Role of human microsomal and human complementary DNA-expressed cytochromes P4501A2 and P4503A4 in the bioactivation of aflatoxin B1.

The metabolism of the carcinogenic mycotoxin aflatoxin B1 (AFB1) was examined in microsomes derived from human lymphoblastoid cell lines expressing transfected CYP1A2 or CYP3A4 complementary DNAs and in microsomes prepared from human liver donors (n = 4). Lymphoblast microsomes expressing only CYP1A2 activated AFB1 to AFB1-8,9-epoxide (AFB1-8,9-epoxide trapped as the glutathione, conjugate) at both 16 microM and 128 microM AFB1 concentrations, whereas activation of AFB1 to the epoxide in lymphoblast microsomes expressing only CYP3A4 was detected only at high substrate concentrations (128 microM AFB1). AFB1 epoxidation was strongly inhibited in CYP1A2 but not CYP3A4 lymphoblast microsomes pretreated with furafylline, a specific mechanism-based CYP1A2 inhibitor, whereas troleandomycin (TAO), a specific CYP3A inhibitor, strongly inhibited AFB1 epoxidation in CYP3A4 but not CYP1A2 microsomes. Formation of the hydroxylated metabolite aflatoxin M1 (AFM1) was observed only in the CYP1A2 microsomes whereas aflatoxin Q1 (AFQ1) production was observed exclusively in the CYP3A4 microsomes. Treatment of individual human liver microsomes (HLM) with TAO resulted in an average 20% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1, whereas incubation of HLM with furafylline at 16 microM AFB1 resulted in an average 72% inhibition of AFB1-8,9-epoxide formation at 16 microM AFB1. TAO was slightly more effective than furafylline in inhibiting AFB1 epoxidation at 128 microM AFB1 (46% inhibition by TAO, 32% inhibition by furafylline) in HLM. AFB1-8,9-epoxide formation was inhibited by 89% at low substrate concentration and 85% at high substrate concentrations when HLM were inhibited with a furafylline/TAO mixture. AFM1 formation was strongly inhibited by furafylline, whereas AFQ1 formation was strongly inhibited by TAO, in all HLM regardless of substrate concentration. Analysis of R-6- and R-10-hydroxywarfarin activities (respective markers of CYP1A2 and CYP3A4 activities) in the complementary DNA-expressed microsomes demonstrated that TAO was less effective than furafylline as a selective P450 isoenzyme inhibitor (60% inhibition of CYP3A4 by TAO as compared to 99% inhibition of CYP1A2 by furafylline). The rates of AFB1 epoxidation and AFQ1 formation in HLM were increased 7- and 18-fold, respectively, at high versus low substrate concentrations. These results are consistent with the hypothesis that CYP1A2 is the high-affinity P450 enzyme principally responsible for the bioactivation of AFB1 at low substrate concentrations associated with dietary exposure. CYP3A4 appears to have a relatively low affinity for AFB1 epoxidation and is primarily involved in AFB1 detoxification through AFQ1 formation in HLM.(ABSTRACT TRUNCATED AT 400 WORDS)

Hormonal effects on tirilazad clearance in women: assessment of the role of CYP3A.

This study assessed whether the previously reported difference in tirilazad clearance between pre- and postmenopausal women is reversed by hormone replacement and whether this observation can be explained by differences in CYP3A4 activity. Ten healthy women from each group were enrolled: premenopausal (ages 18-35), postmenopausal (ages 50-70), postmenopausal receiving estrogen, and postmenopausal women receiving estrogen and progestin. Volunteers received 0.0145 mg/kg midazolam and 3.0 mg/kg tirilazad mesylate intravenously on separate days. Plasma tirilazad and midazolam were measured by HPLC/dual mass spectrophotometry (MS/MS) assays. Tirilazad clearance was significantly higher in premenopausal women (0.51 +/- 0.09 L/hr/kg) than in postmenopausal groups (0.34 +/- 0.07, 0.32 +/- 0.06, and 0.36 +/- 0.08 L/hr/kg, respectively) (p = 0.0001). Midazolam clearance (0.64 +/- 0.12 L/hr/kg) was significantly higher in premenopausal women compared to postmenopausal groups (0.47 +/- 0.11, 0.49 +/- 0.11, and 0.53 +/- 0.19 L/hr/kg, respectively) (p = 0.037). Tirilazad clearance was weakly correlated with midazolam clearance (r2 = 0.129, p = 0.02). Tirilazad clearance is faster in premenopausal women than in postmenopausal women, but the effect of menopause on clearance is not reversed by hormone replacement. Tirilazad clearance in these women is weakly related to midazolam clearance, a marker of CYP3A activity.

Problems associated with in vitro assessment of drug inhibition of CYP3A4 and other P-450 enzymes and its impact on drug discovery.

The cytochromes P-450 recognize and metabolize a broad range of structurally diverse therapeutic agents. As a consequence, many clinically relevant drug--drug interactions (DDI) are associated with inhibition and/or induction of a specific P-450 enzymes (in particular human cytochrome P-450 3A4, CYP3A4). In addition to inhibition and induction, CYP-mediated drug metabolism may be enhanced upon coincubation with certain compounds. Moreover, some of these enzyme-based interactions appear to be substrate specific. In this presentation, several issues associated with the generation of accurate DDI information will be discussed.

Pharmacology of AMG 181, a human anti-α4 ß7 antibody that specifically alters trafficking of gut-homing T cells.

BACKGROUND AND PURPOSE: AMG 181 is a human anti-α4 ß7 antibody currently in phase 1 and 2 trials in subjects with inflammatory bowel diseases. AMG 181 specifically targets the α4 ß7 integrin heterodimer, blocking its interaction with mucosal addressin cell adhesion molecule-1 (MAdCAM-1), the principal ligand that mediates α4 ß7 T cell gut-homing. EXPERIMENTAL APPROACH: We studied the in vitro pharmacology of AMG 181, and the pharmacokinetics and pharmacodynamics of AMG 181 after single or weekly i.v. or s.c. administration in cynomolgus monkeys for up to 13 weeks. KEY RESULTS: AMG 181 bound to α4 ß7 , but not α4 ß1 or αE ß7 , and potently inhibited α4 ß7 binding to MAdCAM-1 (but not vascular cell adhesion molecule-1) and thus inhibited T cell adhesion. Following single i.v. administration, AMG 181 Cmax was dose proportional from 0.01 to 80 mg·kg(-1) , while AUC increased more than dose proportionally. Following s.c. administration, dose-proportional exposure was observed with single dose ranging from 5 to 80 mg·kg(-1) and after 13 weekly doses at levels between 20 and 80 mg·kg(-1) . AMG 181 accumulated two- to threefold after 13 weekly 80 mg·kg(-1) i.v. or s.c. doses. AMG 181 had an s.c. bioavailability of 80%. The linear elimination half-life was 12 days, with a volume of distribution close to the intravascular plasma space. The mean trend for the magnitude and duration of AMG 181 exposure, immunogenicity, α4 ß7 receptor occupancy and elevation in gut-homing CD4+ central memory T cell count displayed apparent correlations. CONCLUSIONS AND IMPLICATIONS: AMG 181 has in vitro pharmacology, and pharmacokinetic/pharmacodynamic and safety characteristics in cynomolgus monkeys that are suitable for further investigation in humans.

Topological alteration of the CYP3A4 active site by the divalent cation Mg(2+).

Phenyldiazene reacted with lymphoblast-expressed CYP3A4 to give a stable phenyl-iron complex that could be induced to rearrange in situ producing approximately equal amounts of four N-phenyl-protoporphyrin IX isomers (N(B):N(A):N(C):N(D), 01:01:02:02). In the presence of 10 mM MgCl(2), the formation profile of the protoporphyrin isomers was markedly altered compared with control, favoring the N(A) isomer (N(B):N(A):N(C):N(D), 01:34:01:02). In addition, an investigation of MgCl(2) effects on CYP3A4-mediated metabolism of triazolam revealed that 10 mM MgCl(2) increased the apparent K(m) of triazolam 4-hydroxylation from 83 to 173 microM and reduced the V(max) for the reaction from 3.4 to 2.4 min(-1). Moreover, when the reaction kinetics of the oxidation of pyrene by CYP3A4 was examined in the absence of MgCl(2), it was found that the substrate-velocity curve was best approximated by a sigmoidal velocity curve (Hill coefficient 1.7 +/- 0.1). However, when the reaction was conducted in the presence of 10 mM MgCl(2), the resulting pyrene kinetics was not sigmoidal but rather biphasic (Hill coefficient 0.80 +/- 0.07). Based on the current results, it appears that CYP3A4 is conformationally sensitive to its in vitro environment and parameters, such as the presence of a divalent magnesium, can have a measurable effect on active site topography and consequently catalytic activity.

Assessment of potential interactions between dopamine receptor agonists and various human cytochrome P450 enzymes using a simple in vitro inhibition screen.

The selectivity of inhibition for four dopamine receptor agonists (pramipexole, ropinirole, pergolide, and bromocriptine) on six human cytochrome P450 enzyme activities were evaluated using a simple in vitro inhibition screen. Drug-P450 interactions characterized as potent (i.e. greater than 50% inhibition of control enzyme activity) were then further examined to determine an IC50 for the interaction. Of the dopamine receptor agonists tested, three drugs, ropinirole, pergolide, and bromocriptine, were found to inhibit the activity of at least one human cytochrome P450 enzyme, while the remaining dopamine agonist, pramipexole, was devoid of any potent P450 interaction. None of the agonists tested inhibited the P450 marker activities of 2C9, 2C19, and 2E1. However, partial inhibition was observed between ropinirole and CYP1A2 and pergolide and CYP3A4. In contrast, potent interactions were observed between CYP2D6 and pergolide and ropinirole, as well as with CYP3A4 and bromocriptine. The results of this study indicate several drug P450 interactions; however, the likelihood of an in vivo interaction with these drugs remains to be established.

Triazolam substrate inhibition: evidence of competition for heme-bound reactive oxygen within the CYP3A4 active site.

In human liver microsomes, triazolam is principally metabolized by CYP3A4 to form two metabolites, 1'-hydroxytriazolam (1'OHTz) and 4-hydroxytriazolam (4OHTz). The velocity of 1'OHTz formation was found to decrease at higher triazolam concentrations (>200 microM), indicative of "substrate inhibition". Coincubation of [(14)C]triazolam with authentic metabolite standards of either 1'OHTz or 4OHTz up to 30 microM did not significantly inhibit the rate of [(14)C]1'OHTz formation. The effects of secondary compounds on triazolam oxidation were shown to be product-specific, producing either activation or inhibition depending on the triazolam metabolite monitored. When human liver microsomes were supplemented with exogenous human cytochrome b(5), it was observed that substrate inhibition was attenuated and the resulting increase in 1'OHTz formation, relative to control (nonsupplemented) incubations, corresponded to a decrease in the ratio of 4OHTz to 1'OHTz. In contrast, when cofactor (e.g., 100 microM NADPH) was rate limiting, the metabolite ratio (4OHTz/1'OHTz) was markedly increased over the entire substrate concentration range (0.5-1000 microM). To explain these kinetic observations, a two-site binding model is proposed in which triazolam is hypothesized to bind within the CYP3A4 active site in spatially distinct orientations, which may lead to the formation of either the 1'-hydroxytriazolam or 4-hydroxytriazolam. Differential inhibition/activation is consistent with this two-site model and substrate inhibition is hypothesized to result from competition between the two sites for reactive oxygen.

Covalent alteration of the CYP3A4 active site: evidence for multiple substrate binding domains.

The inhibition of CYP3A4-mediated oxidation of triazolam and testosterone was assessed in the presence of a selection of known CYP3A4 substrates and inhibitors. Under experimental conditions where the Michaelis-Menten model predicts substrate-independent inhibition ([S] = K(m)), results yielded substrate-dependent inhibition. Moreover, when the same experimental design was extended to a group of structurally similar flavonoids it was observed that flavanone, flavone, 3-hydroxyflavone, and 6-hydroxyflavone (10 microM) activated triazolam metabolism, but inhibited testosterone hydroxylation. In additional studies, residual CYP3A4 activity toward testosterone and triazolam hydroxylation was measured after pretreatment with the CYP3A4 mechanism based inhibitor, midazolam. After midazolam preincubation, CYP3A4 6 beta-hydroxylase activity was reduced by 47% while, in contrast, triazolam hydroxylation was reduced by 75%. These results provide physical evidence, which supports the hypothesis that the active site of CYP3A4 contains spatially distinct substrate-binding domains within the enzyme active site.

Oxidation of the novel oxazolidinone antibiotic linezolid in human liver microsomes.

In vitro studies were conducted to identify the hepatic enzyme(s) responsible for the oxidative metabolism of linezolid. In human liver microsomes, linezolid was oxidized to a single metabolite, hydroxylinezolid (M1). Formation of M1 was determined to be dependent upon microsomal protein and NADPH. Over a concentration range of 2 to 700 microM, the rate of M1 formation conformed to first-order (nonsaturable) kinetics. Application of conventional in vitro techniques were unable to identify the molecular origin of M1 based on the following experiments: a) inhibitor/substrates for various cytochrome P-450 (CYP) enzymes were unable to inhibit M1 formation; b) formation of M1 did not correlate (r(2) < 0.23) with any of the measured catalytic activities across a population of human livers (n = 14); c) M1 formation was not detectable in incubations using microsomes prepared from a baculovirus insect cell line expressing CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5, and 4A11. In addition, results obtained from an in vitro P-450 inhibition screen revealed that linezolid was devoid of any inhibitory activity toward the following CYP enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4). Additional in vitro studies excluded the possibility of flavin-containing monooxygenase and monoamine oxidase as potential enzymes responsible for metabolite formation. However, metabolite formation was found to be optimal under basic (pH 9.0) conditions, which suggests the potential involvement of either an uncharacterized P-450 enzyme or an alternative microsomal mediated oxidative pathway.

Human biotransformation of bropirimine. Characterization of the major bropirimine oxidative metabolites formed in vitro.

Bropirimine (2-amino-5-bromo-6-phenyl-4-pyrimidinone) is a member of a class of antineoplastic agents known as aryl pyrimidinones. In human liver microsomal incubations, bropirimine oxidative metabolism is characterized by the formation of three metabolites. Mass spectrometric analysis of the incubation mixture revealed three bropirimine oxidative metabolites, identified as the bropirimine dihydrodiol, p-hydroxybropirimine, and m-hydroxybropirimine. In vitro studies using human liver microsomes and recombinant cytochrome P450 isoforms were performed to identify the P450 enzyme(s) responsible for bropirimine oxidation. Coincubation with the selective CYP1A2 inhibitor alpha-naphthoflavone abolished bropirimine metabolism in human liver microsomes. Furthermore, when screened against a panel of cDNA expressed cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), bropirimine was metabolized to both p- and m-hydroxybropirimine exclusively in incubations with cDNA-expressed CYP1A2 microsomes. Mechanistic studies using cDNA-expressed CYP1A2 microsomes fortified with microsomal epoxide hydrolase revealed that all three bropirimine oxidative metabolites appear to be the result of a common arene oxide, which serves as a substrate for microsomal epoxide hydrolase to generate the dihydrodiol or rearranges to yield p- and m-hydroxybropirimine.

Identification of cytochrome P4503A as the major subfamily responsible for the metabolism of roquinimex in man.

1. Roquinimex, a novel immunomodulator, is metabolized in liver microsomes from mouse and rat via cytochrome P450s to four hydroxylated and two demethylated metabolites (R1-6). The study investigated which cytochrome P450 enzyme(s) is responsible for the metabolism of roquinimex in man. 2. Enzyme kinetic analysis demonstrated an apparent Km = 1.28-7.00 mM and Vmax = 50-159 pmol x mg(-1) microsomal protein x min(-1) for the primary metabolites in human liver microsomes. The sum of Cl(int) for the primary pathways was 0.167 microl x mg(-1) microsomal protein x min(-1). 3. A correlation between the formation rate of R1-6 and 6beta-hydroxylation of testosterone was obtained within a panel of liver microsomes from 11 individuals (r2 = 0.72-0.97). Furthermore, significant inhibition (>90%) of roquinimex primary metabolism was demonstrated by ketoconazole and troleandomycin, specific inhibitors of CYP3A4 as well as with anti-CYP3A4 antibodies. Moreover, a similar metabolite pattern was produced from roquinimex by incubation with cDNA-expressed CYP3A4 as by human liver microsomes. 4. In conclusion, these data indicate a major role for CYP3A4 in the formation of roquinimex primary metabolites in human liver microsomes.

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.

Bioactivation of the anticancer agent CPT-11 to SN-38 by human hepatic microsomal carboxylesterases and the in vitro assessment of potential drug interactions.

Human hepatic microsomes were used to investigate the carboxylesterase-mediated bioactivation of CPT-11 to the active metabolite, SN-38. SN-38 formation velocity was determined by HPLC over a concentration range of 0.25-200 microM CPT-11. Biphasic Eadie Hofstee plots were observed in seven donors, suggesting that two isoforms catalyzed the reaction. Analysis by nonlinear least squares regression gave KM estimates of 129-164 microM with a Vmax of 5.3-17 pmol/mg/min for the low affinity isoform. The high affinity isoform had KM estimates of 1.4-3.9 microM with Vmax of 1.2-2.6 pmol/mg/min. The low KM carboxylesterase may be the main contributor to SN-38 formation at clinically relevant hepatic concentrations of CPT-11. Using standard incubation conditions, the effects of potential inhibitors of carboxylesterase-mediated CPT-11 hydrolysis were evaluated at concentrations >/= 21 microM. Positive controls bis-nitrophenylphosphate (BNPP) and physostigmine decreased CPT-11 hydrolysis to 1.3-3.3% and 23% of control values, respectively. Caffeine, acetylsalicylic acid, coumarin, cisplatin, ethanol, dexamethasone, 5-fluorouracil, loperamide, and prochlorperazine had no statistically significant effect on CPT-11 hydrolysis. Small decreases were observed with metoclopramide (91% of control), acetaminophen (93% of control), probenecid (87% of control), and fluoride (91% of control). Of the compounds tested above, based on these in vitro data, only the potent inhibitors of carboxylesterase (BNPP, physostigmine) have the potential to inhibit CPT-11 bioactivation if administered concurrently. The carboxylesterase-mediated hydrolysis of alpha-naphthyl acetate (alpha-NA) was used to determine whether CPT-11 was an inhibitor of hydrolysis of high turnover substrates of carboxylesterases. Inhibition of alpha-NA hydrolysis by CPT-11 was determined relative to positive controls BNPP and NaF. Incubation with microsomes pretreated with CPT-11 (80-440 microM) decreased alpha-naphthol formation to approximately 80% of control at alpha-NA concentrations of 50-800 microM. The inhibitors BNPP (360 microM) and NaF (500 microM) inhibited alpha-naphthol formation to 9-10% of control and to 14-20% of control, respectively. Therefore, CPT-11-sensitive carboxylesterase isoforms may account for only 20% of total alpha-NA hydrolases. Thus, CPT-11 is unlikely to significantly inhibit high turnover, nonselective substrates of carboxylesterases.

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.

Warfarin-fluconazole. I. Inhibition of the human cytochrome P450-dependent metabolism of warfarin by fluconazole: in vitro studies.

The antifungal agent fluconazole was found to be a potent inhibitor of cytochrome P450 (P450) 2C9 (Ki = 7-8 microM), the principal enzyme responsible for the clearance (85%) of the more potent anticoagulant (S)-warfarin to the inactive (S)-7- and (S)-6-hydroxywarfarin metabolites in vivo. Fluconazole was also found to be a potent inhibitor of the P4503A4-catalyzed formation of (R)-10-hydroxywarfarin (Ki = 15-18 microM) as well as the low KM P450 enzymes responsible for the formation of (R)-6-, (R)-7-, and (R)-8-hydroxywarfarin (Ki = 2-6 microM). By contrast, experiments with the P4501A2 inhibitor furafylline and cDNA-expressed P4501A2 indicate that fluconazole is a weak inhibitor of this enzyme (Ki > 800 microM), as measured by the inability of fluconazole to significantly suppress the P4501A2-dependent 6-hydroxylation of (R)-warfarin. The prediction generated from these studies, that fluconazole is a potent in vivo inhibitor of warfarin metabolism, , is tested in complementary studies reported in the accompanying article, "Warfarin-Fluconazole II".

Cytochrome P-450-mediated metabolism of the individual enantiomers of the antidepressant agent reboxetine in human liver microsomes.

In vitro studies were conducted to identify the hepatic cytochrome P-450 (CYP) enzymes responsible for the oxidative metabolism of the individual enantiomers of reboxetine. In human liver microsomes, each reboxetine enantiomer was metabolized to one primary metabolite, O-desethylreboxetine, and three minor metabolites, two arising via oxidation of the ethoxy aromatic ring and a third yet unidentified metabolite. Over a concentration range of 2 to 200 microM, the rate O-desethylreboxetine formation for either enantiomer conformed to monophasic Michaelis-Menten kinetics. Evidence for a principal role of CYP3A in the formation of O-desethylreboxetine for (S, S)-reboxetine and (R,R)-reboxetine was based on the results from the following studies: 1) inhibition of CYP3A activity by ketoconazole markedly decreased the formation of O-desethylreboxetine, whereas inhibitors selective for other CYP enzymes did not inhibit reboxetine metabolism, 2) formation of O-desethylreboxetine correlated (r(2) = 0.99; p <.001) with CYP3A-selective testosterone 6-beta-hydroxylase activity across a population of human livers (n = 14). Consistent with inhibition and correlation data, O-desethylreboxetine formation was only detectable in incubations using microsomes prepared from a Baculovirus-insect cell line expressing CYP3A4. Furthermore, the apparent K(M) for the O-desethylation of reboxetine in cDNA CYP3A4 microsomes was similar to the affinity constants determined in human liver microsomes. In addition, (S,S)-reboxetine and (R,R)-reboxetine were found to be competitive inhibitors of CYP2D6 and CYP3A4 (K(i) = 2.5 and 11 microM, respectively). Based on the results of the study, it is concluded that the metabolism of both reboxetine enantiomers in humans is principally mediated via CYP3A.

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.

Biotransformation of tirilazad in human: 4. effect of finasteride on tirilazad clearance and reduced metabolite formation.

The effect of oral finasteride, an inhibitor of 5alpha-reductase, on the clearance of tirilazad, a membrane lipid peroxidation inhibitor, was assessed in eight healthy men who received: 1) 10 mg/kg tirilazad mesylate solution orally on the 7th day of a 10-day regimen of 5 mg finasteride once daily, 2) 10 mg/kg tirilazad mesylate orally, 3) 2 mg/kg tirilazad mesylate i.v. on the 7th day of a 10-day regimen of 5 mg finasteride once daily and 4) 2 mg/kg tirilazad mesylate i.v., in a four-way cross-over design. Plasma concentrations of tirilazad and its active reduced metabolites (U-89678 and U-87999) were measured by liquid chromatography with tandem mass spectrometry (LC-MS-MS). Finasteride increased mean tirilazad areas under the curve by 21 and 29% for i.v. and p.o. tirilazad, respectively. Mean U-89678 areas under the curve were decreased 92 and 75% by finasteride administration with i.v. and p.o. tirilazad, respectively, and decreases of 94 and 85% in mean U-87999 area under the curve values were observed. These differences were statistically significant. These results indicate that finasteride inhibits the metabolism of tirilazad to U-89678. However, this inhibition has only a moderate effect on the overall clearance of tirilazad. These results thus confirm earlier in vitro work that showed that tirilazad is predominantly metabolized by CYP3A4. Although the major circulating metabolites of tirilazad are formed via reduction, this represents a minor route of tirilazad elimination in man.