In vitro evidence for the formation of reactive intermediates of resveratrol in human liver microsomes.

Resveratrol (3,4',5-trihydroxystilbene) is a naturally occurring polyphenolic compound found in a variety of foods and over-the-counter health products. It has gained wide public use due to its potential health properties, and is available over-the-counter at health product stores. Although the safety profile of resveratrol has been minimally investigated in humans, resveratrol has been associated with observations of toxicity in vitro, and has been identified as a mechanism-based inhibitor of cytochrome P450 3A4. In addition, resveratrol has been rationally hypothesized to form reactive quinone methide metabolites, despite experimental evidence supporting this assumption. This work evaluates the potential for resveratrol to form glutathione-trapped reactive intermediates in human liver microsomes utilizing liquid chromatography and electrospray tandem mass spectrometry, and has resulted in the identification of several in vitro products including two hydroxylated metabolites (piceatannol and metabolite 2), and two pairs of regioisomeric glutathione adducts. The parallel metabolism of resveratrol to piceatannol and metabolite 2 (a putative quinone methide) are demonstrated to result in the formation of two putative quinone methide intermediates resulting in divergent mechanisms for formation of each pair of regioisomeric glutathione adducts.

Biotransformation of tirilazad in human: 3. tirilazad A-ring reduction by human liver microsomal 5alpha-reductase type 1 and type 2.

Tirilazad mesylate (FREEDOX), a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage. In humans, tirilazad is cleared almost exclusively via hepatic elimination with a medium-to-high extraction ratio. In human liver microsomal preparations, tirilazad is biotransformed to multiple oxidative products and one reduced, pharmacologically active metabolite, U-89678. Characterization of the reduced metabolite by mass spectrometry and cochromatography with an authentic standard demonstrated that U-89678 was formed via stereoselective reduction of the Delta4 bond in the steroid A-ring. Kinetic analysis of tirilazad reduction in human liver microsomes revealed that kinetically distinct type 1 and type 2 5alpha-reductase enzymes were responsible for U-89678 formation; the apparent KM values for type 2 and type 1 were approximately 15 and approximately 0.5 microM, respectively. Based on pH dependence and finasteride inhibition studies, it was inferred that 5alpha-reductase type 1 was the high affinity/low capacity microsomal reductase that contributed to tirilazad clearance in vivo. In addition, a role for CYP3A4 in the metabolism of U-89678 was established using cDNA expressed CYP3A4 and correlation studies comparing U-89678 consumption with cytochrome P450 activities across a population of human liver microsomes. Collectively, these data suggest that formation of U-89678, a circulating pharmacologically active metabolite, contributes to the total metabolic elimination of tirilazad in humans and that clearance of U-89678 is mediated primarily via CYP3A4 metabolism. Therefore, concurrent administration of therapeutic agents that modulate 5alpha-reductase type 1 or CYP3A activity are anticipated to affect the pharmacokinetics of PNU-89678.

Replacing 14C with stable isotopes in drug metabolism studies.

After administration of a mixed dose of both radioisotope and stable-isotope-labeled tirilazad, we carried out a parallel set of HPLC analyses for drug metabolites in bile samples from monkeys and dogs using either radioactivity monitoring (RAM) for 14C or the chemical reaction interface mass spectrometry technique (CRIMS) to detect 13C or 15N. CRIMS is a novel method where analytes are decomposed in a microwave-induced plasma and the elements contained in the analytes are reformulated into small gaseous species that are detected by a mass spectrometer. The comprehensiveness of detection, chromatographic resolution, sensitivity, signal/noise, and quantitative abilities of CRIMS were compared with RAM and in no case was RAM superior. This implies that stable isotopes may be substituted for radioisotopes in studies of drug metabolism where the ability of the latter approach to detect a label independent of the structures in which the label appears has been the primary reason for continuing to use a hazardous and expensive tracer. With HPLC-CRIMS, stable isotopes such as 13C and 15N can be comprehensively detected and quantitative patterns of drug metabolism from biological fluids can be produced that mirror the results when 14C is used.

Biotransformation of tirilazad in human: 1. Cytochrome P450 3A-mediated hydroxylation of tirilazad mesylate in human liver microsomes.

Tirilazad mesylate (Freedox), a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage. In humans, tirilazad is cleared almost exclusively via hepatic elimination. Characterization of three major microsomal metabolites of tirilazad by mass spectrometry indicated that hydroxylation had occurred in the pyrrolidine ring(s) and at the 6 beta-position of the steroid domain. A role for CYP3A4 in the formation of the three major metabolites (tirilazad hydroxylase activity) was established in human liver microsomal preparations: 1) Tirilazad hydroxylation was potently inhibited by troleandomycin and ketoconazole, specific inhibitors of CYP3A4. 2) The rates of tirilazad hydroxylation within a population of 14 human livers displayed a 9-fold interindividual variation and a significant correlation (r2 = .95) between tirilazad hydroxylation and testosterone 6 beta-hydroxylation. 3) Kinetic analysis of tirilazad hydroxylase activity in three human livers resulted in an apparent Km of 2.12, 1.68 and 1.66 microM, and Vmax = 0.85, 0.44 and 3.45 (nmol/mg protein/min) for HL14, HL17 and HL21, respectively. In addition, an apparent Km of 2.07 microM was established for tirilazad hydroxylation in a cDNA-expressed CYP3A4 microsomal system. Collectively, these data indicate that the metabolic clearance of tirilazad in humans is catalyzed primarily by CYP3A4 and provide an insight into factors (i.e., age, sex, drug-drug interactions) that modulate the metabolic clearance of tirilazad in vivo.

Identification of N-acetylcysteine conjugates of 1,2-dibromo-3-chloropropane: evidence for cytochrome P450 and glutathione mediated bioactivation pathways.

The haloalkane 1,2-dibromo-3-chloropropane (DBCP) is a carcinogen, mutagen, nephrotoxin, and testicular toxin. The identification of N-acetylcysteine conjugates of DBCP provides information on GSH mediated and cytochrome P450 mediated bioactivation pathways in the expression of DBCP-induced toxicities. N-Acetylcysteine conjugates excreted in the urine of male Sprague-Dawley rats administered DBCP, C1D2-DBCP, C2D1-DBCP, C3D2-DBCP, or D5-DBCP (80 mg/kg) were purified by reverse-phase HPLC as their methyl ester derivatives and characterized by fast atom bombardment tandem mass spectrometry. These metabolites were also converted to tert-butyldimethylsilyl ether derivatives and analyzed by gas chromatography-mass spectrometry (GC-MS) to facilitate the identification of N-acetyl-S-(2,3-dihydroxypropyl)cysteine (Ia), an apparent regioisomer of Ia, 2-(S-(N-acetylcysteinyl))-1,3-propanediol (Ib), N-acetyl-S-(3-hydroxypropyl)cysteine (IIa), and N-acetyl-S-(3-chloro-2-hydroxypropyl)-cysteine (III). Metabolites Ia, Ib, and III displayed quantitative retention of deuterium, an observation consistent with the formation of episulfonium ion intermediate(s) in their biogenesis. Mercapturate IIa retained three atoms of deuterium from D5-DBCP, and two atoms of deuterium from the dideuterio analogs (C1D2-DBCP and C3D2-DBCP), thus invoking P450 mediated formation of 2-bromoacrolein (2-BA) as an intermediate in the biogenesis of IIa. A mechanism is proposed in which conjugate addition of GSH to 2-BA, subsequent episulfonium ion formation, and addition of GSH afford 1,2-(diglutathion-S-yl)propanal. Glutathione mediated reduction is invoked to afford S-(3-hydroxypropyl)GSH which would be excreted in the urine as IIa. The quantitative retention of deuterium from C1D2-DBCP or C3D2-DBCP was indicative of isotopically sensitive branching of P450 metabolism at either C1 or C3 to afford 2-BA. C2D1-DBCP showed a 30% retention of 1 deuterium atom in IIa; the loss of the deuterium is consistent with 2-BA formation, whereas the retention of one deuterium atom is indicative of the formation of metabolite IIa through GSH conjugation of either 2,3-dibromopropanal or 2-bromo-3-chloropropanal. These data indicate that IIa is a marker metabolite for the potent direct-acting mutagen, 2-BA, or its metabolic precursors 2,3-dibromopropanal or 2-bromo-3-chloropropanal. Therefore, evidence has been presented for bioactivation of DBCP by glutathione and cytochrome P450 mediated mechanisms.

In vitro metabolism of tirilazad mesylate in male and female rats. Contribution of cytochrome P4502C11 and delta 4-5 alpha-reductase.

Tirilazad mesylate, a potent inhibitor of membrane lipid peroxidation in vitro, is under clinical development for the treatment of subarachnoid hemorrhage and head injury. In rat, tirilazad seems to be highly extracted and is cleared almost exclusively via hepatic elimination. The biotransformation of tirilazad has been investigated in liver microsomal preparations from adult male and female Sprague-Dawley rats. Tirilazad metabolism in male rat liver microsomes resulted in the formation of two primary metabolites: M1 and M2. In incubations with female rat liver microsomes, M2 was the only primary metabolite detected. Structural characterization of M1 and M2 by mass spectrometry demonstrated that M2 was formed by reduction of the delta 4-double bond in the steroid A-ring, whereas M1 arose from oxidative desaturation of one pyrrolidine ring. Further structural analysis of M2 by proton NMR demonstrated that reduction at C-5 had occurred by addition of hydrogen in the alpha-configuration. Using metabolic probes and antibodies specific to individual hepatic microsomal enzymes, CYP2C11 and 3-oxo-5 alpha-steroid:NADP+ delta 4-oxidoreductase (5 alpha-reductase) were identified as responsible for the formation of M1 and M2, respectively. The formation of M1 was inhibited by testosterone, nicotine, cimetidine, and anti-CYP2C11 IgG. The formation of M2 was inhibited by finasteride, a potent inhibitor of 5 alpha-reductase. Kinetic analysis of CYP2C11-mediated M1 formation in male rat liver microsomal incubations revealed that M1 formation occurred through a low-affinity/low-capacity process (KM = 16.67 microM, Vmax = 0.978 nmol/mg microsomal protein/min); the formation of M2 was mediated by 5 alpha-reductase in a high-affinity/low-capacity process (KM = 3.07 microM, Vmax = 1.06 nmol/mg microsomal protein/min). In contrast, the formation of M2 in female rat liver microsomes was mediated by 5 alpha-reductase in a high-affinity/high-capacity process (KM = 2.72 microM, Vmax = 4.11 nmol/mg microsomal protein/min). Comparison of calculated intrinsic formation clearances (Vmax/KM) for M1 and M2 indicated that the female rat possessed a greater in vitro metabolic capacity for tirilazad biotransformation than the male rat. Therefore, the clearance of tirilazad mesylate in the rat is mediated primarily by rat liver 5 alpha-reductase, and the capacity in the female rat is 5-fold the capacity in the male. These observations correlate with documented differences in 5 alpha-reductase activity and predict a gender difference in tirilazad hepatic clearance in vivo.

Inhibition of in vitro lipid peroxidation by 21-aminosteroids. Evidence for differential mechanisms.

In a previous report (Ryan and Petry, Arch Biochem Biophys 300: 699-704, 1993), the effects of two 21-aminosteroids (U-74500A and U-74006F) on the oxidation and reduction of iron in a buffer/organic solvent system were investigated. In those studies, U-74500A was found to be an efficient iron reductant and potential iron chelator, whereas U-74006F had little effect on iron redox chemistry. As an extension of those studies, we now report the effects of U-74006F and U-74500A on lipid peroxidation in systems that are dependent upon iron oxidation/reduction. In liposomes, U-74500A inhibited ADP:Fe(II)-dependent lipid peroxidation in a concentration-dependent manner, whereas U-74006F was minimally effective in this system. The mechanism of U-74500A-dependent inhibition probably involved interactions with iron, as iron oxidation was inhibited in the presence of this compound. No effects on iron oxidation were observed in the presence of U-74006F. Addition of Ferrozine to liposomal incubation mixtures indicated that at least two iron pools were present in samples containing U-74500A, one immediately bound by Ferrozine, and another that was bound more slowly. Furthermore, ADP:Fe(III)/ascorbate-dependent lipid peroxidation was blocked completely by U-74500A, presumably by formation of a redox inert complex upon reduction of the iron. U-74500A partially protected ADP:Fe(II) from oxidation by H2O2 and lipid hydroperoxides, indicating that the U-74500A:iron complex was stable in the presence of biologically relevant oxidants. U-74006F did not markedly affect iron oxidation or reduction when incorporated into phospholipid liposomes. In microsomal lipid peroxidation systems containing ADP:Fe(III) and NADPH, both U-74500A and U-74006F inhibited lipid peroxidation. U-74006F-dependent inhibition of microsomal lipid peroxidation was dependent on both NADPH and Fe(III). Further, it was enhanced when U-74006F was allowed to preincubate in this system prior to iron addition. Preincubation of U-74006F with microsomes, NADPH, and ADP:Fe(III) produced several metabolites detectable by HPLC. These results suggest that U-74500A inhibits lipid peroxidation by directly affecting iron redox chemistry, whereas U-74006F-mediated inhibition is enhanced by preincubation with a metabolically competent microsomal system.

Evaluation of the pharmacokinetics and tolerability of tirilazad mesylate, a 21-aminosteroid free radical scavenger: II. Multiple-dose administration.

The multiple dose tolerability and pharmacokinetics of tirilazad mesylate, a 21-aminosteroid free radical scavenger, were assessed in 50 healthy male volunteers. Volunteers were randomized to receive intravenous normal saline placebo (n = 10), citrate vehicle placebo (n = 10), or 0.5 mg/kg/day (n = 6), 1.0 mg/kg/day (n = 6), 2.0 mg/kg/day (n = 6), 4.0 mg/kg/day (n = 6), or 6.0 mg/kg/day (n = 6) tirilazad mesylate in divided doses every 6 hours for 5 days, for a total of 21 doses. Drug was infused over 10 or 30 minutes. All tirilazad mesylate treatment groups and the citrate vehicle group had significantly more frequent and more intense pain at the injection site than did the saline group, but the pain intensity did not require interruption of dosing. Three episodes of clinical thrombophlebitis were observed. No statistically significant effects of tirilazad mesylate on blood pressure, heart rate, electrocardiograms, or renal function were apparent. Moderate and transient increases in serum alanine transaminase were observed in several subjects. In the 6.0 mg/kg/day group, 50% of the subjects exhibited increased alanine transaminase. Tirilazad mesylate did not significantly affect measures of glucocorticoid activity (blood glucose, adrenocorticotropic hormone, cortisol, eosinophil, or lymphocyte levels). Tirilazad mesylate pharmacokinetics were linear over the dosage range studied. Steady state appeared to be achieved by the fifth day of dosing. After the last dose, a mean terminal half-life of 35 hours was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

A pharmacokinetic evaluation of the combined administration of triazolam and fluoxetine.

The influence of fluoxetine on triazolam pharmacokinetics was studied because of changes in diazepam pharmacokinetics reportedly produced by fluoxetine. Twenty-four healthy volunteers received a single 0.25-mg triazolam tablet alone, and another 0.25-mg tablet after 8 days of fluoxetine therapy 60 mg/day. All subjects received these treatments in the same sequence. Several blood samples were drawn from the subjects after the triazolam doses and were assayed by high-performance liquid chromatography (HPLC). Blood samples were drawn immediately before the last three fluoxetine doses to determine the concentration of fluoxetine and its metabolite norfluoxetine, also by HPLC. The pharmacokinetics of triazolam did not change significantly when the tablets were administered after multiple doses of fluoxetine. These results indicate that no pharmacokinetic interaction exists between triazolam and fluoxetine or norfluoxetine. However, each patient's clinical response to therapy should be monitored when triazolam tablets and fluoxetine capsules are administered concomitantly.

Pharmacokinetic pharmacodynamic evaluation of the combined administration of alprazolam and fluoxetine.

The pharmacokinetic and pharmacodynamic effects of concomitant administration of alprazolam and fluoxetine were studied in this double-blind parallel study in 80 healthy, male volunteers. Subjects were randomly assigned to one of four treatment groups. Drug treatments consisted of 4-day regimens of 1 mg alprazolam four times daily, 60 mg fluoxetine every morning, 1 mg alprazolam four times daily and 60 mg fluoxetine every morning, and placebo four times daily. Psychomotor performance, mood status, and degree of sedation were evaluated at designated times. Combined administration of alprazolam and fluoxetine resulted in an approximate 30% increase in plasma alprazolam concentrations relative to plasma concentrations following the administration of alprazolam alone. There were no significant differences in fluoxetine or norfluoxetine plasma concentrations between the alprazolam/fluoxetine and fluoxetine treatments. Psychomotor decrements increased when fluoxetine was administered with alprazolam relative to alprazolam administration alone. Psychomotor performance of the fluoxetine treatment group was not significantly different from that of the placebo group. No significant changes were observed in mood status, and sedation was minimal in all treatment groups. As when any two psychoactive drugs are administered together, increased patient monitoring and patient education is recommended when alprazolam and fluoxetine are prescribed concurrently.