Prasugrel pharmacokinetics and pharmacodynamics in subjects with moderate renal impairment and end-stage renal disease.

OBJECTIVE: The pharmacokinetic (PK) and pharmacodynamic (PD) responses to prasugrel were compared in three studies of healthy subjects vs. those with moderate or end-stage renal impairment. METHODS: Two of the three protocols were parallel-design, open-label, single dose (60-mg prasugrel) studies in subjects with end-stage renal disease (ESRD; n = 12) or moderate renal impairment (n = 10) and matched healthy subjects with normal renal function (n = 10). The third protocol was an open-label, single-dose escalation (5, 10, 30 and 60 mg prasugrel) study in subjects with ESRD (n = 16) and matched healthy subjects with normal renal function (n = 16). Plasma concentrations of prasugrel's active metabolite were determined and pharmacokinetic parameter estimates were derived. Maximum platelet aggregation (MPA) was measured by light transmission aggregometry using 20 mum adenosine diphosphate as agonist. RESULTS: Across all studies, prasugrel's C(max) and AUC(0-t) were 51% and 42% lower in subjects with ESRD than in healthy subjects. AUC(0-t) did not differ between healthy subjects and subjects with moderate renal impairment. The magnitude of change and time-course profiles of MPA was similar for healthy subjects compared with subjects with moderate renal impairment and those with ESRD. Prasugrel was well-tolerated in all subjects. CONCLUSION: There was no difference in pharmacokinetics or PD responses between subjects with moderate renal impairment and healthy subjects. Despite significantly lower exposure to prasugrel's active metabolite in subjects with ESRD, MPA did not differ between healthy subjects and those with ESRD.

In vitro metabolism of the epoxide substructure of cryptophycins by cytosolic glutathione S-transferase: species differences and stereoselectivity.

The enzyme kinetics of the glutathione (GSH) conjugation of cryptophycin 52 (C52, R-stereoisomer) and cryptophycin 53 (C53, S-stereoisomer) by cytosolic glutathione S-transferases (cGSTs) from human, rat, mouse, dog and monkey liver were studied. Vmax, Km, and CLint values for glutathione conjugation of C52 (R-stereoisomer) were 0.10 +/- 0.01 nmol min-1 mg-1, 3.24 +/- 0.23 microM, and (3.15 +/- 0.09) x 10(-2) ml min-1 mg-1, respectively, in human cytosol. Due to limited solubility relative to the Km, only CLint values were determined in rat ((7.76 +/- 0.10) x 10-2 ml min-1 mg-1) and mouse ((7.61 +/- 0.50) x 10(-2) ml min-1 mg-1) cytosol. Enzyme kinetic parameters could not be determined for C53 (S-stereoisomer). Microsomal GSH conjugation in human, rat, and mouse was attributed to cytosolic contamination. No GSH conjugation was seen in any biological matrix from dog or monkey. There was little GSH conjugation of C53 by cytosol or microsomes from any species. The metabolism of C52 and C53 by epoxide hydrolase was also investigated. No diol product was observed in any biological matrix from any species. Thus, cGSTs are primarily responsible for C52 metabolism.

Conjugation of glutathione with a toxic metabolite of valproic acid, (E)-2-propyl-2,4-pentadienoic acid, catalyzed by rat hepatic glutathione-S-transferases.

The hepatotoxic metabolite of the anticonvulsant drug valproic acid (VPA), namely (E)-2-propyl-2,4-pentadienoic acid (E)-2,4-diene VPA), is known to react with glutathione (GSH) in vivo. Although glutathione-S-transferase (GST) was suspected of being the catalyst for this conjugation reaction, this was yet to be confirmed. In this study, GST activities were detected in the hepatic cytosolic and sonic-disrupted mitoplast fractions isolated from male Sprague-Dawley rats by using 1-chloro-2,4-dinitrobenzene as a substrate. An elevation of GST activities by 45 to 100% was observed after pretreatment of rats with phenobarbital (PB). Subsequently, these apparent GST activities were examined for their effects on the in vivo conjugation of GSH with N-acetyl-S-((E)-2-propyl-2,4-pentadienoyl)cysteamine (2,4-diene VPA-NACA), a structural mimic of (E)-2,4-diene VPA coenzyme A thioester. Reaction products were identified and quantitated by combined liquid chromatography-tandem mass spectrometry. The GST-mediated conjugation of GSH with 2,4-diene VPA-NACA produced two structural isomers via either 5,6- or 1,6-addition of GSH. Only the 1,6- addition product was found for the spontaneous conjugation reaction (control). Quantitatively, GSH conjugates formed in the cytosolic fraction were 23-fold that of control. An additional 1.5-fold enhancement was observed in the cytosolic fraction from PB-treated rats. The production of the GSH conjugates was increased by 2-fold for reactions involving the sonic-disrupted mitoplasts, either from untreated or PB-treated rats. Partially purified GST was found to catalyze the conjugation reactions in a fashion similar to that of the isolated subcellular fractions. No reaction with GSH could be detected for the free acid form of (E)-2,4-diene VPA. As was the case with the in vitro data, two structural isomers of GSH conjugates were detected in the bile of rats that received (E)-2,4-diene VPA. These results indicate that in vivo production of the GSH conjugates of (E)-2,4-diene VPA is most likely catalyzed by GST enzymes, with the esterified diene being essential for the conjugation reaction. In a separate experiment, 2,4-diene VPA-NACA was observed to alkylate reduced oxytocin through one or both cysteine residues. Thus, the toxicity of (E)-2,4-diene VPA might be produced via either GST-promoted depletion of cellular GSH, or a direct modification of key proteins, or both.

Characterization of novel isocyanate-derived metabolites of the formamide N-formylamphetamine with the combined use of electrospray mass spectrometry and stable isotope methodology.

Bioactivation of the formamide N-formylamphetamine (NFA) to 1-methyl-2-phenylethyl isocyanate (MPIC) was investigated in rats by screening bile and urine for conjugates subsequent to the phase I event. NFA was administered to rats as a mixture of protio- and pentadeuteriophenyl analogues to gain insight into the carbamoylating activity of MPIC when traced by electrospray liquid chromatography/mass spectrometry (LC/MS). An LC/MS contour generated by recording the summed mass spectrum as a function of chromatographic retention time allowed four biliary metabolites to be identified from four sets of doublets, with the peak of each doublet offset by 5 amu and ca. 0.07 min. Tandem mass spectrometry experiments allowed these metabolites to be attributed structurally to the glutathione, cysteinylglycine, cysteine, and N-acetylcysteine conjugates of the isocyanate MPIC. These assignments were subsequently validated by comparison of the LC/MS properties of the metabolites to synthetic reference compounds. Only the carbamoylated N-acetylcysteine conjugate was detected in urine. The observed excretion in bile of all metabolites of the mercapturate pathway is novel for formamide metabolism. NFA can thus be added to the short list of compounds that are eliminated in this fashion. Factors envisioned as contributory to this metabolic profile in bile include hepatorenal, enterohepatic, and biliary-hepatic cycling, in addition to possible equilibrium exchange of the isocyanate from thiocarbamate conjugates to endogenous free thiols during the course of biliary transit.

Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: hepatic microvesicular steatosis and glutathione status.

It is postulated that the hepatotoxicity of valproic acid (VPA) results from the mitochondrial beta-oxidation of its cytochrome P450 metabolite, 2-propyl-4-pentenoic acid (4-ene VPA), to 2-propyl-(E)-2,4-pentadienoic acid ((E)-2,4-diene VPA) which, in the CoA thioester form, either depletes GSH or produces a putative inhibitor of beta-oxidation enzymes. In order to test this hypothesis, 2-fluoro-2-propyl-4-pentenoic acid (alpha-fluoro-4-ene VPA) which was expected to be inert to beta-oxidative metabolism was synthesized and its effect on rat liver studied in comparison with that of 4-ene VPA. Similarly, the known hepatotoxicant 4-pentenoic acid (4-PA) and 2,2-difluoro-4-pentenoic acid (F2-4-PA) were compared. Male Sprague-Dawley rats (150-180 g, 4 rats per group) were dosed ip with 4-ene VPA (0.7 mmol/kg per day), 4-PA (1.0 mmol/kg per day), or equivalent amounts of their alpha-fluorinated analogues for 5 days. Both 4-ene VPA and 4-PA induced severe hepatic microvesicular steatosis ( > 85% affected hepatocytes), and 4-ene VPA produced mitochondrial alterations. By contrast, alpha-fluoro-4-ene VPA and F2-4-PA were not observed to cause morphological changes in the liver. The major metabolite of 4-ene VPA in the rat urine and serum was the beta-oxidation product (E)-2,4-diene VPA. The N-acetylcysteine (NAC) conjugate of (E)-2,4-diene VPA was also found in the urine. Neither (E)-2,4-diene VPA nor the NAC conjugate could be detected in the rats administered alpha-fluoro-4-ene VPA. In a second set of rats (3 rats per group), total liver GSH levels were determined to be depleted to 56% and 72% of control following doses of 4-ene VPA (1.4 mmol/kg) and equivalent alpha-fluoro-4-ene VPA, respectively. Mitochondrial GSH remained unchanged in the alpha-fluoro-4-ene VPA treated group but was reduced to 68% of control in the rats administered 4-ene VPA. These results strongly support the theory that hepatotoxicity of 4-ene VPA, and possibly VPA itself, is mediated largely through beta-oxidation of 4-ene VPA to reactive intermediates that are capable of depleting mitochondrial GSH.

Identification of carbamoylated thiol conjugates as metabolites of the antineoplastic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, in rats and humans.

The metabolic fate of 1-(2-chloroethyl)-3-cyclohexyl)-nitrosourea (CCNU) in rats and humans was investigated with a view to characterizing the nature of the carbamoylating species released upon in vivo transformation of the drug. CCNU undergoes oxidation in vivo to afford 4-hydroxy and 3-hydroxy CCNU which, along with the parent drug, decomposes to the corresponding isocyanates. Although the highly reactive nature of the isocyanate species precludes their identification in vivo, their existence as electrophilic intermediates was detected in the likeness of trapped glutathione (GSH) and N-acetylcysteine (NAC) conjugates. Conjugated thiol metabolites were purified by HPLC from the bile and urine of CCNU-dosed rats, and the urine of a patient on CCNU therapy. The metabolites were identified by atmospheric pressure chemical ionization LC/MS and LC/MS/MS. In addition, LC/MS and LC/MS/MS spectra of synthesized authentic standards corroborated the identity of the metabolites. In rats, 4-hydroxycyclohexyl, 3-hydroxycyclohexyl, and cyclohexyl isocyanate were identified as their GSH conjugates in bile and NAC conjugates in urine. In the case of the patient, the NAC conjugates of 4-hydroxycyclohexyl and 3-hydroxycyclohexyl isocyanate were identified as urinary metabolites. The identification of GSH and NAC conjugates reported herein marks a significant advance in the assessment of the in vivo carbamoylating activity of CCNU and its phase I metabolites.

Metabolic profiling of clobazam, a 1,5-benzodiazepine, in rats.

The metabolism of the 1,5-benzodiazepine clobazam (CLBZ) was investigated in the rat and in vitro by GC/MS using stable isotope techniques. Coadministration of CLBZ and pentadeuteriophenyl CLBZ to rats facilitated the identification of 4'-hydroxy CLBZ 7,4'-hydroxy N-desmethylclobazam (4'-hydroxy DMC) 5, 3',4'-dihydroxy CLBZ 13, 4'-hydroxy-3'-methoxy CLBZ 14, 3'-hydroxy-4'-methoxy CLBZ 15, and 4'-hydroxy-3'-methoxy DMC 16 in bile as both glucuronide and sulfate conjugates. Metabolites 7, 13, and 14 were present in urine as sulfate conjugates. 4'-Hydroxy CLBZ and 4'-hydroxy-3'-methoxy CLBZ were the major conjugated metabolites in bile and urine, respectively. An unusual in vivo disposition of CLBZ to the O-methyl catechols was discovered. In bile, the para O-methyl catechol 15 constituted 2% of the O-methyl catechols as a glucuronide conjugate, in contrast to constituting 30% (of the O-methyl catechols) as a sulfate. This marks an unprecedented observation of a different catechol O-methyl isomer ratio within the same biological fluid for different conjugate pools. The isotope effect associated with the microsomal N-demethylation of trideuteriomethyl CLBZ was determined. The values of kH/kD were calculated at 5.07 +/- 0.37 (N = 3) and 3.88 +/- 0.23 (N = 4) for control and induced microsomes, respectively.