite of FK-506 in all types of microsomes examined, was inhibited completely by anti-P450 3A in liver microsomes from dexamethasone-induced rats and erythromycin-induced rabbits but only partially in human and control rat liver microsomes.

Antidiabetic and antisteatotic effects of the selective fatty acid synthase (FAS) inhibitor platensimycin in mouse models of diabetes

Platensimycin (PTM) is a recently discovered broad-spectrum antibiotic produced by Streptomyces platensis. It acts by selectively inhibiting the elongation-condensing enzyme FabF of the fatty acid biosynthesis pathway in bacteria. We report here that PTM is also a potent and highly selective inhibitor of mammalian fatty acid synthase. In contrast to two agents, C75 and cerulenin, that are widely used as inhibitors of mammalian fatty acid synthase, platensimycin specifically inhibits fatty acid synthesis but not sterol synthesis in rat primary hepatocytes. PTM preferentially concentrates in liver when administered orally to mice and potently inhibits hepatic de novo lipogenesis, reduces fatty acid oxidation, and increases glucose oxidation. Chronic administration of platensimycin led to a net reduction in liver triglyceride levels and improved insulin sensitivity in db/+ mice fed a high-fructose diet. PTM also reduced ambient glucose levels in db/db mice. These results provide pharmacological proof of concept of inhibiting fatty acid synthase for the treatment of diabetes and related metabolic disorders in animal models.

Orally bioavailable, indole-based nonpeptide GnRH receptor antagonists with high potency and functional activity

Stereospecific introduction of a methyl group to the indole-3-side chain enhanced activity in our tryptamine-derived series of GnRH receptor antagonists. Further improvements were achieved by variation of the bicyclic amino moiety of the tertiary amide and by adjustment of the tether length to a pyridine or pyridone terminus. These modifications culminated in analogue 24, which had oral activity in a rat model and acceptable oral bioavailability and half-life in dogs and monkeys.

Metabolism and Excretion of Anacetrapib, a Novel Inhibitor of the Cholesteryl Ester Transfer Protein, in Humans

Anacetrapib is a novel cholesteryl ester transfer protein inhibitor being developed for the treatment of primary hypercholesterolemia and mixed dyslipidemia. The absorption, distribution, metabolism, and excretion of anacetrapib were investigated in an open-label study in which six healthy male subjects received a single oral dose of 150 mg and 165 μCi of [14C]anacetrapib. Plasma, urine, and fecal samples were collected at predetermined times for up to 14 days postdose and were analyzed for total radioactivity, the parent compound, and metabolites. The majority of the administered radioactivity (87%) was eliminated by fecal excretion, with negligible amounts present in urine (0.1%). The peak level of radioactivity in plasma (∼2 μM equivalents of [14C]anacetrapib) was achieved ∼4 h postdose. The parent compound was the major radioactive component (79–94% of total radioactivity) in both plasma and feces. Three oxidative metabolites, M1, M2, and M3, were detected in plasma and feces and were identified as the O-demethylated species (M1) and two secondary hydroxylated derivatives of M1 (M2 and M3). Each metabolite was detected at low levels, representing ≤14% of the radioactivity in plasma or fecal samples. In vitro data indicated that anacetrapib is metabolized mainly by CYP3A4 to form M1, M2, and M3. Overall, these data, along with those from other preclinical and clinical studies, indicate that anacetrapib probably exhibits a low-to-moderate degree of oral absorption in humans and the absorbed fraction of the dose is eliminated largely via CYP3A4-catalyzed oxidative metabolism, followed by excretion of metabolites by the biliary-fecal route.

Imidazopyridines: a novel class of hNav1.7 channel blockers.

A series of imidazopyridines were evaluated as potential sodium channel blockers for the treatment of neuropathic pain. Several members were identified with good hNa(v)1.7 potency and excellent rat pharmacokinetic profiles. Compound 4 had good efficacy (52% and 41% reversal of allodynia at 2 and 4h post-dose, respectively) in the Chung rat spinal nerve ligation (SNL) model of neuropathic pain when dosed orally at 10mg/kg.

2-Arylindoles as gonadotropin releasing hormone (GnRH) antagonists: optimization of the tryptamine side chain

A series of 2-arylindoles containing novel heteroaromatic substituents on the tryptamine tether, based on compound 1, was prepared and evaluated for their ability to act as gonadotropin releasing hormone (GnRH) antagonists. Successful modifications of 1 included chain length variation (reduction) and replacement of the pyridine with heteroaromatic groups. These alterations culminated in the discovery of compound 27kk which had excellent in vitro potency and oral efficacy in rodents.

Metabolism of MK-0524, a Prostaglandin D2 Receptor 1 Antagonist, in Microsomes and Hepatocytes from Preclinical Species and Humans

(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopenta[b]indol-3-yl acetic acid (MK-0524) is a potent orally active human prostaglandin D2 receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The major in vitro and in vivo metabolite of MK-0524 is the acyl glucuronic acid conjugate of the parent compound, M2. To compare metabolism of MK-0524 across preclinical species and humans, studies were undertaken to determine the in vitro kinetic parameters (Km and Vmax) for the glucuronidation of MK-0524 in Sprague-Dawley rat, beagle dog, cynomolgus monkey, and human liver microsomes, human intestinal microsomes, and in recombinant human UDP glucuronosyltransferases (UGT). A comparison of Km values indicated that UGT1A9 has the potential to catalyze the glucuronidation of MK-0524 in the liver, whereas UGT1A3 and UGT2B7 have the potential to catalyze the glucuronidation in the intestine. MK-0524 also was subject to phase I oxidative metabolism; however, the rate was significantly lower than that of glucuronidation. The rate of phase I metabolism was ranked as follows: rat ∼ monkey > human intestine > dog > human liver with qualitatively similar metabolite profiles across species. In all the cases, the major metabolites were the monohydroxylated epimers (M1 and M4) and the keto-metabolite, M3. Use of inhibitory monoclonal antibodies and recombinant human cytochromes P450 suggested that CYP3A4 was the major isozyme involved in the oxidative metabolism of MK-0524, with a minor contribution from CYP2C9. The major metabolite in hepatocyte preparations was the acyl glucuronide, M2, with minor amounts of M1, M3, M4, and their corresponding glucuronides. Overall, the in vivo metabolism of MK-0524 is expected to proceed via glucuronidation, with minor contributions from oxidative pathways.

A potent and selective indole N-type calcium channel (Cav2.2) blocker for the treatment of pain

N-type calcium channels (Ca(v)2.2) have been shown to play a critical role in pain. A series of low molecular weight 2-aryl indoles were identified as potent Ca(v)2.2 blockers with good in vitro and in vivo potency.

Absorption, Metabolism, and Excretion of [14C]MK-0524, a Prostaglandin D2 Receptor Antagonist, in Humans

[(3R)-4-(4-Chlorobenzyl)-7-fluoro-5-(methylsulfonyl)-1,2,3,4-tetrahydrocyclopentaindol-3-yl]acetic acid (MK-0524) is a potent orally active human prostaglandin D2 receptor 1 antagonist that is currently under development for the prevention of niacin-induced flushing. The metabolism and excretion of [14C]MK-0524 in humans were investigated in six healthy human volunteers following a single p.o. dose of 40 mg (202 μCi). [14C]MK-0524 was absorbed rapidly, with plasma Cmax achieved 1 to 1.5 h postdose. The major route of excretion of radioactivity was via the feces, with 68% of the administered dose recovered in feces. Urinary excretion averaged 22% of the administered dose, for a total excretion recovery of ∼90%. The majority of the dose was excreted within 96 h following dosing. Parent compound was the primary radioactive component circulating in plasma, comprising 42 to 72% of the total radioactivity in plasma for up to 12 h. The only other radioactive component detected in plasma was M2, the acyl glucuronic acid conjugate of the parent compound. The major radioactive component in urine was M2, representing 64% of the total radioactivity. Minor metabolites included hydroxylated epimers (M1/M4) and their glucuronic acid conjugates, which occurred in the urine as urea adducts, formed presumably during storage of samples. Fecal radioactivity profiles mainly comprised the parent compound, originating from unabsorbed parent and/or hydrolyzed glucuronic acid conjugate of the parent compound. Therefore, in humans, MK-0524 was eliminated primarily via metabolism to the acyl glucuronic acid conjugate, followed by excretion of the conjugate into bile and eventually into feces.

The pharmacokinetics and disposition of MK-0524, a Prostaglandin D2 receptor 1 antagonist, in rats, dogs and monkeys

MK-0524 is a potent, selective and orally active Prostaglandin D2 receptor 1 (DP1) antagonist currently under clinical development for the treatment of niacin-induced flushing. Experiments to study the pharmacokinetics, metabolism and excretion of MK-0524 were conducted in rats, dogs and monkeys. MK-0524 displayed linear kinetics and rapid absorption following an oral dose. Following intravenous (i.v.) administration of MK-0524 to rats and dogs (1 and 5 mg/kg), the mean Clp was ∼2 and ∼6 ml/min/kg, the T1/2 was ∼7 and ∼13 h and the Vdss was ∼1 and ∼5 L/kg, respectively. In monkeys dosed i.v. at 3 mg/kg, the corresponding values were 8 ml/min/kg, 3 h and 1 L/kg, respectively. Following oral dosing of MK-0524 to rats (5, 25 and 100 mg/kg), dogs (5 mg/kg) and monkeys (3 mg/kg), the absorption was rapid with the mean Cmax occurring between 1 and 4 h. Absolute oral bioavailability values in rats, dogs and monkeys were 50, 70 and 8%, respectively. The major circulating metabolite was the acyl glucuronide of MK-0524 (M2), with ratios of glucuronide to the parent aglycone being highest in the monkey followed by dog and rat. In bile duct-cannulated rats and dogs, MK-0524 was eliminated primarily via acyl glucuronidation followed by biliary excretion of the acyl glucuronide, M2, the major drug-related entity in bile.