Photomotor Responses in Zebrafish and Electrophysiology Reveal Varying Interactions of Anesthetics Targeting Distinct Sites on γ-Aminobutyric Acid Type A Receptors.

BACKGROUND: Etomidate, barbiturates, alfaxalone, and propofol are anesthetics that allosterically modulate γ-aminobutyric acid type A (GABAA) receptors via distinct sets of molecular binding sites. Two-state concerted coagonist models account for anesthetic effects and predict supra-additive interactions between drug pairs acting at distinct sites. Some behavioral and molecular studies support these predictions, while other findings suggest potentially complex anesthetic interactions. We therefore evaluated interactions among four anesthetics in both animals and GABAA receptors. METHODS: The authors used video assessment of photomotor responses in zebrafish larvae and isobolography to evaluate hypnotic drug pair interactions. Voltage clamp electrophysiology and allosteric shift analysis evaluated coagonist interactions in α1ß3γ2L receptors activated by γ-aminobutyric acid (GABA) versus anesthetics [log(d, AN):log(d, GABA) ratio]. Anesthetic interactions at concentrations relevant to zebrafish were assessed in receptors activated with low GABA. RESULTS: In zebrafish larvae, etomidate interacted additively with both propofol and the barbiturate R-5-allyl-1-methyl m-trifluoromethyl mephobarbital (R-mTFD-MPAB; mean ± SD α = 1.0 ± 0.07 and 0.96 ± 0.11 respectively, where 1.0 indicates additivity), while the four other drug pairs displayed synergy (mean α range 0.76 to 0.89). Electrophysiologic allosteric shifts revealed that both propofol and R-mTFD-MPAB modulated etomidate-activated receptors much less than GABA-activated receptors [log(d, AN):log(d, GABA) ratios = 0.09 ± 0.021 and 0.38 ± 0.024, respectively], while alfaxalone comparably modulated receptors activated by GABA or etomidate [log(d) ratio = 0.87 ± 0.056]. With low GABA activation, etomidate combined with alfaxalone was supra-additive (n = 6; P = 0.023 by paired t test), but etomidate plus R-mTFD-MPAB or propofol was not. CONCLUSIONS: In both zebrafish and GABAA receptors, anesthetic drug pairs interacted variably, ranging from additivity to synergy. Pairs including etomidate displayed corresponding interactions in animals and receptors. Some of these results challenge simple two-state coagonist models and support alternatives where different anesthetics may stabilize distinct receptor conformations, altering the effects of other drugs.

Binding site location on GABAA receptors determines whether mixtures of intravenous general anaesthetics interact synergistically or additively in vivo.

BACKGROUND AND PURPOSE: General anaesthetics can act on synaptic GABAA receptors by binding to one of three classes of general anaesthetic sites. Canonical drugs that bind selectively to only one class of site are etomidate, alphaxalone, and the mephobarbital derivative, R-mTFD-MPAB. We tested the hypothesis that the general anaesthetic potencies of mixtures of such site-selective agents binding to the same or to different sites would combine additively or synergistically respectively. EXPERIMENTAL APPROACH: The potency of general anaesthetics individually or in combinations to cause loss of righting reflexes in tadpoles was determined, and the results were analysed using isobolographic methods. KEY RESULTS: The potencies of combinations of two or three site-selective anaesthetics that all acted on a single class of site were strictly additive, regardless of which single site was involved. Combinations of two or three site-selective anaesthetics that all bound selectively to different sites always interacted synergistically. The strength of the synergy increased with the number of separate sites involved such that the percentage of each agent's EC50 required to cause anaesthesia was just 35% and 14% for two or three sites respectively. Propofol, which binds non-selectively to the etomidate and R-mTFD-MPAB sites, interacted synergistically with each of these agents. CONCLUSIONS AND IMPLICATIONS: The established pharmacology of the three anaesthetic binding sites on synaptic GABAA receptors was sufficient to predict whether a mixture of anaesthetics interacted additively or synergistically to cause loss of righting reflexes in vivo. The principles established here have implications for clinical practice.

Identifying barbiturate binding sites in a nicotinic acetylcholine receptor with [3H]allyl m-trifluoromethyldiazirine mephobarbital, a photoreactive barbiturate.

At concentrations that produce anesthesia, many barbituric acid derivatives act as positive allosteric modulators of inhibitory GABAA receptors (GABAARs) and inhibitors of excitatory nicotinic acetylcholine receptors (nAChRs). Recent research on [(3)H]R-mTFD-MPAB ([(3)H]R-5-allyl-1-methyl-5-(m-trifluoromethyldiazirinylphenyl)barbituric acid), a photoreactive barbiturate that is a potent and stereoselective anesthetic and GABAAR potentiator, has identified a second class of intersubunit binding sites for general anesthetics in the α1ß3γ2 GABAAR transmembrane domain. We now characterize mTFD-MPAB interactions with the Torpedo (muscle-type) nAChR. For nAChRs expressed in Xenopus oocytes, S- and R-mTFD-MPAB inhibited ACh-induced currents with IC50 values of 5 and 10 µM, respectively. Racemic mTFD-MPAB enhanced the equilibrium binding of [(3)H]ACh to nAChR-rich membranes (EC50 = 9 µM) and inhibited binding of the ion channel blocker [(3)H]tenocyclidine in the nAChR desensitized and resting states with IC50 values of 2 and 170 µM, respectively. Photoaffinity labeling identified two binding sites for [(3)H]R-mTFD-MPAB in the nAChR transmembrane domain: 1) a site within the ion channel, identified by photolabeling in the nAChR desensitized state of amino acids within the M2 helices of each nAChR subunit; and 2) a site at the γ-α subunit interface, identified by photolabeling of γMet299 within the γM3 helix at similar efficiency in the resting and desensitized states. These results establish that mTFD-MPAB is a potent nAChR inhibitor that binds in the ion channel preferentially in the desensitized state and binds with lower affinity to a site at the γ-α subunit interface where etomidate analogs bind that act as positive and negative nAChR modulators.

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.

Allyl m-trifluoromethyldiazirine mephobarbital: an unusually potent enantioselective and photoreactive barbiturate general anesthetic.

We synthesized 5-allyl-1-methyl-5-(m-trifluoromethyl-diazirynylphenyl)barbituric acid (14), a trifluoromethyldiazirine-containing derivative of general anesthetic mephobarbital, separated the racemic mixture into enantiomers by chiral chromatography, and determined the configuration of the (+)-enantiomer as S by X-ray crystallography. Additionally, we obtained the (3)H-labeled ligand with high specific radioactivity. R-(-)-14 is an order of magnitude more potent than the most potent clinically used barbiturate, thiopental, and its general anesthetic EC(50) approaches those for propofol and etomidate, whereas S-(+)-14 is 10-fold less potent. Furthermore, at concentrations close to its anesthetic potency, R-(-)-14 both potentiated GABA-induced currents and increased the affinity for the agonist muscimol in human α1ß2/3γ2L GABA(A) receptors. Finally, R-(-)-14 was found to be an exceptionally efficient photolabeling reagent, incorporating into both α1 and ß3 subunits of human α1ß3 GABA(A) receptors. These results indicate R-(-)-14 is a functional general anesthetic that is well-suited for identifying barbiturate binding sites on Cys-loop receptors.

The antiepileptic drug mephobarbital is not transported by P-glycoprotein or multidrug resistance protein 1 at the blood-brain barrier: a positron emission tomography study.

Aim of this study was to determine whether the carbon-11-labeled antiepileptic drug [(11)C]mephobarbital is a substrate of P-glycoprotein (Pgp) and can be used to assess Pgp function at the blood-brain barrier (BBB) with positron emission tomography (PET). We performed paired PET scans in rats, wild-type (FVB) and Mdr1a/b((-/-)) mice, before and after intravenous administration of the Pgp inhibitor tariquidar (15mg/kg). Brain-to-blood AUC(0-60) ratios in rats and brain AUC(0-60) values of [(11)C]mephobarbital in wild-type and Mdr1a/b((-/-)) mice were similar in scans 1 and 2, respectively, suggesting that in vivo brain distribution of [(11)C]mephobarbital is not influenced by Pgp efflux. Absence of Pgp transport was confirmed in vitro by performing concentration equilibrium transport assay in cell lines transfected with MDR1 or Mdr1a. PET experiments in wild-type mice, with and without pretreatment with the multidrug resistance protein (MRP) inhibitor MK571 (20mg/kg), and in Mrp1((-/-)) mice suggested that [(11)C]mephobarbital is also not transported by MRPs at the murine BBB, which was also supported by in vitro transport experiments using human MRP1-transfected cells. Our results are surprising, as phenobarbital, the N-desmethyl derivative of mephobarbital, has been shown to be a substrate of Pgp, which suggests that N-methylation abolishes Pgp affinity of barbiturates.

Fetal malformations associated with the use of methylphenobarbital and carbamazepine during pregnancy. Two case reports and review of the literature.

The use of antiepileptic drugs (AEDs) during pregnancy has been associated with an increased risk of major and minor fetal malformations. This paper describes 2 infants with malformations born to epileptic mothers who used AEDs throughout pregnancy. In the first case, the AED used for seizure control was methylphenobarbital, while in the second case the patient had been prescribed carbamazepine. We noted major and minor congenital malformations in both infants exposed in utero to these anticonvulsant drugs. Pregnant women still experience poor obstetrical care because they report to tertiary centers at the end of their pregnancy or when in labor, making it difficult to provide proper medical care for both the infant and the mother.

The influence of sulfur substituents on the molecular geometry and packing of thio derivatives of N-methylphenobarbital.

The room-temperature crystal structures of four new thio derivatives of N-methylphenobarbital [systematic name: 5-ethyl-1-methyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trione], C(13)H(14)N(2)O(3), are compared with the structure of the parent compound. The sulfur substituents in N-methyl-2-thiophenobarbital [5-ethyl-1-methyl-5-phenyl-2-thioxo-1,2-dihydropyrimidine-4,6(3H,5H)-dione], C(13)H(14)N(2)O(2)S, N-methyl-4-thiophenobarbital [5-ethyl-1-methyl-5-phenyl-4-thioxo-3,4-dihydropyrimidine-2,6(1H,5H)-dione], C(13)H(14)N(2)O(2)S, and N-methyl-2,4,6-trithiophenobarbital [5-ethyl-1-methyl-5-phenylpyrimidine-2,4,6(1H,3H,5H)-trithione], C(13)H(14)N(2)S(3), preserve the heterocyclic ring puckering observed for N-methylphenobarbital (a half-chair conformation), whereas in N-methyl-2,4-dithiophenobarbital [5-ethyl-1-methyl-5-phenyl-2,4-dithioxo-1,2,3,4-tetrahydropyrimidine-6(5H)-one], C(13)H(14)N(2)OS(2), significant flattening of the ring was detected. The number and positions of the sulfur substituents influence the packing and hydrogen-bonding patterns of the derivatives. In the cases of the 2-thio, 4-thio and 2,4,6-trithio derivatives, there is a preference for the formation of a ring motif of the R(2)(2)(8) type, which is also a characteristic of N-methylphenobarbital, whereas a C(6) chain forms in the 2,4-dithio derivative. The preferences for hydrogen-bond formation, which follow the sequence of acceptor position 4 > 2 > 6, confirm the differences in the nucleophilic properties of the C atoms of the heterocyclic ring and are consistent with the course of N-methylphenobarbital thionation reactions.

[Intoxication due to replacement of the precursor methylphenobarbital by phenobarbital]. / Intoxicatie door het vervangen van de precursor methylfenobarbital door fenobarbital.

An 80-year-old woman with a history of familial primary generalised epilepsy presented to the outpatient clinic with complaints of dizziness, confusion, dullness and feeling of'being worthless'. It turned out that she had been using medication for some time in which the chemical nature of the pharmacologically active ingredient had been changed: methylphenobarbital 60 mg t.i.d. had been replaced by phenobarbital 60 mg t.i.d. The resultant phenobarbital concentration was much higher than the concentration to which she was accustomed. At the same dosage, phenobarbital is more active than methylphenobarbital. When one compound ofa pharmacologically active substance is replaced by a different compound, the dosage should be corrected for both the chemical structure, such as the molecular weight, and the pharmacokinetic properties such as absorption, metabolism and biological availability.

A novel single nucleotide polymorphism (SNP) of the CYP2C19 gene in a Japanese subject with lowered capacity of mephobarbital 4'-hydroxylation.

We sequenced all nine exons and exon-intron junctions of the cytochrome P450 2C19 (CYP2C19) gene from a Japanese subject with a lowered capacity of CYP2C19-mediated 4'-hydroxylation after an oral administration of mephobarbital. We found a novel single nucleotide polymorphism (SNP) of CYP2C19 gene as follows: SNP, 040110MoritaJ001; GENENAME: CYP2C19; ACCESSION NUMBER: NT_030059.8; LENGTH; 25 bases; 5'-GAGGGCCTGGCCC/TGCATGGAGCTGT-3'. The SNP (168946C>T) induced an amino acid alteration (Arg442Cys) located in exon 9 close to the heme-binding region of CYP2C19, which may result in the decrease in the catalytic properties of CYP2C19. A new allele having this SNP was designated as CYP2C19*16.

Pharmacogenetic roles of CYP2C19 and CYP2B6 in the metabolism of R- and S-mephobarbital in humans.

OBJECTIVES AND METHODS: We assessed the relationship between the metabolism of R- and S-mephobarbital (MPB) and genetic polymorphisms of cytochrome P450 (CYP) 2C19 and CYP2B6. Nine homozygous extensive metabolizers (homo-EMs, 2C19*1/2C19*1) of CYP2C19, ten heterozygous EMs (hetero-EMs, 2C19*1/2C19*2, 2C19*1/2C19*3) and eleven poor metabolizers (PMs, 2C19*2/2C19*2, 2C19*3/2C19*3, 2C19*2/2C19*3) recruited from a Japanese population, received an oral 200 mg-dose of racemic MPB. Blood and urine samples were collected, and R-MPB, S-MPB and the metabolites, phenobarbital (PB) and 4'-hydroxy-MPB, were measured. Each subject was also genotyped for CYP2B6 gene. RESULTS: The mean area under the plasma concentration-time curve (AUC) of R-MPB was 92-fold greater in PMs than in homo-EMs. R/S ratios for AUC of MPB were much higher in PMs than in EMs (homo- and hetero-). The cumulative urinary excretion of 4'-hydroxy-MPB up to 24 h postdose was 21-fold less in PMs than in homo-EMs. The metabolic ratio of AUCPB/(AUCS-MPB + AUCR-MPB) was higher in PMs than in EMs (homo- and hetero-). In addition, this metabolic ratio was lower in the carriers of CYP2B6*6 compared with that in its non-carriers. CONCLUSIONS: Our results indicate that the 4'-hydroxylation of R-MPB is mediated via CYP2C19 and that the rapid 4'-hydroxylation of R-MPB results in a marked difference in the pharmacokinetic profiles between R-MPB and S-MPB in the different CYP2C19 genotypic individuals. In addition, a minor fraction of the interindividual variability in PB formation from MPB may be explainable by the CYP2B6*6 allele.

Monolithic silica columns with chemically bonded beta-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals.

An enantioselective silica rod type chiral stationary phase (CSP) is presented; a novel combination of the well known enantiomer separation properties of beta-cyclodextrin and the unique properties concerning the flow behavior of silica monoliths. Two different synthesis routes are described, and it was found that the in situ modification of a plain silica rod column turned out to be the best. The chromatographic behaviour of the beta-cyclodextrin silica rod was studied and compared with a very similar commercially available beta-cyclodextrin bonded particulate material (ChiraDex). Even if the amount of beta-cyclodextrin bound to the silica rod was only about half of the amount of beta-cyclodextrin bound to ChiraDex) particles, good resolutions were achieved for a set of chiral test components like Chromakalin, Prominal, Oxazepam, Methadone and some other drugs. By taking advantage of the unique features of the silica rods relating to their flat H/u (Van Deemter) curves, fast enantiomer separations could be demonstrated.

Determination of two ternary mixtures containing phenobarbitone by second derivative of the ratio spectrum-zero-crossing and HPLC methods.

A spectrophotometric method is developed for the determination of ternary mixtures with overlapping spectra. The method is based on the use of the second derivative of the ratio spectrum with a zero-crossing technique. The ratio spectrum was obtained by dividing the absorption spectrum of the mixture by that of one of the components. The concentration of the other components are then determined from their respective calibration graphs treated similarly. The method is accurate, non-destructive and do not require resolutions of equations. The method has been applied for the resolution of two ternary mixtures, namely, phenobarbitone, methylphenobarbitone and phenytoin (1), and phenobarbitone, papaverine HCl and piperazine acefyllinate (2). Also, a HPLC method was developed for determination of phenobarbitone, papaverine and HCL and piperazine acefyllinate. The HPLC method depends upon using ODS column with a mobile phase consisting of acetonitrile-5 mM aqueous heptane sulfonic acid sodium salt (50:50, v/v) and adjusted to apparent pH 4 using acetic acid. Quantitation was achieved with UV detection at 220 nm based on peak area. The proposed methods were applied for the determination of the two ternary combinations in synthetic mixtures and in commercial pharmaceutical products. The results obtained were precise and accurate.

Prediction of adsorption from multicomponent solutions by activated carbon using single-solute parameters. Part II--Proposed equation.

Prediction of multicomponent adsorption is still one of the most challenging problems in the adsorption field. Many models have been proposed and employed to obtain multicomponent isotherms from single-component equilibrium data. However, most of these models were based on either unrealistic assumptions or on empirical equations with no apparent definition. The purpose of this investigation was to develop a multicomponent adsorption model based on a thermodynamically consistent equation, and to validate that model using experimental data. Three barbiturates--phenobarbital, mephobarbital, and primidone--were combined to form a ternary system. The adsorption of these barbiturates from simulated intestinal fluid (without pancreatin) by activated carbon was studied using the rotating bottle method. The concentrations, both before and after the attainment of equilibrium, were determined with a high-performance liquid chromatography system employing a reversed-phase column. The proposed equation and the competitive Langmuir-like equation were both fit to the data. A very good correlation was obtained between the experimental data and the calculated data using the proposed equation. The results obtained from the original competitive Langmuir-like model were less satisfactory. These results suggest that the proposed equation can successfully predict the trisolute isotherms of the barbituric acid derivatives employed in this study.

Role of CYP2C19 in stereoselective hydroxylation of mephobarbital by human liver microsomes.

The 4-hydroxylation of mephobarbital enantiomers was investigated by using human liver microsomes from the extensive metabolizers (EM) and poor metabolizers of CYP2C19. The 4-hydroxylase activity of R-mephobarbital in the EM microsomes was >10 times higher than that of S-mephobarbital. In the poor metabolizer microsomes, the 4-hydroxylase activity of R-mephobarbital was much lower than that in the EM microsomes, and the ratio of 4-hydroxylase activity of R-mephobarbital to that of S-mephobarbital was also lower than that in the EM microsomes. Moreover, the 4-hydroxylase activity of R-mephobarbital showed a high correlation (r = 0.985, p<0.001) with the 4'-hydroxylase activity of S-mephenytoin in a panel of nine human liver microsomes. Anti-CYP2C antibody inhibited R-mephobarbital 4-hydroxylase activity by 85% of the control activity. R-Mephobarbital competitively inhibited S-mephenytoin 4'-hydroxylase activity (K(i) = 34 microM), while S-mephenytoin inhibited R-mephobarbital 4-hydroxylase activity (K(i) = 103 microM). Among the seven cDNA-expressed CYPs studied, only CYP2C19 catalyzed R-mephobarbital 4-hydroxylation. These findings suggest that the 4-hydroxylation of mephobarbital catalyzed by CYP2C19 is preferential for R-enantiomer in human liver microsomes.

Simultaneous spectrophotometric determination of phenobarbital, phenytoin and methylphenobarbital in pharmaceutical preparations by using partial least-squares and principal component regression multivariate calibration.

Two multivariate calibration methods, partial least squares (PLS-2) and principal component regression (PCR) have been applied to the simultaneous spectrophotometric analysis of ternary mixtures of phenytoin (DPH), phenobarbital (PBT) and methylphenobarbital (MPBT) in the Comital-L pharmaceutical formulation. The PLS-2 and PCR procedures were employed to evaluate the data of a variable number of calibration solutions measured over the wavelength range 400-700 nm. The concentration ranges used to construct the calibration matrix were varied between 5 and 30 microg ml(-1). The proposed methods were validated by applying them to the analysis of the Comital-L pharmaceutical formulation and the average relative errors were less than 6% for each one of the analyzed compounds. The results obtained by both proposed methods have been compared with the results obtained by application of a RPLC reference method.

Prediction of adsorption from multicomponent solutions by activated carbon using single-solute parameters.

The adsorption of 3 barbiturates--phenobarbital, mephobarbital, and primidone--from simulated intestinal fluid (SIF), without pancreatin, by activated carbon was studied using the rotating bottle method. The concentrations of each drug remaining in solution at equilibrium were determined with the aid of a high-performance liquid chromatography (HPLC) system employing a reversed-phase column. The competitive Langmuir-like model, the modified competitive Langmuir-like model, and the LeVan-Vermeulen model were each fit to the data. Excellent agreement was obtained between the experimental and predicted data using the modified competitive Langmuir-like model and the LeVan-Vermeulen model. The agreement obtained from the original competitive Langmuir-like model was less satisfactory. These observations are not surprising because the competitive Langmuir-like model assumes that the capacities of the adsorbates are equal, while the other 2 models take into account the differences in the capacities of the components. The results of these studies indicate that the adsorbates employed are competing for the same binding sites on the activated carbon surface. The results also demonstrate that it is possible to accurately predict multicomponent adsorption isotherms using only single-solute isotherm parameters. Such prediction is likely to be useful for improving in vivo/in vitro correlations.

Automated sample preparation techniques for the determination of drug enantiomers in biological fluids using liquid chromatography with chiral stationary phases.

The determination of drug enantiomers has become of prime importance in the field of pharmaceutical and biomedical analysis. Liquid chromatography (LC) is one of the most frequently used techniques for achieving the separation and quantitation of the enantiomers of drug compounds. In the bioanalytical field, the integrated systems present an interesting alternative to time-consuming sample preparation techniques such as liquid-liquid extraction. Solid phase extraction (SPE) on disposable cartridges, dialysis or column switching are sample preparation techniques that can be fully automated and applied to enantioselective analysis in biological fluids. The selection of the most appropriate LC mode and chiral stationary phase for enantioseparations in bioanalysis is discussed and some aspects of these automated sample preparation procedures are compared, such as selectivity, detectability, elution of the analytes from the extraction sorbent, sample volume and analyte stability.

Role of human CYP2B6 in S-mephobarbital N-demethylation.

The role of cytochrome P-450s (CYPs) in S-mephobarbital N-demethylation was investigated by using human liver microsomes and cDNA-expressed CYPs. Among the 10 cDNA-expressed CYPs studied (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4), only CYP2B6 could catalyze S-mephobarbital N-demethylation. The apparent K(m) values of human liver microsomes for S-mephobarbital N-demethylation were close to that of cDNA-expressed CYP2B6 (about 250 microM). The N-demethylase activity of S-mephobarbital in 10 human liver microsomes was strongly correlated with immunodetectable CYP2B6 levels (r = 0.920, p<.001). Orphenadrine (300 microM), a CYP2B6 inhibitor, inhibited the N-demethylase activity of S-mephobarbital in human liver microsomes to 29% of control activity. Therefore, it appears that CYP2B6 mainly catalyzes S-mephobarbital N-demethylation in human liver microsomes.