Comparison of celecoxib metabolism and excretion in mouse, rabbit, dog, cynomolgus monkey and rhesus monkey.

1. The metabolism and excretion of celecoxib, a specific cyclooxygenase 2 (COX-2) inhibitor, was investigated in mouse, rabbit, the EM (extensive) and PM (poor metabolizer) dog, and rhesus and cynomolgus monkey. 2. Some sex and species differences were evident in the disposition of celecoxib. After intravenous (i.v.) administration of [14C]celecoxib, the major route of excretion of radioactivity in all species studied was via the faeces: EM dog (80.0%), PM dog (83.4%), cynomolgus monkey (63.5%), rhesus monkey (83.1%). After oral administration, faeces were the primary route of excretion in rabbit (72.2%) and the male mouse (71.1%), with the remainder of the dose excreted in the urine. After oral administration of [14C]celecoxib to the female mouse, radioactivity was eliminated equally in urine (45.7%) and faeces (46.7%). 3. Biotransformation of celecoxib occurs primarily by oxidation of the aromatic methyl group to form a hydroxymethyl metabolite, which is further oxidized to the carboxylic acid analogue. 4. An additional phase I metabolite (phenyl ring hydroxylation) and a glucuronide conjugate of the carboxylic acid metabolite was produced by rabbit. 5. The major excretion product in urine and faeces of mouse, rabbit, dog and monkey was the carboxylic acid metabolite of celecoxib.

Pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib in rats.

The pharmacokinetics, tissue distribution, metabolism, and excretion of celecoxib, 4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide, a cyclooxygenase-2 inhibitor, were investigated in rats. Celecoxib was metabolized extensively after i.v. administration of [(14)C]celecoxib, and elimination of unchanged compound was minor (less than 2%) in male and female rats. The only metabolism of celecoxib observed in rats was via a single oxidative pathway. The methyl group of celecoxib is first oxidized to a hydroxymethyl metabolite, followed by additional oxidation of the hydroxymethyl group to a carboxylic acid metabolite. Glucuronide conjugates of both the hydroxymethyl and carboxylic acid metabolites are formed. Total mean percent recovery of the radioactive dose was about 100% for both the male rat (9.6% in urine; 91.7% in feces) and the female rat (10.6% in urine; 91.3% in feces). After oral administration of [(14)C]celecoxib at doses of 20, 80, and 400 mg/kg, the majority of the radioactivity was excreted in the feces (88-94%) with the remainder of the dose excreted in the urine (7-10%). Both unchanged drug and the carboxylic acid metabolite of celecoxib were the major radioactive components excreted with the amount of celecoxib excreted in the feces increasing with dose. When administered orally, celecoxib was well distributed to the tissues examined with the highest concentrations of radioactivity found in the gastrointestinal tract. Maximal concentration of radioactivity was reached in most all tissues between 1 and 3 h postdose with the half-life paralleling that of plasma, with the exception of the gastrointestinal tract tissues.

Metabolism and excretion of [(14)C]celecoxib in healthy male volunteers.

We determined the disposition of a single 300-mg dose of [(14)C]celecoxib in eight healthy male subjects. The [(14)C]celecoxib was administered as a fine suspension reconstituted in 80 ml of an apple juice/Tween 80/ethanol mixture. Blood and saliva samples were collected at selected time intervals after dosing. All urine and feces were collected on the 10 consecutive days after dose administration. Radioactivity in each sample was determined by liquid scintillation counting or complete oxidation and liquid scintillation counting. Metabolic profiles in plasma, urine, and feces were obtained by HPLC, and metabolites were identified by mass spectrometry and NMR. [(14)C]Celecoxib was well absorbed, reaching peak plasma concentrations within 2 h of dosing. [(14)C]Celecoxib was extensively metabolized, with only 2.56% of the radioactive dose excreted as celecoxib in either urine or feces. The total percentage of administered radioactive dose recovered was 84.8 +/- 4.9%, with 27.1 +/- 2.2% in the urine and 57.6 +/- 7.3% in the feces. The oxidative metabolism of celecoxib involved hydroxylation of celecoxib at the methyl moiety followed by further oxidation of the hydroxyl group to form a carboxylic acid metabolite. The carboxylic acid metabolite of celecoxib was conjugated with glucuronide to form the 1-O-glucuronide. The percentages of the dose excreted in the feces as celecoxib and the carboxylic acid metabolite were 2.56 +/- 1.09 and 54.4 +/- 6.8%, respectively. The majority of the dose excreted in the urine was the carboxylic acid metabolite (18.8 +/- 2.1%); only a small amount was excreted as the acyl glucuronide (1.48 +/- 0.15%).

Isolation and identification of metabolites of leukotriene A4 hydrolase inhibitor SC-57461 in rats.

The metabolic fate of SC-57461, N-methyl-N-[3-[4-(phenylmethyl)-phenoxy]propyl]-beta-alanine, a potent and specific inhibitor of the leukotriene A4 hydrolase, was determined by LC/MS/MS, NMR and GC/MS in male Sprague-Dawley rats. The major metabolites of SC-57461 in rats were the desmethyl metabolite, the hydroxylated metabolite, the N-oxide metabolite, the hydroxylamine metabolite, and the propionic acid metabolite. The N-oxide metabolite was found to be stable in the rat plasma and urine, but was unstable in most organic solvents (methanol, acetonitrile, and methylene chloride, etc.) because of the classic Cope reaction of the N-oxide, which led to the formation of the corresponding hydroxylamine product and acrylic acid. The hydroxylamine metabolite and acrylic acid were reactive in the biomatrix and could not be isolated in the in vivo samples. However, formation of the hydroxylamine metabolite and acrylic acid from the N-oxide metabolite in methylene chloride was verified by NMR. The propionic acid metabolite was found to be the common metabolite shared by SC-57461, N-oxide metabolite, as well as the hydroxylamine metabolite, which suggested a sequential metabolism of SC-57461 in rats. The ultimate fate of the propionic acid metabolite was incorporation into rat glycerolipid metabolism as a result of its structural similarity to aryl-substituted propionic acid, a known class of compounds that can be incorporated into rat glycerolipid metabolism. Finally, the isolated hydroxylated metabolite and the N-desmethyl metabolite were found to have excellent inhibitory effects toward leukotriene A4 hydrolase and therefore were the major active metabolites of SC-57461 in rats.

Metabolism of a novel antiarrhythmic agent, bidisomide, in man: use of high resolution mass spectrometry to distinguish desisopropyl bidisomide from desacetyl bidisomide.

1. Metabolism of bidisomide, a novel antiarrhythmic agent, was studied in man, and was not extensive as evidenced by the fact that approximately 60 and 70% of the radioactive doses were recovered as the parent drug after i.v. and oral administration respectively. 2. The mass spectra of bidisomide metabolites indicate that the two major metabolic pathways of bidisomide were hydroxylation of the piperidine ring and N-dealkylation. The latter occurred on the side chain containing the piperidine ring or the isopropyl group. The N-dealkylated metabolite on the side chain containing the piperidine ring was cyclized to result in a pyrrolidone metabolite. 3. The N-dealkylated metabolite, desisopropyl bidisomide, was identified by comparing its high resolution mass spectrum to that of authentic desacetyl bidisomide. 4. In the hydroxylation pathway, both mono- and dihydroxylated metabolites of the piperidine ring were observed. The exact location of the hydroxyl groups on the piperidine ring was not determined.

Metabolism and pharmacokinetics of nufenoxole in animals and humans: an example of stereospecific hydroxylation of an isoquinuclidine ring.

1. Nufenoxole, a novel antidiarrhoeal agent, was well absorbed in rat, monkey and human after oral administration. Systemic availability of nufenoxole was 85% in monkey and 102% in man. 2. The elimination rate was much faster in rat (t1/2 of 1.8 h) and monkey (t1/2 of 4.9 h) compared with human (t1/2 of 35.8 h). 3. After oral and i.v. 14C-nufenoxole, concentrations of 14C in human erythrocytes and saliva were approx. 3- and 4-fold lower, respectively, than plasma concentrations. 4. Nufenoxole was metabolized to metabolites hydroxylated on the methyl substituent and isoquinuclidine ring in rat and monkey. The isoquinuclidine ring hydroxylation, a major pathway in human, was stereospecific. 5. Following oral doses of 14C-nufenoxole the urinary excretion of radioactivity (about 8%) was less than the faecal excretion (66.6%) in rat, while urinary excretion was the major route of drug elimination (about 60%) in man. In monkey, urinary and faecal excretion were equally important.

Identification of N-beta-L-aspartyl-L-phenylalanine as a normal constituent of human plasma and urine.

A new beta-aspartyl dipeptide, N-beta-L-aspartyl-L-phenylalanine (beta-AP), has been isolated and identified in urine and plasma from normal human volunteers. beta-AP was isolated from urine samples by high performance liquid chromatography (HPLC). Its identity and stereochemistry were demonstrated by HPLC and gas chromatography/mass spectrometry (GC-MS). The mean urinary beta-AP concentration in the subjects was 0.63 +/- 0.14 microgram/mg creatinine when averaged over two consecutive days of urine collection. Daily beta-AP excretion, determined from two 24-h urine samples collected from five individuals, was 801 +/- 117 micrograms/d (2.7 mumol/d). No diurnal rhythm was evident within the 24-h collection periods. beta-AP was also isolated from human plasma by HPLC and identified by GC-MS. Plasma from subjects contained approximately 5 ng beta-AP/ml. Furthermore, beta-AP was formed when asparagine and phenylalanine were incubated with an enzyme extract from human kidney. Thus, at least some of the beta-AP present in humans, and presumably other beta-aspartyl dipeptides as well, appears to be synthesized endogenously.

Difference in metabolic profile of potassium canrenoate and spironolactone in the rat: mutagenic metabolites unique to potassium canrenoate.

The metabolic fates of potassium canrenoate (PC) and spironolactone (SP) were compared for the rat in vivo and in vitro. Approximately 18% of an in vivo dose of SP was metabolized to canrenone (CAN) and related compounds in the rat. In vitro, 20-30% of SP was dethioacetylated to CAN and its metabolites by rat liver 9000 g supernatant (S9). Thus, the major route of SP metabolism is via pathways that retain the sulfur moiety in the molecule. PC was metabolized by rat hepatic S9 to 6 alpha, 7 alpha- and 6 beta, 7 beta-epoxy-CAN. The beta-epoxide was further metabolized to its 3 alpha- and 3 beta-hydroxy derivatives as well as its glutathione (GSH) conjugate. Both 3 alpha- and 3 beta-hydroxy-6 beta, 7 beta-epoxy-CAN were shown to be direct acting mutagens in the mouse lymphoma assay, whereas 6 alpha, 7 alpha- and 6 beta, 7 beta-epoxy-CAN were not. These mutagenic metabolites, their precursor epoxides and their GSH conjugates were not formed from SP under identical conditions. The above findings appear to be due to inhibition of metabolism of CAN formed from SP by SP and/or its S-containing metabolites, since the in vitro metabolism of PC by rat hepatic microsomes was appreciably reduced in the presence of SP. The hypothesized mechanism(s) for this inhibition is that SP and its S-containing metabolites specifically inhibit an isozyme of hepatic cytochrome P-450 or SP is a preferred substrate over PC/CAN for the metabolizing enzymes. Absence of the CAN epoxide pathway in the metabolism of SP provides a possible explanation for the observed differences in the toxicological profiles of the two compounds.

The pH dependence of disobutamide-induced clear cytoplasmic vacuoles in cultured cells.

Cellular uptake of disobutamide (D), and clear cytoplasmic vacuoles (CCV) induction by D in cultured rat urinary bladder carcinoma cells were dependent on the culture medium pH. At pH 6.0-6.7, drug uptake was slow and no CCV formed in 24 hr. At pH 7.0-8.0, the rate of D uptake and early appearance of CCV were directly proportional to increased basicity. This was explained by the increasing fraction of un-ionized D molecules at increasing basicity of the culture medium. It is only these electrically neutral D molecules which can penetrate the lipoidal cell membrane to induce formation of CCV. Intracellular presence of D was demonstrated by mass spectrometry methods. The results indicate that D is incorporated intracellularly, that D and not its metabolite(s) is in cells, and suggest that CCV are a result of drug sequesteration.

The analysis and source of 1-diphenylmethyl-4-nitrosopiperazine in 1-diphenylmethyl-4-[(6-methyl-2-pyridyl) methyleneamino]piperazine: a case history.

Analytical methods were developed to determine whether there was less than one ppm of II in IV. Purified IV contained a compound with the same GC retention time as authentic II on OV-17 and Silar 10C columns, using a FID or a nitrogen/phosphorus detector. The GC-coupled mass spectra contained major peaks at m/e 251 and 167 and the GC-coupled methane CI spectra gave the quasi-molecular ion, m/e 282. However, the CI selected ion monitor indicated three compounds at m/e 282. The nitrosamine II was separated from IV by HPLC on muBondapak C18. The GC-coupled mass spectra of the HPLC nitrosamine fraction had peaks at m/e 251 and 167 and other peaks for a compound of greater MW than II, although the GC retention times were the same. Evidence for II and a decrease in the detection limit to one ppm was obtained with a TEA interfaced with a HPLC. To determine if II was formed from IV, it was exposed to ozone at -78 degrees C in methylene chloride. The DSC, IR and NMR of the product were coincident with those of the standard of II. In other experiments, IV in methylene chloride was exposed to light and dry air in the presence and absence of methylene blue. The PMR and 13C NMR of the product formed in the presence of methylene blue were the same as that of II. It is postulated that this nitrosamine was formed by a singlet oxygen mechanism.

The identification of some human metabolites of norgestrel, a new progestational agent.

PIP: Norgestrel 1 (racemic 13-beta-ethyl-17-alpha-hydroxygon-4-en-3-one) a progestational agent with an angular ethyl group between Rings C and D, was studied by mass spectrometry to discover its structural characteristics. Synthesis of postulated metabolites of Norgestrel 1 for use in identification is described and structural formulas are given. Urine was used as a source to characterize fractions via mass spectra, and the fraction spectra are listed. The major metabolite was 13-beta-ethyl-17-alpha-ethynl-5-beta-gonan-3-alpha-1m-beta-diol 8c.^ieng