Flow cytometric assay of modulation of P-glycoprotein function in whole blood by the multidrug resistance inhibitor GG918.

We sought to develop an assay for measuring the inhibition of P-glycoprotein (Pgp) function in whole blood as an indicator of in vivo drug activity. Since the CD56(+) subset of peripheral blood lymphocytes (PBLs) has been shown to express functional Pgp, the changes in rhodamine 123 (R123) uptake by CD56(+) PBLs from GG918-treated and untreated whole blood were used as the basis for these studies. In an ex vivo study, heparin-treated whole blood was obtained from normal volunteers, and GG918 and R123 were added at various concentrations for time course analyses of dye loading. GG918 concentrations from 2.5 to 800 nm were tested in incubations ranging from 15 min to 3 h prior to R123 addition. R123 loading times ranged from 0 to 80 min. Flow cytometric analyses of the CD56(+) PBLs indicated that the resolution of Pgp inhibition was dependent on inhibitor concentration and time of R123 loading and independent of the R123 concentrations tested. In this ex vivo assay model, a dose-dependent response was seen for GG918 with a 2-fold increase in cellular R123 intensity being produced at a drug concentration of 80 nm. When this assay method was applied to blood samples from volunteers dosed p.o. with GG918, similar shifts in R123 fluorescence of the CD56(+) PBLs were observed with significant increases in R123 intensity occurring at serum concentrations as low as 40 nm. In contrast to assays in which target cell populations are enriched prior to testing, the addition of the substrate (R123) directly to the blood sample combined with the segregation of the target cells by specific immunofluorescence provides the investigator an indication of in situ activity of circulating drug. Thus, CD56(+) PBLs may prove useful as a surrogate target for monitoring multidrug resistance inhibitor activity in situ.

Measurement of in vivo microsomal epoxide hydrolase activity in white subjects.

An impairment or hereditary defect in microsomal epoxide hydrolase is considered a possible risk factor for drug and chemical toxicity. However, nothing is known about variability of in vivo epoxide hydrolase activity in humans. Our objectives were to develop and test a simple pharmacokinetic approach for measuring microsomal epoxide hydrolase activity in a population. After administration of carbamazepine-10,11-epoxide (100 mg), oral clearance showed a nearly linear relationship to the log (transdihydrodiol/epoxide) urine ratio in the 24- to 36-hour interval (log metabolic ratio). Intrasubject variability was assessed by administering the epoxide twice to 13 subjects (1- to 4-month interval); the log metabolic ratio did not change significantly (mean difference, 11%; paired t test, p = 0.79). In 110 healthy white adults, the log metabolic ratio ranged from 1.28 to 2.05 (mean +/- SD, 1.68 +/- 0.155). Outliers indicating enzyme-deficient phenotypes were not observed, and the frequency distribution was unimodal normal. The log metabolic ratio detected pronounced inhibition of epoxide hydrolase by valpromide (six subjects; median ratio, 0.91) and induction by phenobarbital/phenytoin (six subjects; median ratio, 2.42). We conclude that distribution of microsomal epoxide hydrolase activity in a study group can be measured pharmacokinetically by use of carbamazepine epoxide.

Inhibition of human liver microsomal epoxide hydrolase by valproate and valpromide: in vitro/in vivo correlation.

On the basis of drug interactions with carbamazepine epoxide, it has been hypothesized that valproic acid and valpromide are inhibitors of epoxide hydrolase, but the role of epoxide hydrolase in these interactions has not been clearly established. In this study, therapeutic concentrations of valproic acid (less than 1 mmol/L) and valpromide (less than 10 mumol/L) inhibited hydrolysis of carbamazepine epoxide and styrene oxide in human liver microsomes and in preparations of purified human liver microsomal epoxide hydrolase. Valpromide (KI = 5 mumol/L) was 100 times more potent than valproic acid (KI = 550 mumol/L) as an inhibitor of carbamazepine epoxide hydrolysis in microsomes. After administration of carbamazepine epoxide to volunteers, the transdihydrodiol formation clearance was decreased 20% by valproic acid (blood concentration approximately 113 mumol/L) and 67% by valpromide (blood concentration less than 10 mumol/L). For both valproic acid and valpromide, a striking similarity exists between in vitro and in vivo inhibitory potencies. Valproic acid and valpromide are the first drugs known to inhibit microsomal epoxide hydrolase, an important detoxification enzyme, at therapeutic concentrations.

Clinical and experimental studies linking oxidative metabolism to phenytoin-induced teratogenesis.

The research presented in this article concerns the proposed mechanism of phenytoin-induced teratogenicity that focuses on oxidative metabolites as sources of reactive species in clinical studies and by testing paradigms in animal models. The clinical aspect involved determining whether at-risk fetuses could be detected prenatally on the basis of low or deficient epoxide hydrolase activity. In 19 pregnancies monitored by amniocentesis, we predicted an adverse outcome in four infants on the basis of low enzyme activity. When examined neonatally, all four infants had the dysmorphic features of the "fetal hydantoin syndrome." In an animal model of phenytoin-induced teratogenesis, the level of fetal exposure to oxidative metabolites was decreased by coadministration of the cytochrome P-450-inhibiting antiepileptic drug stiripentol, which significantly reduced the incidence of phenytoin-induced congenital malformations in two of the three inbred mouse strains, thus providing support for the hypothesis that oxidative metabolites are critical in mediating phenytoin teratogenesis.

Delavirdine: clinical pharmacokinetics and drug interactions.

Delavirdine, a non-nucleoside reverse transcriptase inhibitor (NNRTI), is a potent and specific inhibitor of HIV-1 reverse transcriptase. The approved therapeutic regimen for delavirdine is 400mg 3 times daily in combination with appropriate antiretroviral agents; however, a dose of 600mg twice daily appears to provide similar systemic exposure. The steady-state pharmacokinetics of delavirdine are not appreciably affected by food. Delavirdine undergoes extensive metabolism by cytochrome P450 (CYP) with little urinary excretion of unchanged drug. Metabolic drug interactions between delavirdine and nucleoside reverse transcriptase inhibitors are unlikely as their metabolic pathways differ; delavirdine has no effect on the pharmacokinetics of zidovudine. Concomitant use of CYP inducers, such as rifampicin (rifampin), rifabutin, phenytoin, phenobarbital or carbamazepine, should be avoided since delavirdine plasma concentrations are significantly lowered. Reduction in gastric acidity (pH > 3) decreases the extent of delavirdine absorption, so administration of antacids and the buffered formulations of didanosine should be separated from that of delavirdine by at least 1 hour. Delavirdine, unlike other currently available NNRTI agents, is an inhibitor rather than an inducer of CYP isozymes. Consequently, the drug interaction profile and rationale for combining delavirdine with other antiretroviral agents is unique among the current NNRTI agents. Delavirdine inhibits the CYP3A4-mediated metabolism of HIV protease inhibitors and thereby increases systemic exposure to protease inhibitors. The ability of delavirdine to enhance the pharmacokinetic profiles of protease inhibitors may permit the use of simplified administration regimens. Combining delavirdine and indinavir removes the food restrictions during indinavir administration. Furthermore, the superior virological response observed in antiretroviral regimens containing delavirdine and protease inhibitors has been attributed to the favourable pharmacokinetic interactions and the introduction of a new drug class in NNRTI-naïve therapy-experienced patients. Pharmacokinetic drug interactions are an important consideration in selecting an HIV treatment regimen, due to the multiplicity of drugs that are coadministered and the varying direction and magnitude of interaction that can occur. Considerations for utilising delavirdine in a treatment regimen are different than for other NNRTI agents due to the unique drug interaction profile of delavirdine.

Human liver carbamazepine metabolism. Role of CYP3A4 and CYP2C8 in 10,11-epoxide formation.

A number of drugs inhibit the metabolism of carbamazepine catalyzed by cytochrome P450, sometimes resulting in carbamazepine intoxication. However, there is little information available concerning the identity of the specific isoforms of P450 responsible for the metabolism of this drug. This study addressed the role of CYP3A4 in the formation of carbamazepine-10,11-epoxide, the major metabolite of carbamazepine. Results of the study showed that: (1) purified CYP3A4 catalyzed 10,11-epoxidation; (2) cDNA-expressed CYP3A4 catalyzed 10,11-epoxidation (Vmax = 1730 pmol/min/nmol P450, Km = 442 microM); (3) the rate of 10,11-epoxidation correlated with CYP3A4 content in microsomes from sixteen human livers (r2 = 0.57, P < 0.001); (4) triacetyloleandomycin and anti-CYP3A4 IgG reduced 10,11-epoxidation to 31 +/- 6% (sixteen livers) and 43 +/- 2% (four livers) of control rates, respectively; and (5) microsomal 10,11-epoxidation but not phenol formation was activated 2- to 3-fold by alpha-naphthoflavone and progesterone and by carbamazepine itself (substrate activation). These findings indicate that CYP3A4 is the principal catalyst of 10,11-epoxide formation in human liver. Experiments utilizing a panel of P450 isoform selective inhibitors also suggested a minor involvement of CYP2C8 in liver microsomal 10,11-epoxidation. Epoxidation by CYP2C8 was confirmed in incubations of carbamazepine with cDNA-expressed CYP2C8. The role of CYP3A4 in the major pathway of carbamazepine elimination is consistent with the number of inhibitory drug interactions associated with its clinical use, interactions that result from a perturbation of CYP3A4 catalytic activity.

Clinical pharmacokinetics of carbamazepine.

The relationship between daily dose and plasma levels of carbamazepine is poor. The slope of that relationship varies with the patient population. Children require twice as much of the drug as adults do to achieve a given level. Also, dosage requirements increase in the presence of other drugs that are enzyme inducers. Recent reports suggest that the 10,11-epoxide metabolite of carbamazepine can contribute to the neurotoxicity attributed to carbamazepine. Carbamazepine is a broad-spectrum enzyme inducer. Three reports in the past 2 years show that haloperidol levels are reduced by approximately 50% when carbamazepine is introduced. Those interactions may complicate the interpretation of clinical trials of carbamazepine.

Differences in the patterns of phenytoin-induced malformations following stiripentol coadministration in three inbred mouse strains.

Differences in the patterns of congenital malformations observed in three inbred mouse strains (SWV, LM/Bc, and C57BL/6J) were compared following exposure to phenytoin monotherapy and a polytherapeutic regimen of phenytoin and stiripentol. Treatment groups containing no fewer than 10 dams were chronically exposed to the test compound(s) prior to and throughout gestation. The pattern of fetal defects observed included abnormalities of the neural, cardiac, urogenital, and skeletal systems. The coadministration of the cytochrome P-450-inhibiting antiepileptic drug stiripentol significantly reduced the incidence of fetal malformations in all three strains, primarily by reducing phenytoin's deleterious effects on congenital abnormalities related directly to fetal growth and development. In the SWV fetuses, there were significantly more soft tissue defects (neural and renal) than were evident in the LM/Bc fetuses. Overall, the C57BL/6J fetuses were the most sensitive to the induction of skeletal defects, with a preponderance of defects in the ossification of the craniofacial bones. It is hypothesized that the reduction in fetal defects was the result of limiting the biotransformation of phenytoin to highly teratogenic oxidative metabolites, which interfere with normal fetal growth.

Pharmacokinetics of old, new, and yet-to-be-discovered antiepileptic drugs.

The pharmacokinetics of antiepileptic drugs have been investigated extensively during the last two decades. It has become evident that the therapeutic management of epileptic patients would be significantly improved with the elimination of the drawbacks associated with existing antiepileptic drugs. Based on the information accumulated to date, the following set of principles is proposed: (1) The requirement of central nervous system (CNS) penetration automatically creates vulnerability to CNS toxicity. (2) The narrower the therapeutic range of a given drug, the more limiting its pharmacokinetic properties become. (3) The requirements of chronic administration and compliance place narrow limits on the optimum half-life (24 h). (4) The practice of polytherapy and the potential for pharmacodynamic and pharmacokinetic interactions make it necessary to simplify metabolic schemes (i.e., use of metabolically directed design approaches). (5) New antiepileptic drugs have shown that widely different structures are associated with anticonvulsant properties--it becomes important to minimize toxicity and optimize pharmacokinetic characteristics early in the development of future antiepileptic drugs. (6) Yet-to-be-discovered antiepileptic drugs will probably include biotechnology-based pharmaceuticals. Such biologic agents as neuropeptides present a new set of pharmacokinetic requirements because they are generally receptor targeted, they do not cross absorption barriers easily, and they are labile and subject to degradation.

Pharmacokinetics of bismuth and ranitidine following multiple doses of ranitidine bismuth citrate.

1. The pharmacokinetics of bismuth and ranitidine derived from oral doses of ranitidine bismuth citrate 800 mg given twice daily for 28 days were examined in this double-blind, placebo-controlled, parallel-group study in 27 healthy subjects. 2. Bismuth accumulation in plasma reflected its multicompartmental disposition, achieving the majority of predicted steady state within 14-28 days. Bismuth absorption from ranitidine bismuth citrate is limited (< 0.5% of the dose), and bismuth elimination is predominantly renal secretion. Peak plasma concentrations did not exceed 19 ng ml-1, remaining well below those associated with bismuth toxicity. Bismuth was measurable at low concentrations in plasma and urine for up to 5 months after the last dose. Plasma bismuth concentration-time data and urinary excretion data were best described by separate multicompartmental models, with terminal half-lives averaging 21 days and 45 days, respectively. 3. The pharmacokinetics of ranitidine derived from ranitidine bismuth citrate were similar to those of ranitidine administered alone. Ranitidine did not appreciably accumulate in plasma. 4. Ranitidine bismuth citrate was well-tolerated during 28 days of repeated dosing.

Stiripentol in atypical absence seizures in children: an open trial.

Stiripentol (STP) was added to the antiepileptic drug (AED) regimen of 10 patients with uncontrolled atypical absence seizures (more than one seizure a day). Seven boys and three girls aged 6-16 years participated in the study. Concomitant AEDs included various combinations of phenobarbital (PB), phenytoin (PHT), carbamazepine (CBZ), and valproate (VPA). Parents counted daily seizures over a 4-week baseline period before institution of STP, and in a 20-week period during STP therapy. To compensate for drug interactions, doses of other AEDs were adjusted during STP administration to keep serum levels close to levels of the baseline period. Maintenance doses of STP were 1,000-3,000 mg/day, giving serum levels of 4-22 micrograms/mL. All patients experienced a decrease in atypical absence seizures. Average decrease was 70% (range 5-95%). Side effects experienced by some patients were dose related and included anorexia, nausea, vomiting, and lethargy. In only 1 patient did an adverse effect (vomiting) require discontinuation of STP. We conclude that STP shows promise in treatment of atypical absence seizures in children, and further trials are warranted.

Carbamazepine dose requirements during stiripentol therapy: influence of cytochrome P-450 inhibition by stiripentol.

The inhibitory effect of stiripentol (STP) on disposition of carbamazepine (CBZ) and carbamazepine-10,11-epoxide (CBZE) was quantitated to establish CBZ dosage reduction guidelines for future clinical add-on efficacy trials of STP. In seven epileptic patients, STP (1,500-3,000 mg/day for 2 weeks) inhibited CBZ clearance by 50 +/- 16% (p = 0.001) and reduced the CBZE/CBZ plasma ratio by 45 +/- 14% (p = 0.0005). The inhibitory effect was gradually manifested over a period of 7-10 days after initiation of STP therapy. In contrast to inhibition of CBZE formation, STP had no effect (p greater than 0.05) on elimination clearance or half-life (t1/2) of CBZE in six healthy volunteers. STP most likely exerts inhibitory effects through inhibition of cytochrome P-450. This hypothesis was confirmed in the present study by the finding that a therapeutic concentration of STP (7 micrograms/mL) inhibited 10,11-epoxidation of CBZ in human liver microsomes by 40-50%. On the basis of results from this study, we propose that (a) CBZ dosage should be reduced in steps over a period of 7-10 days after initiation of STP, and (b) a CBZ dosage of 4.3 to 8.7 mg/kg/day will maintain therapeutic CBZ plasma levels of 5-10 micrograms/mL.

A population pharmacokinetic analysis of nelfinavir mesylate in human immunodeficiency virus-infected patients enrolled in a phase III clinical trial.

A population pharmacokinetic analysis was conducted on nelfinavir in patients infected with human immunodeficiency virus (HIV) who were enrolled in a phase III clinical trial. The data consisted of 509 plasma concentrations from 174 patients who received nelfinavir at a dose of 500 or 750 mg three times a day. The analysis was performed using nonlinear mixed-effect modeling as implemented in NONMEM (version 4.0; double precision). A one-compartment model with first-order absorption best described the data. The timing and small number of early postdose blood levels did not allow accurate estimation of volume of distribution (V/F) and the absorption rate constant (k(a)). As a result, two models were used to analyze the data: model 1, in which oral clearance (CL/F), V/F, and k(a) were estimated, and model 2, in which V/F and k(a) were fixed to known values and only CL/F was estimated. Estimates of CL/F ranged from 41. 9 to 45.1 liters/h, values in close agreement with previous studies. Neither body weight, age, sex, race, dose level, baseline viral load, metabolite-to-parent drug plasma concentration ratio, history of liver disease, nor elevated results of liver function tests appeared to be significant covariates for clearance. The only significant covariate-parameter relationship was concomitant use of fluconazole on CL/F, which was associated with a modest reduction in interindividual variability of CL/F. Patients who received concomitant therapy with fluconazole had a statistically significant reduction in nelfinavir CL/F of 26 to 30%. Since serious dose-limiting toxicity and concentration-related toxicities are not apparent for nelfinavir, this effect of fluconazole is unlikely to be of clinical significance.

Protection from phenytoin-induced congenital malformations by coadministration of the antiepileptic drug stiripentol in a mouse model.

To test the hypothesis that the cytochrome P-450-inhibiting antiepileptic drug (AED) stiripentol (STP) can reduce the incidence of phenytoin (PHT) induced congenital malformations, we chronically administered the AEDs to three inbred mouse strains. The frequency of congenital defects in PHT-treated animals was dosage dependent, ranging from 7 to 55%. When STP was coadministered orally with PHT, there was a significant reduction in fetal defects in SWV and C57BL/6J mouse strains. A replication of the experiment was conducted with addition of a seventh group of mice that received the high dosage of PHT together with STP. In the replicate, all three strains demonstrated a significant reduction in fetal defects in fetuses exposed to PHT (60 mg/kg) and STP (200 mg/kg) as compared with PHT (60 mg/kg) monotherapy. These results strongly suggest that oxidative metabolites activated by cytochrome P-450 are the primary teratogenic molecules involved in PHT-induced teratogenesis and that clinical management of pregnant epileptic patients may be improved through heightened awareness of these drug interactions.

Chemical aspects of propranolol metabolism. Synthesis and identification of 3-(4-hydroxy-1-naphthoxy)propane-1,2-diol as a metabolite of propranolol in the dog, in man and in the rat liver 9000g supernatant fraction.

4-Hydroxypropranolol glycol (2), a suspected metabolite of propranolol was synthesized from 4-allyloxy-1-naphthaldehyde (4). Osmium tetroxide oxidation of 4 afforded a glycol (5) and subsequent Baeyer-Villiger rearrangement afforded 2. Its presence as a biliary metabolite in a dog maintained on a constant infusion of pseudoracemic propranolol (made up of equal molar 2S-propranolol-3',3'-d2 and 2R-propranolol-d0) into the portal vein was confirmed based on GC-MS data of the TFA and TMS derivatives of the standard and biliary metabolites. Greater amounts of 2 arose from 2R-propranolol than from 2S-propranolol (1.5:1). Similarly, 2 was formed as a urinary metabolite in one subject maintained on oral propranolol, 80 mg every 6 hours. Compound 2 was also formed when propranolol and propranolol glycol were incubated in the presence of the rat liver 9000g supernatant fraction. In addition to 2, an isomeric ring-hydroxylated propranolol glycol, perhaps 7-hydroxypropranolol glycol, was formed when propranolol glycol was the substrate.

Pharmacokinetic interactions between nelfinavir and 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors atorvastatin and simvastatin.

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are effective agents in lowering cholesterol and triglycerides and are being used by human immunodeficiency virus-positive patients to treat the lipid elevation that may be associated with antiretroviral therapy. Many HMG-CoA reductase inhibitors and protease inhibitors are metabolized by the same cytochrome P450 enzyme 3A4 (CYP3A4). In addition, many protease inhibitors are potent inhibitors of CYP3A4. Therefore, coadministration of these two classes of drugs may cause significant drug interactions. This open-label, multiple-dose study was performed to determine the interactions between nelfinavir, a protease inhibitor, and two HMG-CoA reductase inhibitors, atorvastatin and simvastatin, in healthy volunteers. Thirty-two healthy subjects received either atorvastatin calcium (10 mg once a day) or simvastatin (20 mg once a day) for the first 14 days of the study. Nelfinavir (1,250 mg twice a day) was added on days 15 to 28. Pharmacokinetic assessment was performed on days 14 and 28. The study drugs were well tolerated. Nelfinavir increased the steady-state area under the plasma concentration-time curve during one dosing period (AUC(tau)) of atorvastatin 74% and the maximum concentration (C(max)) of atorvastatin 122% and increased the AUC(tau) of simvastatin 505% and the C(max) of simvastatin 517%. Neither atorvastatin nor simvastatin appeared to alter the pharmacokinetics of nelfinavir. It is recommended that coadministration of simvastatin with nelfinavir should be avoided, whereas atorvastatin should be used with nelfinavir with caution.

Three transcriptionally distinct forms of Epstein-Barr virus latency in somatic cell hybrids: cell phenotype dependence of virus promoter usage.

Phenotypically distinct human B cell lines display two transcriptionally distinct forms of Epstein-Barr virus (EBV) latency. Latency I (Lat I) in group I Burkitt's lymphoma (BL) cell lines is characterized by selective expression of the virus-coded nuclear antigen EBNA 1 from a uniquely spliced mRNA driven by the Fp promoter. Latency III (Lat III) in group III BL and EBV-transformed lymphoblastoid cell lines (LCLs) is characterized by expression of EBNAs 1, 2, 3a, 3b, 3c, and -LP from mRNAs driven by the Cp or Wp promoter and of the latent membrane proteins (LMPs 1, 2A, and 2B) from mRNAs driven by the LMP promoters. Here we have altered the group I BL and LCL phenotypes by cell hybridization and screened for attendant changes in EBV latency by PCR analysis of viral mRNAs and immunoblotting of viral proteins. Fusion of group I BL cells with LCLs activated the BL virus genome from a Lat I to Lat III pattern of gene expression. Fusion of LCLs with nonlymphoid lines repressed virus gene expression from Lat III either to Lat I or to another form of latency (Lat II) hitherto not seen in vitro and characterized by selective expression of the Fp-driven EBNA 1 mRNA and of the LMP 1, 2A, and 2B transcripts. There are therefore three forms of EBV latency which can be interconverted by altering cellular phenotype and thereby virus promoter usage.

Multiple forms of cytochrome P450 are involved in the metabolism of ondansetron in humans.

Ondansetron is cleared primarily by metabolism in humans, with hydroxylation of the indole moiety in the 7- and 8-positions being the major identified phase I pathways. In vitro studies using lymphoblastoid cell lines expressing single human cytochrome P450 forms and hepatic microsomes were undertaken to investigate the forms involved in the metabolism of ondansetron in humans. The cell lines that expressed CYP1A1, CYP1A2, and CYP2D6 were shown to be capable of metabolizing [14C]ondansetron. Studies with human hepatic microsomes and the specific inhibitors furafylene, quinidine, and ketoconazole confirmed the role of CYP1A2 and CYP2D6 and also demonstrated the involvement of the CYP3A subfamily. The data in this study collectively indicate that multiple cytochrome P450 forms, including CYP1A1, CYP1A2, CYP2D6, and the CYP3A subfamily, are probably involved in the clearance of ondansetron in humans, with no single form of cytochrome P450 dominating the overall metabolism of ondansetron. The role played by CYP2D6 in the metabolism of [14C]ondansetron by human hepatic microsomes in vitro was shown to be minor. This finding is consistent with the lack of bimodality in the clinical pharmacokinetics of ondansetron. It is therefore concluded that ondansetron is metabolized by multiple forms of cytochrome P450, and this limits the likelihood of a clinically relevant interaction with ondansetron by a modulator of a single form of cytochrome P450.

Characterization of the selectivity and mechanism of human cytochrome P450 inhibition by the human immunodeficiency virus-protease inhibitor nelfinavir mesylate.

In vitro studies with human liver microsomes and P450 probe substrates were performed to characterize selectivity and mechanism of cytochrome P450 inhibition by nelfinavir mesylate. At therapeutic concentrations (steady-state plasma concentrations approximately 4 microM), nelfinavir was found to be a competitive inhibitor of only testosterone 6beta-hydroxylase (CYP3A4) with a Ki concentration of 4. 8 microM. At supratherapeutic concentrations, nelfinavir competitively inhibited dextromethorphan O-demethylase (CYP2D6), S-mephenytoin 4-hydroxylase (CYP2C19), and phenacetin O-deethylase (CYP1A2) with Ki concentrations of 68, 126, and 190 microM, respectively. Nelfinavir did not appreciably inhibit tolbutamide 4-hydroxylase (CYP2C9), paclitaxel 6alpha-hydroxylase (CYP2C8), or chlorzoxaxone 6beta-hydroxylase (CYP2E1) activities. The inhibitory potency of nelfinavir toward CYP3A4 suggested the possibility of in vivo inhibition of this isoform, whereas in vivo inhibition of other P450s was considered unlikely. In a one-sequence crossover study in 12 healthy volunteers, nelfinavir inhibited the elimination of the CYP3A substrate terfenadine and the carboxylate metabolite of terfenadine. The 24-hr urinary recoveries of 6beta-hydroxycortisol were reduced by an average of 27% during nelfinavir treatment, consistent with CYP3A inhibition by nelfinavir. Inhibition of CYP3A4 by nelfinavir in vitro was NADPH-dependent requiring the catalytic formation of a metabolite or a metabolic intermediate. The catechol metabolite of nelfinavir (M3) was considered unlikely to be responsible for inhibition as the addition of catechol O-methyl transferase, S-adenosyl methionine, and ascorbic acid to the preincubation mixture did not protect against the loss of testosterone 6beta-hydroxylase activity. Also, the addition of M3 to human liver microsomes did not inhibit CYP3A4. Although incubations with nelfinavir showed a time- and concentration-dependent loss of CYP3A4 activity, the partial or complete recovery of enzyme activity upon dialysis indicated that inhibition was reversible. Microsomal incubations with nelfinavir and NADPH did not result in a loss of spectral P450 content compared with the NADPH control. Glutathione, N-acetylcysteine, and catalase did not attenuate CYP3A4 inhibition by nelfinavir. Collectively, these results suggest that the probable mechanism for CYP3A4 inhibition by nelfinavir is a transient metabolic intermediate or stable metabolite that coordinates tightly but reversibly to the heme moiety of the P450.