(-)-N-3-Benzylphenobarbital Is Superior to Omeprazole and (+)-N-3-Benzylnirvanol as a CYP2C19 Inhibitor in Suspended Human Hepatocytes.

Early assessment of metabolism pathways of new chemical entities guides the understanding of drug-drug interactions. Selective enzyme inhibitors are indispensable in CYP reaction phenotyping. The most commonly applied CYP2C19 inhibitor, omeprazole, lacks selectivity. Two promising alternatives, (+)-N-3-benzylnirvanol and (-)-N-3-benzylphenobarbital, are already used as CYP2C19 inhibitors in some in vitro studies with suspended human hepatocytes. However, a full validation proving their suitability in terms of CYP and non-CYP selectivity has not been presented in literature. The present study provides a thorough comparison between omeprazole, (+)-N-3-benzylnirvanol, and (-)-N-3-benzylphenobarbital in terms of potency and selectivity and shows the superiority of (-)-N-3-benzylphenobarbital as a CYP2C19 inhibitor in suspended human hepatocytes. Furthermore, we evaluated the application of (-)-N-3-benzylphenobarbital to predict the in vivo contribution of CYP2C19 to drug metabolism [fraction metabolized (fm) of CYP2C19, fmCYP2C19]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fmCYP2C19 data was evaluated. fmCYP2C19, which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fmCYP2C19 The use of a different hepatocyte batch with a different CYP3A4/CYP2C19 activity ratio showed the impact of intrinsic CYP activities on the determination of fmCYP2C19 Overall, this study confirms the selective CYP2C19 inhibition by (-)-N-3-benzylphenobarbital over other CYP isoforms (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2D6, and CYP3A4) and clinically relevant non-CYP enzymes [aldehyde oxidase, flavin-containing monooxygenase 3, N-acetyltransferase 2, uridine diphosphate glucuronosyltransferase (UGT) 1A1, UGT1A4, UGT2B7, UGT2B15] in suspended human hepatocytes. (-)-N-3-benzylphenobarbital is therefore the preferred CYP2C19 inhibitor to assess fmCYP2C19 in suspended human hepatocytes in comparison with omeprazole and (+)-N-3-benzylnirvanol. SIGNIFICANCE STATEMENT: (-)-N-3-Benzylphenobarbital is a more potent and selective inhibitor of CYP2C19 in suspended human hepatocytes than omeprazole and (+)-N-3-benzylnirvanol. (-)-N-3-Benzylphenobarbital can be used to predict the fraction metabolized by CYP2C19 in suspended human hepatocytes.

Drug interaction considerations in the therapeutic use of carbonic anhydrase inhibitors.

INTRODUCTION: Carbonic anhydrase inhibitors (CAIs) of the sulfonamide and sulfamate type are clinically used drugs as diuretics, antiglaucoma, antiepileptic, antiobesity and anti-high altitude disease agents. Anticancer agents based on CAIs are also in clinical development for the management of hypoxic, metastatic tumors. Acetazolamide, methazolamide, dichlorophenamide, dorzolamide and brinzolamide are mainly used as antiglaucoma drugs, sulthiame, topiramate and zonisamide as antiepileptic/antiobesity agents, celecoxib and polmacoxib are dual carbonic anhydrase/cycloxygenase inhibitors. Girentuximab, a monoclonal antibody and SLC-0111, a sulfonamide inhibitor, are in clinical trials as anticancer agents. AREAS COVERED: The drug interactions with many classes of pharmacological agents are reviewed. Some of these drugs, such as acetazolamide, topiramate and celecoxib show a large number of interactions with non-steroidal anti-inflammatory drugs (NSAIDs), diuretics, antiepileptics, immunosupressants, anticholinesterase drugs, ß-blockers, anesthetics, oral contraceptives, anticancer agents, antifungals, anti-mycobacterials, lithium, metformin and clopidogrel. EXPERT OPINION: The multiple drug interactions in which CAIs are involved should be carefully considered when such drugs are used in combination with the drug classes mentioned above, as the risks of developing toxicity and serious side effects if the dosages are not adjusted are high. There are also synergistic effects between CAIs and some NSAIDs, anticancer agents and benzodiazepines for the management of cystoid macular edema, some tumor types and neuropathic pain, respectively.

Contrasting actions of a convulsant barbiturate and its anticonvulsant enantiomer on the α1 ß3 γ2L GABAA receptor account for their in vivo effects.

KEY POINTS: Most barbiturates are anaesthetics but unexpectedly a few are convulsants whose mechanism of action is poorly understood. We synthesized and characterized a novel pair of chiral barbiturates that are capable of photolabelling their binding sites on GABAA receptors. In mice the S-enantiomer is a convulsant, but the R-enantiomer is an anticonvulsant. The convulsant S-enantiomer binds solely at an inhibitory site. It is both an open state inhibitor and a resting state inhibitor. Its action is pH independent, suggesting the pyrimidine ring plays little part in binding. The inhibitory site is not enantioselective because the R-enantiomer inhibits with equal affinity. In contrast, only the anticonvulsant R-enantiomer binds to the enhancing site on open channels, causing them to stay open longer. The enhancing site is enantioselective. The in vivo actions of the convulsant S-enantiomer are accounted for by its interactions with GABAA receptors. ABSTRACT: Most barbiturates are anaesthetics but a few unexpectedly are convulsants. We recently located the anaesthetic sites on GABAA receptors (GABAA Rs) by photolabelling with an anaesthetic barbiturate. To apply the same strategy to locate the convulsant sites requires the creation and mechanistic characterization of a suitable agent. We synthesized enantiomers of a novel, photoactivable barbiturate, 1-methyl-5-propyly-5-(m-trifluoromethyldiazirinyl) phenyl barbituric acid (mTFD-MPPB). In mice, S-mTFD-MPPB acted as a convulsant, whereas R-mTFD-MPPB acted as an anticonvulsant. Using patch clamp electrophysiology and fast solution exchange on recombinant human α1 ß3 γ2L GABAA Rs expressed in HEK cells, we found that S-mTFD-MPPB inhibited GABA-induced currents, whereas R-mTFD-MPPB enhanced them. S-mTFD-MPPB caused inhibition by binding to either of two inhibitory sites on open channels with bimolecular kinetics. It also inhibited closed, resting state receptors at similar concentrations, decreasing the channel opening rate and shifting the GABA concentration-response curve to the right. R-mTFD-MPPB, like most anaesthetics, enhanced receptor gating by rapidly binding to allosteric sites on open channels, initiating a rate-limiting conformation change to stabilized open channel states. These states had slower closing rates, thus shifting the GABA concentration-response curve to the left. Under conditions when most GABAA Rs were open, an inhibitory action of R-mTFD-MPPB was revealed that had a similar IC50 to that of S-mTFD-MPPB. Thus, the inhibitory sites are not enantioselective, and the convulsant action of S-mTFD-MPPB results from its negligible affinity for the enhancing, anaesthetic sites. Interactions with these two classes of barbiturate binding sites on GABAA Rs underlie the enantiomers' different pharmacological activities in mice.

End of the barbexaclone era: an experience of treatment withdrawal.

Barbexaclone is a salt compound of phenobarbital and propylhexedrine (a drug with indirect sympathomimetic properties). Due to the presence of the psychostimulating agent, propylhexedrine, this drug has less of a sedative effect and is well tolerated, compared to phenobarbital. Barbexaclone was widely used in Turkey until 2009 when its production ended, however, it gave rise to an epidemic for which we were not prepared. Since then, no standardised management protocol has been developed and each patient has been evaluated individually, thereby creating tailor-made solutions based on the extent of each patient's supply of remaining drug (from a few tablets to a stock which might last for six months). The rate of seizure freedom was 37.7% under barbexaclone treatment and dropped to 32.2% in the follow-up period after discontinuation of the drug. In the majority of cases, a new antiepileptic drug was added and this was commonly levetiracetam, a more expensive drug. In this article, we share our experiences of a general problem: the withdrawal of an antiepileptic drug from the market. Although there was prior notification regarding barbexaclone withdrawal, it was not possible to contact all patients since such a database is not available in Turkey. Although no conclusions regarding the efficacy of the drug or comparison of efficacy with other antiepileptic drugs is provided, it is nonetheless noteworthy to share these experiences since some patients had lost seizure control for reasons that could not be explained.

First HPLC-UV method for rapid and simultaneous quantification of phenobarbital, primidone, phenytoin, carbamazepine, carbamazepine-10,11-epoxide, 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine, lamotrigine, oxcarbazepine and licarbazepine in human plasma.

A sensitive and fast high-performance liquid chromatographic method coupled with ultraviolet detection is herein reported for the simultaneous determination of human plasma concentration of six antiepileptic drugs frequently used in clinical practice [phenobarbital (PB), primidone (PRM), phenytoin (PHT), carbamazepine (CBZ), lamotrigine (LTG), oxcarbazepine (OXC)] and some of their main metabolites, carbamazepine-10,11-epoxide (CBZ-E), 10,11-trans-dihydroxy-10,11-dihydrocarbamazepine (trans-diol) and licarbazepine (Lic). Sample preparation consisted of a deproteinization step with methanol followed by a solid-phase extraction procedure. Chromatographic separation was achieved in approximately 15 min on a reversed-phase C18 column using a mobile phase composed by water-methanol-acetonitrile-triethylamine (68.7:25:6:0.3, v/v/v/v; pH 6.5) pumped isocratically at 1.0 mL/min. The detector was set at 237 nm. Calibration curves were linear with regression coefficients greater than 0.992 over the concentration ranges 0.25-100 µg/mL for PB, 0.4-50 µg/mL for PRM, 0.5-50 µg/mL for PHT, 0.1-50 µg/mL for CBZ, LTG and CBZ-E, 0.1-25 µg/mL for OXC, 0.25-10 µg/mL for trans-diol and 0.15-80 µg/mL for Lic. Inter- and intra-day imprecision did not exceed 12.15% and inaccuracy was within ±14.91%. Absolute mean recoveries ranged from 78.49 to 101.04% and no interferences were observed at the retention times of the analytes and internal standard (ketoprofen). This bioanalytical method was successfully applied to real plasma samples from epileptic patients and it seems to be a suitable tool for routine therapeutic drug monitoring and also to support other clinical pharmacokinetic-based studies.

How did phenobarbital's chemical structure affect the development of subsequent antiepileptic drugs (AEDs)?

Phenobarbital has been in clinical use as an antiepileptic drug (AED) since 1912. The initial clinical success of phenobarbital and other barbiturates affected the design of subsequent AEDs (e.g., phenytoin, primidone, ethosuximide), developed between 1938 and 1962, the chemical structures of which resemble that of phenobarbital. However, the empirical discovery of carbamazepine (1962) and the serendipitous discovery of valproic acid (1967) led to subsequent AEDs having chemical structures that are diverse and completely different from that of phenobarbital. Sixteen AEDs were introduced between 1990 and 2012. Most of these AEDs were developed empirically, using mechanism-unbiased anticonvulsant animal models. The empirical nature of the discovery of these AEDs, coupled with their multiple mechanisms of action, explains their diverse chemical structures. The antiepileptic market is therefore crowded. Future design of new AEDs must have a potential for treating nonepileptic central nervous system (CNS) disorders (e.g., bipolar disorder, neuropathic pain, migraine prophylaxis, or restless legs syndrome). The barbiturates were once used as sedative-hypnotic drugs, but have been largely replaced in this role by the much safer benzodiazepines. In contrast, phenobarbital is still used worldwide in epilepsy. Nevertheless, the development of nonsedating phenobarbital derivatives will answer a clinical unmet need and might make this old AED more attractive.

Sample stacking microemulsion electrokinetic capillary chromatography induced by reverse migrating pseudostationary phase for the quantification of phenobarbital and its p-hydroxyphenobarbital metabolite in rat urine.

For the first time, a capillary electrophoretic (CE) method with sample stacking induced by a reverse migrating pseudostationary phase (SRMP) technique has been developed and validated for sensitive determination of phenobarbital (PB) and its p-hydroxyphenobarbital (PHPB) metabolite in rat urine samples. Separation and determination were optimized on a fused-silica capillary with a total length of 50 cm (effective length 40 cm) and 75 µm ID. The microemulsion background electrolyte consisted of 0.8% (v/v) ethyl acetate, 6.6% (v/v) butan-2-ol, 1.0% (v/v) acetonitrile, 2.0% (w/v) sodium n-dodecyl sulfate (SDS), and 89.6% (v/v) of 7.5 mM ammonium formate at pH 8. When this preconcentration technique was used, the sample stacking and the separation processes took place successively with changing the voltage with an intermediate polarity switching step. For practical application, a solid-phase extraction (SPE), C(18) sorbent with n-hexane/ethyl acetate (1 : 1%, v/v) as the elution solvent was used for sample purification and concentration. The SPE method gave good extraction yields for all the analytes, with absolute recovery values of 96.9% and 99.1% for PB and PHPB, respectively. The regression equations for PB and PHPB showed excellent linearity over a concentration range of 55-1386 ng mL(-1) for PB and PHPB (r = 0.998). The developed microemulsion electrokinetic capillary chromatography (MEEKC) method for separation of the studied compounds with SRMP as the electrophoretic preconcentration technique allowed detection limits in urine samples at 16.8 ng mL(-1) for PB and PHPB which are 15-fold lower than the reported CE method in the literature. The precision results, expressed by the intra-day and inter-day relative standard deviation (RSD) values range from 3.6 to 7.1% (repeatability) and from 3.2 to 7.2% (intermediate precision) for PB and PHPB, respectively, which were in line with Food and Drug Administration (FDA) criteria.

Transport of 5,5-diphenylbarbituric acid and its precursors and their effect on P-gp, MRP2 and CYP3A4 in Caco-2 and LS180 cells.

AIM: To examine the transport of 5,5-diphenylbarbituric acid sodium (T2007) and its mono- (MMMDPB) and di- (T2000) methoxymethylated precursors and their inducibility potential in Caco-2 and LS180 cells. METHODS: Transport studies of T2000, MMMDPB and T2007 in Caco-2 cells were performed in Transwells. P-gp and CYP3A4 activities were assayed by [(3)H]digoxin and rhodamine 123 cellular retention and testosterone 6ß-hydroxylation, respectively. Expressions of PXR, VDR and CAR mRNA and CYP3A4, MDR1/P-gp and MRP2 mRNA and protein were determined by qPCR and Western blotting, respectively. PXR siRNA was used to assess the involvement of PXR. RESULTS: The P(app(A→B))s and P(app(B→A))s of T2000, MMMDPB and T2007 were similar (30-35×10(-6)cm/s) in Caco-2 cells. Treatment for 3 days with T2000 (15µM), MMMDPB (70µM) and T2007 (300µM) generally furnished a greater induction in LS180 cells over the Caco-2 cells due to the higher, natural abundance of PXR. Changes in expression were confined mostly to MDR1 and CYP3A4: in LS180 cells, treatment for 3 days increased MDR1 and CYP3A4 but not MRP2 mRNA, and elevated P-gp and CYP3A4 protein expression that led to decreased cellular accumulation of [(3)H]digoxin and rhodamine 123, and enhanced testosterone 6ß-hydroxylase activity towards T2007, respectively. The silencing of PXR by PXR siRNA in LS180 cells significantly attenuated the induction of MDR1 and CYP3A4. CONCLUSIONS: T2000, MMMDPB, and T2007 exhibited high permeabilities but are not P-gp substrates. T2007 and its analogs upregulated CYP3A4 and MDR1 modestly via the PXR.

Barbexaclone abuse in a cannabis ex-user.

Abuse of drugs including addictive ingredients is common among patients with initial addiction history. This article reports a patient who had experienced a panic attack due to cannabis intoxication and has began to abuse an antiepileptic drug barbexaclone after he had quitted cannabis.

Hydrolysis of 2,4-dithiophenobarbital.

Hydrolysis of 2,4-dithiophenobarbital in aqueous solutions of pH 2-12 was investigated at 40 and 60 degrees C using UV spectrophotometry. The values of reaction order, rate constants, pKa1 and pKa2 and activation energy were determined. The preliminary estimation of degradation products was accomplished using thin layer chromatography. The major products were isolated by circular chromatography and identified by spectroscopic and classical methods.

Treatment of essential tremor with the barbiturate T2000 (1,3-dimethoxymethyl-5,5-diphenyl-barbituric acid).

The effect of the barbiturate T2000 (1,3-dimethoxymethyl-5,5-diphenyl-barbituric acid; DMMDPB) on essential tremor, given in twice daily doses of 400 and 300 mg, was assessed in two brief, randomized, placebo-controlled, parallel-group, double-blinded, single-center trials in 12 and 22 patients, respectively. These trials represent the first clinical use of T2000 for a specific indication. The primary endpoint was the change in the mean scores of the treated and control groups based on the Fahn-Tolosa-Marin tremor scale. In the first study of 12 patients treated with 400 mg or placebo twice daily for 14 days, the mean change from baseline at day 14 was 19.3 (P < 0.0001) in the treated group and 9.0 (P = 0.0121) in the control group. Using a two-factor mixed ANOVA model to evaluate within group and between group changes, the effect of T2000 was significantly different from that of the placebo group (P = 0.03). In the second study of 22 patients treated with 300 mg of T2000 or placebo twice daily for 20 days, statistically significant changes were seen in treated patients compared to baseline, but the ANOVA model did not demonstrate a significant treatment effect of T2000 compared to placebo. When the treated groups from each study are compared, the 800-mg daily group is significantly different from the 600-mg daily group (P = 0.02). Some treated patients in each study, but no placebo patients, experienced marked improvement. These results support further evaluation of T2000 in the treatment of essential tremor.

Estimating the attitude of immigrants toward primary prevention of the hemoglobinopathies.

OBJECTIVES: We conducted population specific confidential enquiries among immigrants who had never experienced hemoglobinopathies, to study the reliability of this approach in estimating the wish for primary prevention by prenatal diagnosis and selective abortion. METHODS: We collected data from Surinamese Hindustanis (n = 119), Surinamese and Antillean Afro-Americans (n = 105) and North Africans (mainly Moroccans) (n = 102), living in Holland. We also interviewed 105 informed individuals of different ethnicities, all members of the multi-ethnic patients and carriers' organization 'OSCAR Nederland'. RESULTS: On average, 68% of the Surinamese Hindustanis and 42% of the Surinamese Afro-Americans were in favor of selective abortion in case of affected pregnancy. Remarkably, 77% of the last group wanted to be tested for carrier diagnostics and 67% declared to have knowledge of the disease before they were informed. Only 16% of the Moroccans were in favor of selective abortion in case of an affected fetus, while 79% wanted to have blood analysis to establish their carrier status. CONCLUSIONS: The apparently limited wish for selective abortion expressed by Moroccans is in contrast with the high number of illegal abortions reported among married women in Morocco (39%). The wish for selective abortion among informed members of the patients' organization was more than 80%.

Stability of 2-thiophenobarbital and its N-methyl derivatives in the presence of beta-cyclodextrin.

The rates of hydrolysis of thiophenobarbital and its N-mono- and N,N'-dimethyl-derivatives were determined under different conditions of pH and temperature using UV spectroscopy. They were compared with those obtained in the presence of different concentrations of beta-cyclodextrin. It was found that the compounds degrade with different rates and beta-cyclodextrin retards the hydrolysis. The formation of complexes between the investigated compounds and beta-cyclodextrin was proved by 13C NMR and ROESY spectra and molecular modeling. The inclusion with the phenyl substituent into the beta-cyclodextrin cavity is preferred.

Validation of (-)-N-3-benzyl-phenobarbital as a selective inhibitor of CYP2C19 in human liver microsomes.

(-)-N-3-Benzyl-phenobarbital (NBPB) was reported to be a potent and selective inhibitor of CYP2C19. To validate the selectivity of NBPB toward CYP2C19 in human liver microsomes, the inhibitory effects on major cytochrome P450 isoform-specific reactions were evaluated in the present study. In human liver microsomes, NBPB showed potent competitive inhibition on CYP2C19-mediated S-mephenytoin 4'-hydroxylation with an IC(50) value of 0.25 microM and K(i) value of 0.12 microM, whereas weak inhibition was observed for CYP1A2-, CYP2A6-, CYP2B6-, CYP2C8-, CYP2C9-, CYP2D6-, and CYP3A4-mediated reactions with IC(50) values >100, >100, 62, 34, 19, >100, and 89 microM, respectively. Importantly, its selectivity toward CYP2C19 among the CYP2C subfamily was demonstrated. Therefore, NBPB can be used as a potent and selective inhibitor to establish the relative contribution of CYP2C19 for in vitro reaction phenotyping studies. This compound can also serve as a positive control inhibitor of CYP2C19 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source.

Opposite effects of depressant and convulsant barbiturate stereoisomers on acetylcholine release from the rat hippocampus in vivo.

BACKGROUND: It has been shown that the R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) induces loss of the righting reflex (LRR), while S(+)-MPPB causes pure excitatory effects, including convulsions, in vivo. METHODS: We studied the effects of the depressant and convulsant MPPB stereoisomers on rat hippocampal acetylcholine (ACh) release in vivo, using a brain microdialysis technique in freely moving animals. RESULTS: R(-)-MPPB 60 and 90 mg x kg(-1) i.p. decreased ACh release from the rat hippocampus by 44.1 (8.2)% and 60.8 (8.2)%, respectively. In the hippocampus, the local application of bicuculline, a gamma-aminobutyric acid (GABA)(A) receptor antagonist, 1 micromol litre(-1) antagonized the inhibitory effects of R(-)-MPPB 90 mg x kg(-1) i.p. In contrast, R(-)-MPPB, S(+)-MPPB 60 and 90 mg x kg(-1) i.p. increased ACh release to 151.8 (6.8)% and 169.6 (11.1)% of the basal release, respectively. CONCLUSIONS: Our results demonstrated that R(-)-MPPB decreased, while S(+)-MPPB increased, rat hippocampal ACh release and that the inhibitory effects of R(-)-MPPB may involve the GABA(A) receptor in vivo. These data imply that changes in hippocampal ACh due to these agents may be related to their central inhibitory and stimulatory actions in vivo.

Hydrolysis of N,N- and N,S-dimethyl derivatives of 2-thiophenobarbital.

Kinetics of hydrolysis of N,N- and N,S-dimethyl-2-thiophenobarbital and products of this reaction were investigated. The UV spectroscopy served as a tool for kinetic investigations and chromatography was used to separate and isolate the main products of hydrolysis. These products were identified by spectroscopic methods and the course of hydrolysis of both isomers was compared.

Hydrolysis of N- and S-monomethyl derivatives of 2-thiophenobarbital.

Kinetics of hydrolysis of S-methyl-2-thiophenobarbital in aqueous solutions was investigated using the UV spectroscopic method within the pH range 1.5-12.9 at 60 degrees C. Chromatography was used to separate and isolate the products of hydrolysis of this compound and its N-methyl isomer. The products were identified by spectroscopic methods and the course of hydrolysis of both isomers were compared.

Mechanisms of impaired biliary excretion of acetaminophen glucuronide after acute phenobarbital treatment or phenobarbital pretreatment.

Previous studies have demonstrated that phenobarbital (PB) significantly impairs the biliary excretion of acetaminophen glucuronide (AG) in rats. Studies also suggested that Mrp2 mediates AG biliary excretion, and Mrp3 is involved in AG basolateral export. It was hypothesized that inhibition of Mrp2-mediated AG transport by PB or PB metabolites, and PB induction of Mrp3, may contribute to the impaired biliary excretion of AG by PB. In the present study, the hepatobiliary transport of AG in single-pass isolated perfused Wistar and TR(-) rat livers was investigated. The AG biliary clearance was markedly decreased, and the AG basolateral clearance was significantly increased in TR(-) rat livers. Uptake of AG by Mrp2 and Mrp3, and inhibition of Mrp2- and Mrp3-mediated transport by PB and major PB metabolites, were investigated with rat Mrp2- or Mrp3-expressing Sf9 cell plasma membrane vesicles (Sf9-PMVs). AG was transported by Mrp3 (K(m) approximately 0.91 mM). Net ATP-dependent AG uptake into Mrp2-expressing Sf9-PMVs could not be detected directly. However, AG significantly inhibited Mrp2-mediated 5-(and 6)-carboxy-2',7'-dichlorofluorescein (CDF) transport. p-Hydroxyphenobarbital glucuronide (p-OHPBG), but not PB or p-hydroxyphenobarbital, significantly inhibited Mrp2-mediated CDF transport. The IC(50) values for p-OHPBG inhibition of Mrp2-mediated CDF uptake and Mrp3-mediated AG transport were similar (approximately 0.68 and 0.46 mM, respectively). PB treatment (80 mg/kg/day x 4 days) markedly increased hepatic Mrp3 expression in Wistar rats. In conclusion, inhibition of Mrp2-mediated AG transport by p-OHPBG provided one possible explanation for the impaired biliary excretion of AG after acute PB treatment. However, impaired biliary excretion of AG after PB pretreatment may be attributed primarily to the induction of hepatic Mrp3 by PB.

(+)-N-3-Benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital: new potent and selective in vitro inhibitors of CYP2C19.

Highly potent and selective CYP2C19 inhibitors are not currently available. In the present study, N-3-benzyl derivatives of nirvanol and phenobarbital were synthesized, their respective (+)- and (-)-enantiomers resolved chromatographically, and inhibitor potencies determined for these compounds toward CYP2C19 and other human liver cytochromes P450 (P450s). (-)-N-3-Benzyl-phenobarbital and (+)-N-3-benzyl-nirvanol were found to be highly potent, competitive inhibitors of recombinant CYP2C19, exhibiting K(i) values of 79 and 250 nM, respectively, whereas their antipodes were 20- to 60-fold less potent. In human liver preparations, (-)-N-3-benzyl-phenobarbital and (+)-N-3-benzyl-nirvanol inhibited (S)-mephenytoin 4'-hydroxylase activity, a marker for native microsomal CYP2C19, with K(i) values ranging from 71 to 94 nM and 210 to 280 nM, respectively. At single substrate concentrations of 0.3 microM [(-)-N-3-benzyl-phenobarbital] and 1 microM [(+)-N-3-benzyl-nirvanol] that were used to examine inhibition of a panel of cDNA-expressed P450 isoforms, neither CYP1A2, 2A6, 2C8, 2C9, 2D6, 2E1, nor 3A4 activities were decreased by greater than 16%. In contrast, CYP2C19 activity was inhibited approximately 80% under these conditions. Therefore, (+)-N-3-benzyl-nirvanol and (-)-N-3-benzyl-phenobarbital represent new, highly potent and selective inhibitors of CYP2C19 that are likely to prove generally useful for screening purposes during early phases of drug metabolism studies with new chemical entities.