Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

Substance P receptor antagonist I: conversion of phosphoramidate prodrug after i.v. administration to rats and dogs.

A water-soluble phosphoramidate prodrug (L-758,298, compound I) of the potent and selective human Substance P receptor antagonist L-754, 030 (compound II) is under development as an i.v. drug for treatment of emesis, migraine, and chronic pain. Compound I undergoes hydrolysis readily to II under acidic conditions. In the studies reported herein, we investigated the stability of I in blood and hepatic subcellular fractions from rats, dogs, and humans as well as the conversion of I to II in rats and dogs after i.v. dosing. Compound I was converted to II rapidly in rat blood but was stable in dog and human blood. However, the conversion was rapid in liver microsomes prepared from dogs and humans. As expected from the results of in vitro studies, the in vivo conversion of I to II was rapid after i.v. dosing of I to rats and dogs. The relative extent of exposure of II after i.v. dosing of I was estimated by comparing the dose-adjusted area under the plasma concentration versus time curve values of II after i.v. dosing of I with those after i.v. dosing of II. In rats, the extent of exposure was estimated to be approximately 90 and approximately 100% at 1 and 8 mg/kg, respectively; in dogs, that was approximately 59% at 0.5 mg/kg. A nonproportional increase in the area under the concentration versus time curve value of II with dose was observed after i.v. administration of I in dogs from 0.5 to 32 mg/kg, suggesting that the elimination of II might have been saturated at higher doses.

Identification of cytochrome P4503A4 as the major enzyme responsible for the metabolism of ivermectin by human liver microsomes.

1. Ivermectin was extensively metabolized by human liver microsomes to at least 10 metabolites. The structure of many of them (mostly hydroxylated and demethylated) was determined by 1H-NMR and LC/MS. 2. To determine which human cytochrome P450 isoform(s) is responsible for the metabolism of ivermectin, chemical inhibitors including sulphaphenazole, quinidine, furafylline, troleandomycin (TAO) and diethyldithiocarbamate (DDC) were used to evaluate their effect on ivermectin metabolism. TAO, a specific inhibitor of cytochrome P4503A4, was the most potent inhibitor, inhibiting the total metabolism as well as formation of each metabolite. Metabolism was also inhibited by an anti-human cytochrome 3A4 antibody by 90%. 3. When ivermectin was incubated with microsomes from cells expressing CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 or 3A4 at 4 mg/ml protein concentrations, metabolic activity was only detected with the microsomes containing CYP3A4. The metabolic profile from cDNA-expressed CYP3A4 microsomes was qualitatively similar to that from human liver microsomes. 4. Thus, cytochrome P4503A4 is the predominant isoform responsible for the metabolism of ivermectin by human liver microsomes.

Orally active inhibitors of human leukocyte elastase. II. Disposition of L-694,458 in rats and rhesus monkeys.

The disposition of L-694,458, a potent monocyclic beta-lactam inhibitor of human leukocyte elastase, was studied in male Sprague-Dawley rats and rhesus monkeys. After iv dosing, L-694,458 exhibited similar pharmacokinetic parameters in rats and rhesus monkeys. The mean values for its plasma clearance, terminal half-life, and volume of distribution at steady state were 27 ml/min/kg, 1.8 hr, and 4.0 liters/kg in rats and 34 ml/min/kg, 2.3 hr, and 5 liters/kg in rhesus monkeys. The bioavailability of a 10 mg/kg oral dose was higher in rats (65%) than in rhesus monkeys (39%). In both species, concentrations of L-694,458 in plasma increased more than proportionally when the oral dose was increased from 10 mg/kg to 40 mg/kg. In monkeys a protracted plasma concentration-time profile was observed at 40 mg/kg, characterized by a delayed T(max) (8-24 hr) and a long terminal half-life (6 hr). [3H]L-694,458 was well absorbed after oral dosing to rats at 10 mg/kg, as indicated by the high recovery of radioactivity in bile (83%) and urine (6%) of bile duct-cannulated rats. Only approximately 5% or less of the radioactivity in bile, urine, and feces was a result of intact L-694,458, indicating that the compound was being eliminated by metabolism, followed by excretion of the metabolites in feces, via bile. Demethylenation of the methylenedioxyphenyl group resulting in the catechol was the primary metabolic pathway in human and rhesus monkey liver microsomes. In rat liver microsomes, the major metabolite was the N-oxide of the methyl-substituted piperazine nitrogen. In rats dosed iv and orally with [3H]L-694,458, concentrations of radioactivity were highest in the lung (the primary target tissue), adrenals, and liver. L-694,458 was unstable in rat blood and plasma, degrading via a pathway believed to be catalyzed by B-esterases and to involve cleavage of the beta-lactam ring and loss of the methylpiperazine phenoxy group. In vitro studies indicated that in human liver, L-694,458 was metabolized by CYP3A and 2C isozymes, and in both monkey and human liver microsomes the compound acted as an inhibitor of testosterone 6beta-hydroxylation.

Orally active inhibitors of human leukocyte elastase. III. Identification and characterization of metabolites of L-694,458 by liquid chromatography-tandem mass spectrometry.

The in vitro and in vivo metabolism of N-[1(R)-(1,3-benzodioxol-5-yl)butyl]-3,3-diethyl-2(S)-[4-[(4-methy l-1-piperazinyl)carbonyl]phenoxy]-4-oxo-1-azetidinecarboxamide (L-694,458) was studied in male Sprague-Dawley rats and rhesus monkeys. Analysis by LC-MS/MS and NMR revealed that the major metabolite generated in incubations with rat liver microsomes resulted from N-oxidation of the piperazine group, while the major metabolite generated in monkey liver microsomes was the catechol that resulted from O-dealkylation of the methylenedioxyphenyl group. Other metabolites observed in these incubations include the piperazine N-desmethyl, several monohydroxylated derivatives of the parent compound, and three products that resulted from cleavage of the beta-lactam ring. Incubations of parent compound with rat hepatocytes in culture generated two major metabolites that resulted from cleavage of the piperazine ring with the loss of an ethylene group from one side of the ring; one of these metabolites retained the piperazine N-methyl group, while the other did not. The metabolite profiles in vivo were similar to those observed in vitro, but they were much more complex owing to secondary and, in some cases, tertiary biotransformations of many of the primary metabolites. Bile obtained from orally dosed rats contained more than 40 parent-related components, and many of these metabolites had arisen from piperazine ring cleavage.

Orally active inhibitors of human leukocyte elastase. I. Disposition of L-683,845 in rats and rhesus monkeys.

L-683,845 is an orally active inhibitor of human leukocyte elastase. Its disposition was studied in rats and rhesus monkeys after dosing with a 3H- or 14C-labeled compound intravenously at 5 mg/kg and orally at 10 mg/kg. L-683,845 exhibited different pharmacokinetics in these two species. In rats, L-683,845 was well-absorbed after oral dosing, with a maximum concentration of 6 microg/ml at 2 hr and bioavailability of approximately 100%. After intravenous dosing, it was cleared slowly at approximately 3 ml/min/kg, with a terminal half-life of approximately 7 hr and a volume of distribution at steady-state of 1 liter/kg. After both intravenous and oral dosing, L-683,845 comprised 50-95% of plasma radioactivity. About 75% of the intravenous and 87% of the oral dose were recovered in the feces as parent and/or conjugates, with the remaining fraction recovered in the urine as polar components. In rhesus monkeys, maximum concentration after oral dosing was only 0.25 microg/ml, and bioavailability was 50%. Plasma clearance was 8-fold higher, at 23 ml/min/kg, and volume of distribution at steady-state larger, at 2 liters/kg, than in rats. The terminal half-life of L-683,845 could not be determined accurately after intravenous dosing, but seemed to be long in orally dosed animals, approximately 13 hr. Intact L-683,845 was a minor component in plasma comprising only approximately 20% of the radioactivity at most time points. Moreover, persistent levels of radioactivity were detected in plasma and urine of rhesus monkeys even at 1-month postdose, and > or = 25% of the radioactivity in plasma was irreversibly bound to proteins at the later time points. Recovery of the radioactivity was incomplete, with only 77% of the intravenous and 43% of the oral dose recovered over a 4-day period. L-683,845-derived radioactivity distributed to all major rat tissues, with highest levels in the liver followed by the small intestine, adrenals, kidneys, and lungs. Radioactivity concentrations in the liver were high even at 24 hr, 22.7 microg eq/g. A large portion of the intravenous dose was recovered in the small intestine, approximately 40% at 2 hr, indicating rapid and extensive biliary excretion. L-683,845 was metabolized primarily to the acyl glucuronide, which was very unstable in rat plasma, and was subject to hydrolysis to L-683,845 and rearrangement. The glucuronide and L-683,845 were degraded in rat plasma by opening the beta-lactam ring and loss of the C4 substituent followed by decarboxylation to give an olefin and/or decomposition to the monosubstituted urea. Based on inhibition by organophosphorus compounds, it is speculated that their degradation is catalyzed by a type B esterase.