Oral bioavailability and disposition of [14C]omapatrilat in healthy subjects.

The objective of this study was to determine the absolute oral bioavailability and disposition of omapatrilat. This single-dose, randomized, crossover study of 20 mg intravenous and 50 mg oral [14C]omapatrilat was conducted in 12 healthy male subjects to determine the disposition and oral bioavailability of omapatrilat, an orally active vasopeptidase inhibitor. Blood samples were collected up to 120 hours, and the excreta were collected over 168 hours postdose. Plasma concentrations of omapatrilat were determined by a validated LC/MS/MS procedure. Radioactivity in blood, plasma, urine, and feces was determined by liquid scintillation counting. Urinary excretion of radioactivity averaged 80% and 64% of intravenous and oral doses, respectively; < 1% of oral dose was excreted unchanged in urine. The absolute oral bioavailability of omapatrilat averaged 31%. Total body clearance of omapatrilat (80 L/h) exceeded liver plasma flow. Apparent steady-state volume of distribution of omapatrilat (21 L/kg) was extremely high compared with total body water. Omapatrilat undergoes substantial presystemic first-pass metabolism after oral administration. Omapatrilat is eliminated primarily by metabolism, and its metabolites are eliminated primarily in urine. Extrahepatic organs may be involved in the elimination of omapatrilat. Plasma concentrations of omapatrilat exhibit a prolonged terminal elimination phase, which represents elimination from a deep compartment.

Metabolism of [(14)C]omapatrilat, a sulfhydryl-containing vasopeptidase inhibitor in humans.

Omapatrilat, a potent vasopeptidase inhibitor, is currently under development for the treatment of hypertension and congestive heart failure. This study describes the plasma profile along with isolation and identification of urinary metabolites of omapatrilat from subjects dosed orally with 50 mg of [(14)C]omapatrilat. Only a portion of the radioactivity in plasma was unextractable (40-43%). Prominent metabolites identified in plasma were S-methyl omapatrilat, acyl glucuronide of S-methyl omapatrilat, and S-methyl (S)-2-thio-3-phenylpropionic acid. Omapatrilat accounted for less than 3% of the radioactivity. However, after dithiothreitol reduction all of the radioactivity was extractable and was characterized to be omapatrilat and its hydrolysis product (S)-2-thio-3-phenylpropionic acid, both apparently bound to proteins via reversible disulfide bonds. Urinary profile of radioactivity showed no parent compound but the presence of several metabolites that can be grouped into three categories. 1) Three metabolites, accounting for 56% of the urinary radioactivity, resulted from the hydrolysis of the exocyclic amide bond of omapatrilat. Two metabolites were diastereomers of S-methyl sulfoxide of (S)-2-thio-3-phenylpropionic acid, and the third was the acyl glucuronide of S-methyl (S)-2-thio-3-phenylpropionic acid. 2) One disulfide, identified as the L-cysteine mixed disulfide of omapatrilat, accounted for 8% of the radioactivity in the urine. 3) Five metabolites, derived from omapatrilat, accounted for 30% of the radioactivity in the urine. Two of these metabolites were mixtures of diastereomers of S-methyl sulfoxide of omapatrilat and the third was the S-methyl omapatrilat ring sulfoxide. The other two metabolites were S-methyl omapatrilat and its acyl glucuronide. These results indicate that omapatrilat undergoes extensive metabolism in humans.

Bioactivation mechanisms of haloalkene cysteine S-conjugates modeled by gas-phase, ion-molecule reactions.

Glutathione conjugate formation plays important roles in the detoxification and bioactivation of xenobiotics. A range of nephrotoxic haloalkenes undergo bioactivation that involves glutathione and cysteine S-conjugate formation. The cysteine S-conjugates thus formed may undergo cysteine conjugate beta-lyase-catalyzed biotransformation to form cytotoxic thiolates or thiiranes. In the studies presented here, cysteine conjugate beta-lyase-catalyzed biotransformations were modeled by anion-induced elimination reactions of S-(2-bromo-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, S-(2-chloro-1,1, 2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester, and S-(2-fluoro-1,1,2-trifluoroethyl)-N-acetyl-L-cysteine methyl ester in the gas phase. Examination of these processes in the gas phase allowed direct observation of the formation of cysteine S-conjugate-derived thiolates and thiiranes, whose formation is inferred from condensed-phase results. The cysteine S-conjugates of these haloethenes exhibit distinctive patterns of mutagenicity that are thought to be correlated with the nature of the products formed by their cysteine conjugate beta-lyase-catalyzed biotransformation. In particular, S-(2-bromo-1,1,2-trifluoroethyl)-L-cysteine is mutagenic, whereas the chloro and fluoro analogues are not. It has been proposed that the mutagenicity of S-(2-bromo-1,1, 2-trifluoroethyl)-L-cysteine is correlated with the greater propensity of the bromine-containing cysteine S-conjugate to form a thiirane compared with those of the chlorine- or fluorine-containing conjugates. The ease of thiirane formation is consistent with the gas-phase results presented here, which show that the bromine-containing conjugate has a greater propensity to form a thiirane on anionic base-induced elimination than the chloro- or fluoro-substituted analogues. The blocked cysteine S-conjugates were deprotonated by gas-phase ion-molecule reactions with hydroxide, methoxide, and ethoxide ions and then allowed to decompose. The mechanisms for these decompositions are discussed as well as the insights into the bioactivation of these cysteine S-conjugates provided by the further decompositions of thiolate intermediates.

Fate and toxicity of 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A)-derived mercapturates in male, Fischer 344 rats.

BACKGROUND: 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is formed in the anesthesia circuit by the degradation of sevoflurane. Compound A is nephrotoxic in rats and undergoes metabolism by the mercapturic acid pathway in rats and humans to yield the mercapturates S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L -cysteine (compound 3) and S-[2(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl-L-cys teine (compound 5). These experiments were designed to examine the fate and nephrotoxicity of compound A-derived mercapturates in rats. METHODS: The deacetylation of compounds 3 and 5 by human and rat kidney cytosol and with purified acylases I and III was measured, and their nephrotoxicity was studied in male Fischer 344 rats. The metabolism of the deuterated analogs of compounds 3 and 5, [acetyl-2H3]S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl ]-N-acetyl-L-cysteine (compound 3-d3) and [acetyl-2H3]S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N -acetyl-L-cysteine (compound 5-d3), respectively, was measured. RESULTS: Compound 5, but not compound 3, was hydrolyzed by human and rat kidney cytosols and by acylases I and III. 19F nuclear magnetic resonance spectroscopic analysis showed no urinary metabolites of compound 3, but unchanged compound 5 and its metabolites 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4-carboxylic acid were detected in urine. Compound 5 (250 microM/kg) produced clinical chemical and morphologic evidence of renal injury in two of three animals studied. CONCLUSIONS: Compounds 3 and 5 underwent little metabolism. Compound 5, but not compound 3, was mildly nephrotoxic. These results indicate that compound A-derived mercapturate formation constitutes a detoxication pathway for compound A.

Cysteine conjugate beta-lyase-dependent metabolism of compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluoro-1-propene) in human subjects anesthetized with sevoflurane and in rats given compound A.

BACKGROUND: Sevoflurane undergoes Baralyme- or soda lime-catalyzed degradation in the anesthesia circuit to yield compound A (2-[fluoromethoxy]-1,1,3,3,3-pentafluroro-1-propene), which is nephrotoxic in rats and undergoes metabolism via the cysteine conjugate beta-lyase pathway in those animals. The objective of these experiments was to test the hypothesis that compound A undergoes beta-lyase-dependent metabolism in humans. METHODS: Human volunteers were anesthetized with sevoflurane (1.25 minimum alveolar concentration, 3%, 2 l/min, 8 h) and thereby exposed to compound A. Urine was collected at 24-h intervals for 72 h after anesthesia. Rats, which served as a positive control, were given compound A intraperitoneally, and urine was collected for 24 h afterward. Human and rat urine samples were analyzed by 19F nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry for the presence of compound A metabolites. RESULTS: Analysis of human and rat urine showed the presence of the compound A metabolites S-[2(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-N-acetyl-L- cysteine, (E)- and (Z)-S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-N-acetyl- L-cysteine, 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid, 3,3,3-trifluorolactic acid, and inorganic fluoride. The presence of 2-(fluoromethoxy)3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in human urine was confirmed by gas chromatography-mass spectrometry. CONCLUSIONS: The formation of compound A-derived mercapturates shows that compound A undergoes glutathione S-conjugate formation. The identification of 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid and 3,3,3-trifluorolactic acid in the urine of humans anesthetized with sevoflurane shows that compound A undergoes beta-lyase-dependent metabolism. Metabolite formation was qualitatively similar in both human volunteers anesthetized with sevoflurane, and thereby exposed to compound A, and in rats given compound A, indicating that compound A is metabolized by the beta-lyase pathway in both species.

Cyclic hydroxamic acid inhibitors of prostate cancer cell growth: selectivity and structure activity relationships.

BACKGROUND: Clinical symptoms of prostatitis, prostatodynia, and benign prostatic hyperplasia are relieved by the pollen extract cernilton, and the water-soluble fraction of this extract selectively inhibits growth of some prostate cancer cells. A cyclic hydroxamic acid, DIBOA, has been isolated from this extract and mimics its cell growth-inhibitory properties, but the specificity of DIBOA for inhibition of prostate cell growth has not been reported. METHODS: The in vitro growth inhibitory effects of DIBOA and nine structurally related compounds on DU-145 prostate cancer cells, MCF-7 breast cancer cells, and COS-7 monkey kidney cells were determined by treatment of the cells with various concentrations of the compounds for 2-6 days. RESULTS: The compounds exhibited a wide range of potencies, but none of them exhibited selective inhibition of DU-145 cell growth. MCF-7 cells were more sensitive to DIBOA than either DU-145 cells or COS-7 cells. 3,4-dihydroquinoline-2(1H)-one, compound (4), and 1-hydroxy-6-chloro-3,4-dihydroquinolin-2(1H)-one, compound (7), selectively inhibited MCF-7 cell growth at a concentration of 10 micrograms/ml. 1-hydroxy-3,4-dihydroquinolin-2(1H)-one, compound (3), and compound 7 were the most potent inhibitors of DU-145 cell growth. Treatment of DU-145 cells with 3 (100 micrograms/ml) substantially decreased the number of viable cells within 2 days, and no viable cells remained in the culture by day 4. CONCLUSIONS: It is unlikely that DIBOA, compound (1), is responsible for the selective growth inhibition of prostate cancer cells by the water-soluble fraction of the pollen extract cernilton. Cell morphology results indicate that the growth-inhibitory effects of DIBOA and structurally related agents on DU-145 cells are due to their ability to cause cell death.

Cysteine conjugate beta-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A).

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (1, Compound A) is a fluoroalkene formed by the base-catalyzed degradation of sevoflurane that is nephrotoxic in rats. Fluoroalkene 1 is a structural analog of other nephrotoxic haloalkenes that undergo glutathione S-conjugate formation and cysteine S-conjugate beta-lyase-dependent bioactivation to reactive intermediates. The present experiments were designed to study the beta-lyase-dependent biotransformation of S-[2-(fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine (4) and S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine (5) by 19F NMR and UV spectroscopy and GC/MS. Incubation of cysteine S-conjugate 4 with rat kidney cytosol or a pyridoxal model system showed the formation of inorganic fluoride, pyruvate, and 2-(fluoromethoxy)-3,3,3-trifluoropropanoic acid (9), the expected products of a beta-lyase-catalyzed reaction. The ratio of fluoride to pyruvate ranged from 2.3 to 2.5. The amount of acid 9 formed in the rat kidney cytosol and the pyridoxal model system was, however, less than 5% of the amount of pyruvate formed. Incubation of conjugate 4 with rat kidney cytosol and analysis by 19F NMR spectroscopy showed resonances that were assigned to 3,3,3-trifluorolactic acid (10); the formation of acid 10 was observed in the pyridoxal model only after prolonged incubation (> 18 h). Lactic acid 10 was identified as a degradation product of acid 9. Cysteine S-conjugate 5 was not stable in pH 7.4 buffer and underwent a rapid cyclisation reaction (t1/2 approximately 5 min) to form 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1,3-thiazol e-4 -carboxylic acid (14). These data show that fluoroalkene 1-derived cysteine S-conjugates are substrates for renal beta-lyase and that acid 9 is formed as a terminal product. Acid 9 is, however, unstable and affords lactic acid 10 as a degradation product.

Heterocyclic N-acetoxyarylamines, models for the putative ultimate carcinogens of aromatic amines: 2-acetoxyamino-5-phenylpyridine and 2-acetoxyaminopyridine.

The structures of O-acetyl-N-(5-phenyl-2-pyridyl)-hydroxylamine, C13H12N2O2, (I), and O-acetyl-N-(2-pyridyl)hydroxylamine, C7H8N2O2. (II), have been determined in order to confirm earlier structure assignments based on spectroscopic information. Compound (I) is the probable mutagenic metabolite of the phenylalanine pyrolysis product 2-amino-5-phenyl-pyridine. The crystal structures of (I) and (II) are the first reported for heterocyclic N-acetoxyarylamines, the corresponding homocyclic arylamine derivatives being extremely unstable. In the solid state, both (I) and (II) exist as hydrogen-bonded dimers, with the arylamine N atom acting as donor and the pyridine N atom of a neighboring inversion-related molecule as acceptor; the distance between donor and acceptor N atoms is 3.007(2) in (I) and 2.956(2) A in (II). This orientation of the N-H bond results in the rotation of the acetoxy group out of the plane of the pyridine ring by 22.5(2) in (I) and 27.4(2) degrees in (II).

Nephrotoxicity of the glutathione and cysteine S-conjugates of the sevoflurane degradation product 2-(fluoromethoxy)-1,1,3,3, 3-pentafluoro-1-propene (Compound A) in male Fischer 344 rats.

2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (Compound A) is a halogenated alkene that is nephrotoxic in rats when administered by inhalation or intraperitoneally. Compound A undergoes glutathione-dependent metabolism: Compound A-derived glutathione S-conjugates and mercapturates are excreted in the bile and urine, respectively, of rats given Compound A. The present experiments were designed to study the nephrotoxicity of the Compound A-derived glutathione and cysteine S-conjugates, S-[2-(fluoromethoxy)-1,1,3,3, 3-pentafluoropropyl]glutathione , S-[2-(fluoromethoxy)-1,3,3, 3-tetrafluoro-1-propenyl]glutathione , S-[2-(fluoromethoxy)-1,1,3,3, 3-pentafluoropropyl]-L-cysteine and S-[2-(fluoromethoxy)-1,3,3, 3-tetrafluoro-1-propenyl]-L-cysteine . Conjugates , and given intraperitoneally produced dose-dependent nephrotoxicity that was characterized by diuresis, increased excretion of glucose and protein, elevated blood urea nitrogen concentrations and severe morphological changes in the kidneys, particularly in the proximal tubules. Glutathione S-conjugate , at a dose of 500 micromol/kg, was hepatotoxic. Cysteine S-conjugate was not nephrotoxic, apparently because of its facile cyclization to the thiazoline 2-[1-(fluoromethoxy)-2,2,2-trifluoroethyl]-4,5-dihydro-1, 3-thiazole-4-carboxylic acid, which is not a beta-lyase substrate. Also, the alpha-methyl analog of cysteine S-conjugate S-[2-(fluoromethoxy)-1,1,3,3, 3-pentafluoropropyl]-DL-alpha-methylcysteine, which cannot undergo beta-lyase-dependent bioactivation, was not nephrotoxic. These in vivo data show that Compound A-derived S-conjugates are nephrotoxic and that the toxicity is associated with beta-lyase-dependent bioactivation.

Cysteine conjugate beta-lyase-dependent biotransformation of the cysteine S-conjugates of the sevoflurane degradation product compound A in human, nonhuman primate, and rat kidney cytosol and mitochondria.

BACKGROUND: 2-(Fluoromethoxy)-1,1,3,3,3-pentafluoro-1-propene (compound A) is a fluorinated alkene formed by the degradation of sevoflurane in the anesthesia circuit. Compound A is toxic to the kidneys in rats and undergoes glutathione-dependent metabolism in vivo. Several nephrotoxic halogenated alkenes also undergo cysteine conjugate beta-lyase-dependent biotransformation. These experiments were designed to test the hypothesis that cysteine S-conjugates of compound A undergo beta-lyase-dependent biotransformation. METHODS: S-[2-(Fluoromethoxy)-1,1,3,3,3-pentafluoropropyl]-L-cysteine 4, S-[2-(fluoromethoxy)-1,3,3,3-tetrafluoro-1-propenyl]-L-cysteine 5, and S-(2-chloro-1,1,2-trifluoroethyl)-L-cysteine 11 were incubated with rat, human, and nonhuman primate (cynomolgus, rhesus, and marmoset) kidney cytosol and mitochondria. beta-Lyase activity was determined by measuring pyruvate formation. RESULTS: Compound A-derived conjugates 4 and 5 as well as conjugate 11, a positive control, were substrates for cytosolic and mitochondrial beta-lyase from human, nonhuman primate, and rat kidney. For all substrates, beta-lyase activity was highest in the rat and lowest in the human and was higher in cytosol than in mitochondria. Conjugate 11 was a much better substrate than conjugates 4 or 5. The biotransformation of conjugates 4, 5, and 11 was inhibited by the beta-lyase inhibitor (aminooxy)acetic acid and was stimulated by the amino group acceptor 2-keto-4-methylthiolbutyric acid, indicating a role for beta-lyase. CONCLUSIONS: These data confirm the presence of beta-lyase activity in human and rat kidney and show that activity is also present in kidney tissue from nonhuman primates. The data also show that compound A-derived conjugates 4 and 5 undergo beta-lyase-catalyzed biotransformation. beta-Lyase activity in rat and nonhuman primate kidney tissue was 8 to 30 times and one- to three times, respectively, higher than in human kidney tissue.