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W F Trager

Publications and source records attributed to W F Trager.

At least 37 records · Page 2Linked to original sources
PubMed1997

Mechanism-based inactivation of human liver cytochrome P450 2A6 by 8-methoxypsoralen.

The P450 2A6 catalyzed 7-hydroxylation of coumarin proceeded with a mean Km of 0.40 (+/-0.13) microM and Vmax of 6.34 nmol/nmol P450/min (36-fold variation) in microsomal preparations from a panel of 12 human livers. Substrate depletion was avoided during the kinetic determinations. 8-Methoxypsoralen (8-MOP) is a potent mechanism-based inactivator of human liver P450 2A6 and reconstituted purified recombinant P450 2A6 based on the following evidence: 1) 8-MOP causes time, concentration, and NADPH-dependent loss of P450 2A6 activity that is not reversed by potassium ferricyanide or extensive dialysis, 2) loss of P450 2A6 activity is associated with a loss of spectrally observable P450, 3) addition of nucleophiles or reactive oxygen scavengers to the incubations does not prevent inactivation of P450 2A6, and 4) 8-MOP-dependent P450 2A6 inactivation is inhibited (concentration dependent) by the addition of a competitive inhibitor (pilocarpine). Inactivation is selective for P450 2A6 at low concentrations of 8-MOP (2.5 microM) after short incubation time periods (3 min) and was characterized by a KI of 0.8 and 1.9 microM in a reconstituted and microsomal system, respectively, and a kinact of 1 min-1 and 2 min-1 in a reconstituted and microsomal system, respectively. A substrate depletion partition ratio of 21 was calculated for the inactivation of recombinant P450 2A6. Potency and selectivity suggest that 8-MOP could be a useful tool in vitro for evaluating P450 2A6 activity in various enzyme preparations.

Adolescent↗
PubMed1996

Allelic variants of human cytochrome P450 2C9: baculovirus-mediated expression, purification, structural characterization, substrate stereoselectivity, and prochiral selectivity of the wild-type and I359L mutant forms.

The purpose of the present studies was to define the role of the I359L allelic variant of CYP2C9 in the metabolism of the low therapeutic index anticoagulant warfarin, by performing in vitro kinetic studies with the two enantiomers of the drug. To obtain sufficient quantities of these variants to perform kinetic studies at physiologically relevant substrate concentrations, methodology was established for the high-level expression, purification, and structural characterization of wild-type CYP2C9 and CYP2C9V1 using the baculovirus system. Both forms were expressed at levels up to 250 nmol/liter and purified in 50-55% yield to specific contents of 13-14 nmol holoenzyme/mg protein. The purified preparations were characterized by Edman degradation and electrospray-mass spectrometry. Both forms of the enzyme metabolized the pharmacologically more potent (S)-enantiomer of warfarin with the same regioselectivity; however, CYP2C9V1 exhibited a fivefold lower Vmax and a fivefold higher Km compared to the wild-type enzyme for this substrate. Neither form of the enzyme formed significant quantities of the (R)-warfarin phenols. Additional studies performed with prochiral arylalkyl sulfides provided confirmation of the low turnover rates catalyzed by CYP2C9V1 and demonstrated further that sulfoxide product stereochemistry did not differ significantly between the two variants. Therefore, decreased catalytic efficiency rather than a gross alteration in substrate orientation appears to be the consequence of this putative active-site mutation. The greatly decreased catalytic efficiency of the I359L variant suggests that leucine homozygotes would eliminate (S)-warfarin, and probably many other CYP2C9 substrates, at much slower rates in vivo than individuals expressing the wild-type enzyme.

Alleles↗
PubMed1996

Formation of (R)-8-hydroxywarfarin in human liver microsomes. A new metabolic marker for the (S)-mephenytoin hydroxylase, P4502C19.

Kinetic studies demonstrate that two forms of human liver cytochrome P450 are responsible for the formation of (R)-8-hydroxywarfarin: a low-affinity enzyme (KM approximately 1.5 mM), previously identified as P4501A2; and a high-affinity enzyme (KM = 330 microM), now identified as P4502C19 on the basis of the following evidence. In crossover inhibition studies with P4501A2-depleted human liver microsomes between (R)-warfarin and (S)-mephenytoin, reciprocal competitive inhibition was observed. Apparent KM values for (S)-mephenytoin-4'-hydroxylation (52-67 microM) were similar to the determined Ki values (58-62 microM) for (S)-mephenytoin inhibition of (R)-8-hydroxywarfarin formation. Similarly, the apparent KM for (R)-warfarin 8-hydroxylation in furafylline-pretreated microsomes (KM = 289-395 microM) was comparable with the Ki values (280-360 microM) for (R)-warfarin inhibition of (S)-4'-hydroxymephenytoin formation. Inhibition studies with tranylcypromine, a known inhibitor of (S)-mephenytoin hydroxylase activity, and either substrate in three different microsomal preparations yielded nearly identical inhibitory constants: Ki = 8.7 +/- 1.6 microM for inhibition of (S)-4'-hydroxymephenytoin formation and 8.8 +/- 2.5 microM for inhibition of (R)-8-hydroxywarfarin formation. In addition, fluconazole, a potent inhibitor of (R)-warfarin 8-hydroxylation, Ki = 2 microM, was found to inhibit (S)-mephenytoin hydroxylation with an identical Ki (2 microM). Finally, a strong correlation between (S)-mephenytoin 4-hydroxylation and (R)-warfarin 8-hydroxylation activities in furafylline-pretreated microsomes was demonstrated in 14 human liver microsomal preparations (r2 = 0.97).

Aryl Hydrocarbon Hydroxylases↗
PubMed1996

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".

Anticoagulants↗
PubMed1996

Warfarin-fluconazole. II. A metabolically based drug interaction: in vivo studies.

Consistent with expectations based on human in vitro microsomal experiments, administration of fluconazole (400 mg/day) for 6 days to six human volunteers significantly reduced the cytochrome P450 (P450)-dependent metabolic clearance of the warfarin enantiomers. In particular, P4502C9 catalyzed 6- and 7-hydroxylation of (S)-warfarin, the pathway primarily responsible for termination of warfarin's anticoagulant effect, was inhibited by approximately 70%. The change in (S)-warfarin pharmacokinetics caused by fluconazole dramatically increased the magnitude and duration of warfarin's hypoprothrombinemic effect. These observations indicate that co-administration of fluconazole and warfarin will result in a clinically significant metabolically based interaction The major P450-dependent, in vivo pathways of (R)-warfarin clearance were also strongly inhibited by fluconazole. 10-Hydroxylation, a metabolic pathway catalyzed exclusively by P4503A4, was inhibited by 45% whereas 6-, 7-, and 8-hydroxylations were inhibited by 61, 73, and 88%, respectively. The potent inhibition of the phenolic metabolites suggests that enzymes other than P4501A2 (weakly inhibited by fluconazole in vitro) are primarily responsible for the formation of these metabolites in vivo as predicted from in vitro kinetic studies. These data suggest that fluconazole can be expected to interact with any drug whose clearance is dominated by P450s 2C9, 3A4, and other as yet undefined isoforms. Overall, the results strongly support the hypothesis that metabolically based in vivo drug interactions may be predicted from human in vitro microsomal data.

Adult↗
PubMed1996

Warfarin-fluconazole. III. A rational approach to management of a metabolically based drug interaction.

The results of studies of the effect of fluconazole on cytochrome P450 (P450) 2C9 activity in vivo and in vitro are used to develop an approach to the safe management of the warfarin-fluconazole drug interaction. This approach begins with a determination of an in vitro Ki value (22 microM), which may be used to relate fluconazole plasma concentrations to inhibitory effect on P4502C9 activity and (S)-warfarin half-life. A means for adding fluconazole to a therapeutic regimen of warfarin is proposed that involves a stepped reduction of the warfarin dose over 5 days to a final target daily dose that is determined by the fluconazole dose level. The effect of interindividual pharmacokinetic variability on outcome quality is explored in simulation studies that indicate that a stepped-dose reduction schedule will be superior to a one-time dose reduction. The in vivo K, was found to predict accurately the magnitude of the fluconazole interaction study with another P4502C9 substrate tolbutamide.

Anticoagulants↗
PubMed1996

Substrate probe for the mechanism of aromatic hydroxylation catalyzed by cytochrome P450.

The effect of branch pathways on the observed intramolecular isotope effect and deuterium retention associated with 6- and 7-hydroxylation of selectively monodeuterated (R)- and (S)-warfarin with cytochrome P450 (CYP) 2C9 and CYP1A2 were studied. cDNA-expressed CYP2C9 was incubated with enantiomerically pure (S)-7d1- and (S)-6d1-warfarin, and expressed CYP1A2 was incubated with enantiomerically pure (R)-7d1- and (R)-6d1-warfarin. A high degree of deuterium retention was observed in all metabolites, independent of the stereochemistry of the substrate or CYP isoform. No deuterium kinetic isotope effect was observed for the formation of 6-hydroxy- or 7-hydroxywarfarin in the case of the (S)-6d1-warfarin metabolism by CYP2C9, or for the formation of 6-hydroxy-, 7-hydroxy-, and 8-hydroxywarfarin in the case of the (R)-6d1-warfarin metabolism by CYP1A2. Deuterium isotope effects of 1.17 and 1.23 accompanied formation of 7-hydroxywarfarin from (S)-7d1-warfarin by CYP2C9 and from (R)-7d1-warfarin by CYP1A2, respectively. These observations are consistent with the addition-rearrangement pathway for aromatic hydroxylation, in which a triplet-like active oxygen species initially adds to the pi system, resulting in a tetrahedral intermediate. The intermediate subsequently rearranges to generate the phenol, the final product of the reaction.

Aryl Hydrocarbon Hydroxylases↗
PubMed1995

Isotope effect studies on the cytochrome P450 enzymes.

Isotope effect experiments provide a powerful tool for study of the fundamental aspects of the enzymology of the cytochrome P450 enzymes. Competition between alternate pathways not only allows P450 isotope effects to be observed, but also provides mechanistic information on both oxygen activation and substrate oxidation. Indeed, the kind of knowledge that isotope effect studies can provide is not readily obtainable by other methodologies.

Animals↗
PubMed1995

Inhibition of (S)-warfarin metabolism by sulfinpyrazone and its metabolites.

Sulfinpyrazone markedly potentiates the anticoagulant effect of warfarin. The increased clotting time is accompanied by a marked decrease in the clearance of (S)-warfarin by virtue of a decrease in the P4502C9-catalyzed formation clearance to its major and inactive metabolite (S)-7-hydroxywarfarin. These data suggested that the mechanism of the drug interaction might be mediated through the inhibition of the catalytic activity of P4502C9 by sulfinpyrazone. However, initial human liver microsomal studies indicated that the in vitro Ki, for inhibition of (S)-7-hydroxywarfarin formation by sulfinpyrazone is at least 25-fold higher than the therapeutic concentration of sulfinpyrazone in vivo. This result implied that other inhibitors probably contribute to the interaction. Kinetic studies conducted on sulfinpyrazone and two major metabolites, sulfinpyrazone sulfide and sulfinpyrazone sulfone, in microsomes prepared from three human livers give mean Ki's of 230 microM, 17 microM, and 73 microM respectively. Because sulfinpyrazone and its sulfide metabolite attain comparable plasma concentrations during the course of therapy, our inhibition results suggest that the sulfide metabolite is likely to be the primary species responsible for the inhibition of P4502C9-catalyzed formation of (S)-7-hydroxywarfarin and the decrease in (S)-warfarin clearance in vivo.

Drug Synergism↗
PubMed1995

Stereoselectivity and isotope effects associated with cytochrome P450-catalyzed oxidation of (S)-nicotine. The possibility of initial hydrogen atom abstraction in the formation of the delta 1', 5-nicotinium ion.

The stereochemical course of cytochromes P450 [P4501A1, P4502B1, P4502B4, and P450101 (P450cam)] catalyzed alpha-carbon oxidations of the cis-(Z)- and trans-(E)-5'-d1 diastereomers of (S)-nicotine has been examined. All enzyme preparations led to the stereoselective abstraction of the 5'-hydrogen atom trans to the pyridine ring with P450101 and human liver microsomal preparations displaying the highest (90%) and P4502B1 the lowest (67%) degree of stereoselectivity. No isotope effect was detected for any of the enzyme-catalyzed reactions, although the existence of an intrinsic isotope effect was inferred by the observation of an intramolecular isotope effect of 2-2.6 observed for the N-demethylation of (S)-N'-dideuteromethylnornicotine. Evidence for P450101-catalyzed N'-oxidation was sought but could not be found at higher than trace levels. These results, together with those obtained by computational methods, are interpreted in terms of an alpha-carbon oxidative pathway involving hydrogen atom abstraction rather than single electron transfer as the initiating event in the P450-catalyzed oxidation of (S)-nicotine to its delta 1',5'-iminium ion metabolite.

Animals↗
PubMed1995

Catalytic role of cytochrome P4503A4 in multiple pathways of alfentanil metabolism.

The synthetic opioid alfentanil (ALF) undergoes extensive metabolism via two major pathways: piperidine nitrogen dealkylation to noralfentanil (NA) and amide nitrogen dealkylation to N-phenylpropionamide (AMX). It is unknown whether AMX results from amide N-dealkylation of ALF directly, or indirectly from NA, the major metabolite of ALF. The major objectives of this investigation were to determine the metabolic origin of AMX and to identify the cytochrome P450 isoforms in human liver microsomes catalyzing ALF metabolism. Metabolites were quantitated by GC/MS. Significant amide N-dealkylation of ALF but not of NA by human liver microsomes was observed, indicating that AMX is derived directly from ALF and that there are two primary routes of ALF metabolism. Three strategies were used to identify the P450 isoform(s) catalyzing each of the two metabolic pathways: effect of isoform-selective inhibitors on metabolite formation catalyzed by human liver microsomes, correlation of metabolite formation rate with microsomal P450 isoform protein content and catalytic activity in a population of human livers, and metabolism by cDNA-expressed P450 isoforms. The mechanism-based P4503A4 inhibitor, troleandomycin, significantly inhibited formation of both NA and AMX. Other P4503A4 inhibitors, including midazolam, erythromycin, and ketoconazole, also diminished ALF metabolism to both metabolites. Formation rates of both NA and AMX were significantly correlated with microsomal P4503A4 protein content and catalytic activity. Of six expressed human P450 isoforms (P450s 1A2, 2A6, 2B6, 2D6, 2E1, and 3A4), only P4503A4 exhibited significant catalytic activity toward ALF dealkylation to NA and AMX. These results indicate the predominant role of P4503A4 in both major pathways of ALF metabolism.

Alfentanil↗
PubMed1994

Application of negative-ion chemical ionization isotope dilution gas chromatography--mass spectrometry to single-dose bioavailability studies of mefloquine.

An electron-capture negative-ion chemical ionization gas chromatographic-mass spectrometric assay for mefloquine, an antimalarial drug used in the treatment of drug-resistant Plasmodium falciparum malaria, is described. The method, developed in support of bioavailability studies involving the co-administration of different tableted formulations of the drug and an aqueous solution of its 13C3-labeled analog, enables quantification of both dosage forms. Quantitative analysis of extracted plasma samples was performed on the O-tert.-butyldimethylsilyl (t-BDMS) derivative of the drug by selected-ion monitoring, using a VG Trio 2000 quadrupole mass spectrometer and monitoring the [M-t-BDMSOH]-. ions of the analytes. The method, incorporating [2H6]mefloquine as an internal standard, demonstrated good accuracy and precision over the 1-200 ng ml-1 range, with correlation coefficients greater than 0.990 for all standard curves and a detection level of 50 fg on-column. Replicate analysis of plasma samples over a 90-day period exhibited a mean intra-day and inter-day variation of less than 4.5% and 5.5%, respectively. The high stability and sensitivity of the assay, combined with the inherent selectivity of mass spectrometric detection, make the method well-suited for such studies.

Biological Availability↗
PubMed1994

Disposition of drugs in cystic fibrosis. VI. In vivo activity of cytochrome P450 isoforms involved in the metabolism of (R)-warfarin (including P450 3A4) is not enhanced in cystic fibrosis.

OBJECTIVE: To determine whether the activity of cytochrome P450 isoforms involved in the metabolism of (R)-warfarin is enhanced in cystic fibrosis. DESIGN: Six adult subjects with cystic fibrosis and six healthy control subjects, matched by age and sex, were administered (R)-warfarin as a single intravenous bolus dose (0.375 mg/kg), and urine and plasma samples were collected for 192 hours. The concentration of (R)-warfarin in plasma and the concentration of (R)-warfarin and its metabolites in urine were determined by HPLC and GC/MS, respectively. Plasma protein binding of (R)-warfarin was measured by ultrafiltration. RESULTS: The unbound plasma clearance of (R)-warfarin was not significantly (p > 0.05) different between the cystic fibrosis and the control groups (cystic fibrosis, 997 +/- 483 ml/hr/kg; control, 788 +/- 219 ml/hr/kg). The unbound metabolic clearances of (R)-warfarin to its oxidative metabolites--6-hydroxywarfarin, 7-hydroxywarfarin, 8-hydroxywarfarin, and 10-hydroxywarfarin (mediated by P450 3A4)--were also similar (p > 0.05) in the two groups (6-hydroxywarfarin: cystic fibrosis: 124.2 +/- 72.8 ml/hr/kg, control: 99.4 +/- 37.3 ml/hr/kg; 7-hydroxywarfarin: cystic fibrosis: 43.8 +/- 32.2 ml/hr/kg, control: 34.5 +/- 10.6 ml/hr/kg; 8-hydroxywarfarin: cystic fibrosis: 80.4 +/- 85.4 ml/hr/kg, control: 69.5 +/- 39.5 ml/hr/kg; 10-hydroxywarfarin: cystic fibrosis: 4.38 +/- 2.72 ml/hr/kg, control: 16.28 +/- 13.71 ml/hr/kg). CONCLUSION: The in vivo activity of cytochrome P450 isoforms involved in the metabolism of (R)-warfarin, including P450 3A4, is not enhanced in cystic fibrosis.

Adult↗
PubMed1994

Use of midazolam as a human cytochrome P450 3A probe: I. In vitro-in vivo correlations in liver transplant patients.

The clearance of midazolam (MDZ) in humans is principally due to metabolic biotransformation catalyzed by CYP3A isoforms. A study was conducted in patients who had undergone liver transplants that provides evidence that MDZ can be used as an in vivo probe of interindividual hepatic CYP3A variability. The clearance of MDZ and cyclosporine after i.v. administration were determined in 10 patients approximately 10 days after transplant surgery. Liver biopsy specimens were obtained within 24 hr of the pharmacokinetic study and CYP3A content and MDZ 1'-hydroxylation activity were measured in 13,000 x g tissue supernatants (S-13). The in vitro rate of 1'-hydroxy-MDZ formation was found to correlate significantly with the total CYP3A content in hepatic S-13 fractions (r = .84, P < .01). The total MDZ clearance measured in vivo was highly correlated with the hepatic CYP3A content measured in vitro (r = .93, P < .001) and with in vivo cyclosporine clearance (r = .81, P < .001). For five of the patients, the intrinsic clearance of midazolam to 1'-hydroxy-MDZ (Vmax/Km) in vitro measured in S-13 preparations was scaled for total liver mass and applied to the well stirred model of hepatic clearance to yield a prediction of MDZ clearance in vivo. The mean MDZ clearance predicted from in vitro 1'-hydroxylation data was identical to the mean clearance observed in vivo (0.60 +/- 0.24 versus 0.59 +/- 0.25 liter/min). Together, the results suggest that variability in hepatic CYP3A expression in liver transplant recipients, and possibly in other populations, can be determined by the measurement of MDZ metabolic clearance.

Cyclosporine↗
PubMed1994

Use of midazolam as a human cytochrome P450 3A probe: II. Characterization of inter- and intraindividual hepatic CYP3A variability after liver transplantation.

Immunosuppression therapy with cyclosporine is often hampered by significant interindividual variability in the metabolic clearance of the drug. It has been suggested that much of the variability in cyclosporine clearance is due to differences in the cytochrome P450 3A4 (CYP3A4) content in the liver and intestinal mucosa. A study was conducted in liver transplant recipients to characterize hepatic CYP3A variability during the first 10 days after surgery. The formation of 1'-hydroxymidazolam (1'-OH MDZ) was followed in the plasma after i.v. midazolam (MDZ) administration to 21 multiple-organ donors and to recipients of 10 of the 21 donor livers. Liver biopsy tissue was obtained from donors and recipients after the in vivo pharmacokinetic test. For liver donors, the plasma 1'-OH MDZ/MDZ concentration ratio 30 min after the i.v. MDZ dose was well correlated with the hepatic CYP3A4 content (r = .87, P < .001). Much of the variability in the two parameters was attributed to the administration of enzyme-inducing drugs before organ procurement. The mean hepatic CYP3A4 content and plasma 1'-OH MDZ/MDZ concentration ratio in six inducer-treated donors was 4.7-fold and 2.3-fold higher than the respective mean value for all other donors. The hepatic CYP3A4 content and plasma 1'-OH MDZ/MDZ ratio for liver recipients, studied on postoperative day 10, was negatively correlated with the respective parameter measured in donors on day 0 (r = -0.60 for CYP3A4 and r = -0.79 for 1'-OH MDZ/MDZ; P < .05 and P < .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Studies on the mechanism of aromatase and other cytochrome P450 mediated deformylation reactions.

Aromatase is a microsomal cytochrome P450 that converts androgens to estrogens by three sequential oxidations. The isolation of the 19-hydroxy and 19-oxo androgens suggests that the first two oxidations occur at the C19 carbon. However, the mechanism of the third oxidation, which results in C10--C19 bond cleavage, has not been determined. Two proposed mechanisms which remain viable involve either initial 1 beta-hydrogen atom abstraction or addition of the ferric peroxy anion from aromatase to the C19 aldehyde. Semiempirical molecular orbital calculations (AM1) were used to study potential reaction mechanisms initiated by initial 1 beta-hydrogen atom abstraction. Initially, the energetics of carbon--carbon bond cleavage of the keto and enol forms of C1-radicals were studied and were found to be energetically similar. A mechanism was proposed in which the 19-oxo intermediate is subject to initial nucleophilic attack by the protein. The geometry of the A-ring in the androgens is between that for the 1-radicals and estrogen, suggesting that some transition state stabilization for the homolytic cleavage reaction can occur. More recently, studies on liver microsomal cytochrome P450 mediated deformylation of xenobiotic aldehydes supports mechanisms involving an alkyl peroxy intermediate formed by addition of the ferric peroxy anion from aromatase to the C19 aldehyde. Although this intermediate could proceed through several different concerted or non-concerted pathways, one non-concerted pathway involves the heterolytic cleavage of the dioxygen bond resulting in an active oxygenating species (iron-oxene) and a diol. The diol could then undergo hydrogen atom abstraction followed by homolytic carbon--carbon bond cleavage as in the mechanisms modeled previously. When this cleavage was modeled for seven aldehydes, a good correlation with reported experimental aldehyde turnover numbers was obtained. However, when dialkoxy derivatives of the aldehydes are subject to microsomal metabolism, the rates of carbon-carbon cleavage products do not approach the rates of deformylation of the aldehyde analog.

Aldehydes↗