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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, fm CYP2C19 ]. A set of 10 clinically used CYP2C19 substrates with reported in vivo fm CYP2C19 data was evaluated. fm CYP2C19 , which was predicted using data from suspended human hepatocyte incubations, underestimated the in vivo fm CYP2C19 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 fm CYP2C19 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 fm CYP2C19 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., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

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, β-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.

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 β3 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., (© 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.)

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.

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., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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., (Wiley Periodicals, Inc. © 2012 International League Against Epilepsy.)

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.

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 6β-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 6β-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., (Copyright © 2010 Elsevier B.V. All rights reserved.)

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 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.

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.

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%., (Copyright 2005 John Wiley & Sons, Ltd.)

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.

(-)-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.

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.

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.

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.

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.

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.

The aim of this study was to evaluate the influence of primidone (PRM) nanoencapsulation on its metabolism. Suspensions of PRM powder and PRM-loaded poly-epsilon-caprolactone nanocapsules were administered orally in the same way to rats. Primidone-loaded poly-epsilon-caprolactone nanocapsules were prepared according to the interfacial deposition technique. Free PRM suspensions were obtained by addition of PRM powder to a suspension of 0.212% carboxymethylcellulose CMC 12H in water. The dose was 20 mg/kg, n = 6, for each experiment. Urinary and faecal levels of PRM and of its three major metabolites, phenylethylmalonamide (PEMA), phenobarbital (PB), and p-hydroxyphenobarbital (p-HO-PB), were determined. Concentrations were evaluated by high-performance liquid chromatography (HPLC) according to a validated analytical method. After PRM nanocapsule administration, non-metabolised PRM urinary levels were increased compared to those observed after administration of a suspension of primidone powder (43.7+/-8.8% after PRM-loaded nanocapsule and 37.7+/-8.1% after free PRM administration). For phenylethylmalonamide, no difference was observed in urinary excretion in the two cases. For two of the oxidised metabolites, PB and p-HO-PB, excretion was delayed and shortened. The amount of these oxidised metabolites was lowered from 0.95% after free PRM administration to 0.25% after PRM-loaded nanocapsule administration. No difference was noted in non-metabolised primidone excretion in faeces. These results suggest that primidone-loaded nanocapsules could be used as a vehicle for oral primidone administration in order to minimise the phenobarbital metabolic pathway.

Mammalian and hepatic aspartate transcarbamylase is inhibited by phenobarbital p-nitrophenylhydrazone in a reversible and non-competitive type with Ki values 8.45 x 10(-5) and 9.64 x 10(-5) M in the reactions toward carbamyl phosphate and aspartate, respectively. In vivo inhibition occurred in a dose-dependent manner in which less than 50% of the activity was retained. These observations suggest that this inhibitor may interfere with the in vivo regulation of this enzyme and lead to an additional biological effect of phenobarbitals.

Four chemiluminescent N-sulfonylacridinium-9-carboxamide active esters (17-20) were prepared from the corresponding acids and coupled to both of the aminated phenobarbital (13) and N-(6-aminohexyl)phenytoin (16) haptens. The level of signal produced by chemiluminescent N-sulfonylacridinium-9-carboxamide phenobarbital and phenytoin tracers in a solid-phase immunoassay format was found to be modulated by at least 20-fold by the judicious choice of the reactive acridinium-hapten linking group.

Unlabelled: Neuronal nicotinic acetylcholine receptors (neuronal nAchRs) are sensitive to many anesthetics, including barbiturates, which suggests that these receptors are potential sites for anesthetic action. Subtle changes in molecular structures of the anesthetic barbiturates can produce compounds with potent convulsant activity. Whereas R(-) isomer of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) exerts anesthetic action, S(+)MPPB exhibits pure excitatory effects, including convulsion. 5-(2-cyclohexilidene-ethyl)-5-ethyl barbituric acid is another example of a convulsant barbiturate. We compared the effects of depressant and convulsant barbiturates on the neuronal nAchR-mediated current to determine whether inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates. Whole cell nicotine-induced currents were recorded in PC12 derived from rat pheochromocytoma, using the conventional whole cell patch clamp technique in the presence and absence of barbiturates. Both depressant and convulsant barbiturates inhibited the nicotine-induced inward current reversibly and in a dose-dependent manner when co-applied with nicotine. All barbiturates accelerated the current decay. There was no significant difference between the concentrations for 50% inhibition for MPPB isomers. There was no correlation between inhibition of ganglionic nAchRs and anesthetic effects of the barbiturates. These results strongly oppose the idea that inhibition of neuronal nAchRs contributes to the anesthetic action of barbiturates., Implications: We found that both convulsant and depressant barbiturates inhibit the current mediated through ganglionic nicotinic acetylcholine receptors in PC12 cells. This finding suggests that the inhibition of neuronal nicotinic acetylcholine receptors does not contribute to the anesthetic action of barbiturates.

Background: Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors mediate fast excitatory synaptic transmission in the central nervous system. Although barbiturates have been shown to suppress the AMPA receptor-mediated responses, it is unclear whether this effect contributes to the anesthetic action of barbiturates. The authors compared the effects of depressant [R(-)] and convulsant [S(+)] stereoisomers of 1-methyl-5-phenyl-5-propyl barbituric acid (MPPB) on the AMPA and gamma-aminobutyric acid type A (GABA(A)) receptor-mediated currents to determine if the inhibitory effects on AMPA receptors correlate to the in vivo effects of the isomers., Method: The authors measured whole-cell currents in the rat cultured cortical neuron at holding potential of -60 mV. Kainate 500 microM was applied as the agonist for AMPA receptors. Thiopental (3-300 microM), R(-)-MPPB or S(+)-MPPB (100-1,000 microM) was coapplied with kainate under the condition in which the GABA(A) receptor-mediated current was blocked. Effects of MPPB isomers on the current elicited by GABA 1 microM were studied in the separate experiments., Results: Thiopental inhibited the kainate-induced current reversibly and in a dose-dependent manner, with a concentration for 50% inhibition of 49.3 microM. Both R(-)-MPPB and S(+)-MPPB inhibited the kainate-induced current with a little stereoselectivity. R(-)-MPPB was slightly but significantly more potent than S(+)-MPPB. In contrast, R(-)-MPPB enhanced but S(+)-MPPB reduced the GABA-induced current., Conclusions: Both convulsant and depressant stereoisomers of the barbiturate inhibited the AMPA receptor-mediated current despite of their opposite effects on the central nervous system in vivo. Although thiopental exhibited a considerable inhibition of AMPA receptors, the results suggest that the inhibition of AMPA receptors contributes little to the hypnotic action of the barbiturates.

A new high-performance liquid chromatographic method for simultaneous determination of primidone (PRM) and of its three major metabolites, phenobarbital (PB), p-hydroxyphenobarbital (p-HO-PB) and phenylethylmalonamide (PEMA), in rat urine, was developed. After acid hydrolysis, these compounds were extracted from urine by means of a Bond Elut Certify LRC column with good clean-up. The extracts were chromatographed on a C18 reversed-phase column using isocratic elution at 40 degrees C, with UV detection at 227 nm. The limit of detection was 0.5 mg/ml for the four compounds. Good linearity (r2>0.99) was observed within the calibration ranges studied: 37.4-299.3 microg/ml for PRM, 26.4-211.2 microg/ml for PB, 12.5-100.2 microg/ml for p-HO-PB and 12.1-97.0 microg/ml for PEMA. Repeatability was in the range 3.1-6.8%. This method constitutes a useful tool for studies on the influence of various parameters on primidone metabolism.

A method is described for the measurement of 5,5-diphenylbarbituric acid in plasma using high-performance liquid chromatography with UV detection. Briefly, the compounds are separated on a C18 reversed-phase column using a mobile phase of 50 mM sodium acetate (pH 4.5) and methanol. The flow-rate is 1.0 ml/min and 25 microl are injected and detected at 215 nm. The method is specific and sensitive in the range of concentrations tested, with a limit of quantification of 0.25 microg/ml. The calibration curves are linear for concentrations between 0.25 and 10 microg/ml. Intra-day and inter-day coefficients of variation are less than 8.5 and 10.5%, respectively, over the linear range. Intra-day and inter-day bias are less than 7.0 and 8.0%, respectively. A pharmacokinetic study conducted in male Beagle dogs administered 10 mg/kg of 1,3-dimethoxymethyl-5,5-diphenylbarbituric acid or 8 mg/kg of 5,5-diphenylbarbituric acid intravenously demonstrates the utility of this method.

To study the specific target to which phenobarbital (PB) binds, resulting in the induction of cytochrome P450, we prepared two azido-PBs (AZPBs) as photoaffinity ligands. The azido substituent was introduced at the para- or meta-position of the PB aromatic ring. In this study, we estimated the utility of these compounds by examining their inducing activities in vivo in rats. Induction was assessed by immunoblotting with anti-CYP2B1/2 antibody and measuring testosterone-metabolizing activity, using hepatic microsomes. Administration of p-AZPB to rats increased hepatic CYP2B1/2 protein and testosterone 16beta-hydroxylase activity, although the effects were less than those of unmodified PB. m-AZPB showed no effect in the induction of CYP2B1/2. To assess the specificity of the effects of substituents, we compared the inducing activities of p/m-nitro-PBs, p/m-amino-PBs, and p/m-hydroxy-PBs with those of AZPBs. The results showed that p-nitro-PB, m-amino-PB, and p-hydroxy-PB were also potent inducers for CYP2B1/2, with lower activity than that of unmodified PB, whereas the other three isomers had no effect. These results suggest that 1) the absence of any substituents on the aromatic ring of PB is needed for maximal inducing activity and 2) substitution at the meta-position of the PB aromatic ring tends to reduce effectiveness as an inducer more than does substitution at the para-position. Because p-amino-PB and p-acetylamino-PB, the minor and major metabolites of p-AZPB, respectively, were without effect in the induction of CYP2B1/2, the effect of p-AZPB was considered to be due to the unchanged compound itself. The present study demonstrates that, based on the weak but positive ability to induce CYP2B1/2, p-AZPB may be a useful tool for identifying the putative PB receptor.

A HPLC assay using UV detection and post-column alkalinization was developed to quantify possible urinary excretion products of phenobarbital in human urine. After filtration the urine was injected directly onto the HPLC column for analysis of phenobarbital, p-hydroxyphenobarbital, phenobarbital N-glucosides and phenobarbital N-glucuronides. The accuracy and precision of the assay were within +/- 15% and the limit of detection (LOD) was 1 microM, suitable for pharmacokinetic studies. Phenobarbital was administered orally to five male subjects and urine was collected for a period of 96-108 h. Phenobarbital, p-hydroxyphenobarbital, and phenobarbital N-glucosides were detected and quantified in the urine of all five subjects. The phenobarbital N-glucuronides were not detected in the urine. This assay provides a rapid method with improved selectivity to analyze urine for phenobarbital and its metabolites.

The relationship between barbiturate structures and their effects on induction of rat cytochrome P450 forms was studied in primary cultured hepatocytes. Treatment of hepatocytes cultured on matrigel with 1 mM barbital, N-methylbarbital, cyclobarbital, hexobarbital, phenobarbital (PB), or mephobarbital (N-methyl-PB) resulted in increased amounts of CYP2B1/2 and CYP2C6 forms. Microsomal CYP3A content was also enhanced by treatment with these barbiturates, except for barbital. Although no relationship was observed between the levels of CYP2B1/2 and CYP3A, ratios of CYP3A/CYP2B1 plus CYP2B2 contents were invariably higher with hepatocytes treated with N-methylated barbiturates than with the nonmethylated analogs. Consistent results were also observed in vivo in rats treated with PB and N-methyl-PB. These results indicate the difference in the structure requirement for induction of CYP2B and CYP3A. In addition, N-methyl-PB was found to suppress PB-mediated induction of CYP2B1. Hepatic levels of CYP2B1 mRNA and protein were increased by treatment with PB or N-methyl-PB alone, but decreased by cotreatment with 1 mM PB and N-methyl-PB. The suppression has been shown to occur at the transcriptional level of the CYP2B1 gene by using a chloramphenicol acetyltransferase reporter-CYP2B1 fused gene system.

The temperature dependence of [35S]-t-butylbicyclophosphorothionate (TBPS) binding to the convulsant sites of the GABAA receptor complex was studied in membrane preparations of rat forebrain. Although specific [35S]TBPS binding was maximal around 20 degrees C, the rate constants of dissociation decreased monotonously between 37 degrees C and 2 degrees C. The displacing potencies of the convulsant S(+) enantiomer of 1-methyl-5-phenyl-5-propyl-barbituric acid (MPPB) (IC50 = 1250 +/- 30 microM) and the depressant R(-) MPPB (IC50 = 310 +/- 5 microM) did not show significant changes between 19 degrees C and 37 degrees C. Therefore barbiturate binding seems to be driven by entropic, rather than enthalpic changes. An excess of MPPB enantiomers elicited accelerated and polyphasic dissociations of [35S]TBPS as compared to the monophasic dissociation by TBPS. Arrhenius analysis was applied to the measurable initial rate constants of dissociation. Arrhenius plots were linear between 2 degrees C and 37 degrees C. Activation parameters were similar when [35S] TBPS dissociation was triggered by the convulsants TBPS and S(+) MPPB. It can be attributed to similar conformations of the closed ionophore complex. In contrast, the depressant R(-) MPPB strongly decreased the activation energy of TBPS dissociation from the open ionophore ternary complex. In whole-cell patch-clamp experiments R(-) MPPB, but not S(+) MPPB, elicited chloride currents in rat primary cortical cultures with an EC50 value of 560 +/- 30 microM and a Hill coefficient of 2.9 +/- 0.2. These currents were similar to those elicited by GABA and blocked by TBPS. A kinetic scheme is proposed for the dissociation of TBPS and to explain the different effects of MPPB enantiomers. Submillimolar R(-) MPPB is supposed to bind to (about three) barbiturate sites on GABAA-ionophores and to open them in a cooperative manner to result in a decreased activation energy for accelerated displacement of convulsant binding.

In order to test the validity of the use of hepatocytes as an alternative to studies of metabolism in live animals, phenobarbital (PB) was applied at 37 degrees C to isolated rat hepatocytes, which had been previously induced by PB. The metabolites were extracted by ethyl acetate, at various pH, and identified and evaluated by GC-MS. PB was not metabolized to p-hydroxyphenobarbital as is usually the case in living animals, but it was transformed into sulphoconjugated 2-phenyl-gamma-butyrolactone. This pathway, beta-hydroxylation of the ethyl side chain followed by lactonization, previously described as a secondary metabolic pathway occurring during intoxications, was here the only observed biodegradation. These results show that it is not possible to use hepatocytes as an alternative to live animals.

Using normal conditions (phosphate buffer pH 7.4, room temperature) and 1% human serum albumin (HSA), (R)-(-)-1-methyl-5-phenyl-5-propyl-barbiturate = (R)-(-)-1) as compared to (S)-(+)-1 was preferentially bound to albumin: the difference in the enantioselective binding was large (factor of 2) with low substrate concentrations and much smaller (factor of 1.3) with high substrate concentrations (range 5-620 micrograms/ml); with (S)-(+)-1 the decrease of the percent binding values observed with increasing concentrations was only moderately marked. The stereoselective binding difference ((R)-(-)- being greater than (S)-(+)-1) was always present independent of the albumin concentration (range 0.5-10%), the pH value (range 5-10) and the temperature (range 5-50 degrees C). Using 20 micrograms/ml as substrate concentration the stereospecific binding difference was large in solutions with low HSA concentration, in acidic and neutral pH range, and in experiments with low temperature. In contrast to HSA, bovine as well as rabbit serum albumin bound (S)-(+)-1 to a higher percentage than (R)-(-)-1; using rat serum albumin an enantioselective binding difference was not obtained. Taking into account for each of three mammalian albumins the respective number of binding classes and sites in each class, the affinity constant and total binding constant, it becomes evident that they bind (S)-(+)- and (R)-(-)-1 quantitatively differently in a species-specific manner.

The effects of felbamate on the pharmacokinetics of phenobarbital and one of its main metabolites, parahydroxyphenobarbital, were assessed in a parallel-group, placebo-controlled, double-blind study, in 24 healthy volunteers. Pharmacokinetic parameters of phenobarbital and parahydroxyphenobarbital were determined from plasma and urine samples obtained after 28 days of daily administration of 100 mg phenobarbital and after a further 9 days of phenobarbital plus 2400 mg/day felbamate or placebo. Felbamate increased phenobarbital values for area under the plasma concentration-time curve from 0 to 24 hours and maximum concentration by 22% and 24%, respectively, whereas placebo had no effect. This increase was caused by a reduction in parahydroxylation of phenobarbital and possibly through effects on other metabolic pathways. Because felbamate inhibits the S-mephenytoin hydroxylase (CYP2C19) isozyme in vitro, it appears that phenobarbital hydroxylation is mediated in part by this isozyme.

The synthesis and characterization of barbital, phenobarbital, metharbital, and mephobarbital glucuronides is reported. The condensation of per(trimethylsilyl)-barbital and -phenobarbital with methyl 1,2,3,4-tetra-O-acetyl-beta-D-glucopyranuronate in the presence of trimethylsilyl trifluoromethanesulfonate gave moderate yields of the N1-(beta-D-glucopyranosyluronate) barbiturate derivatives. The diastereomers of the phenobarbital derivatives were resolved by use of C18 reversed-phase HPLC. The homologous N3-methyl barbiturate N1-glucuronates were prepared by reaction of the barbital and phenobarbital N1-glucuronate derivatives with diazomethane. The absolute configuration of the phenobarbital N1-beta-D-glucopyranuronate epimers was determined by oxidative removal of the glycon from the mephobarbital N1-beta-D-glucopyranuronate epimers to give the optical isomers of mephobarbital. The spectroscopic data for this series of compounds will facilitate the characterization of N-glycosylated imide xenobiotics that may be detected as mammalian metabolites in biodisposition studies.

The abilities of structural congeners of phenobarbital to induce immunoreactive hepatic cytochrome P450 2B (CYP2B) protein and associated catalytic activity (benzyloxyresorufin O-dealkylation) in the male B6C3F1 mouse were examined. Interspecies differences in inducing ability were examined through comparison of the results with induction data obtained previously with the male F344/NCr rat. The congeners were administered in the diet for 2 weeks at concentrations equimolar to 500 ppm of the prototype CYP2B inducer, phenobarbital. Of the series of compounds tested, phenobarbital was the most effective inducer of benzyloxyresorufin O-dealkylation and immunoreactive CYP2B protein, with 2-ethyl-2-phenylsuccinimide, 5-ethyl-5-phenylhydantoin, primidone, and glutethimide being only 19-42% as effective. 5-Ethyl-5-phenyloxazolidinedione and the ring-opened and decarboxylated congeners, N-(2-ethyl-2-phenylacetyl)urea and 2-ethyl-2-phenylmalonamide, displayed minimal induction of these catalytic activities. Dose-response experiments performed with 5-ethyl-5-phenylhydantoin indicated that the intrinsic CYP2B-inducing activity of this congener was as great as that of phenobarbital in the mouse, although a fourfold greater dietary concentration of this hydantoin (2000 ppm) was required to elicit a response equivalent to that caused by 500 ppm phenobarbital. When extent of induction was related to serum total xenobiotic concentration rather than to administered dietary concentration, the potencies of the two congeners were determined to be more similar (58 vs. > or = 78 microM for phenobarbital and 5-ethyl-5-phenylhydantoin, respectively).

Phenobarbitone (PB) produced a dose- and time-dependent decrease in gap junctional intercellular communication (GJIC) (up to 25.0 +/- 5.3% inhibition) in rat hepatocyte couplets (4 h cultures). The effect was reversible and independent of protein synthesis. This inhibition was exacerbated (to 53.3 +/- 5.4% inhibition) by depletion of intracellular glutathione following pretreatment with diethylmaleate (0.5 microM, 15 min). Inhibition was also significantly enhanced by addition of the cytochrome P450 inhibitors SKF 525A (25 microM) and metyrapone (20 nM). In contrast, hepatocyte couplets derived from rats pretreated with PB (0.1% w/v in drinking water) for up to 28 days were fully functional regarding GJIC and were found to be refractory to the effects of PB added in vitro. This, coupled with the lack of effect of p-hydroxy-PB, suggests that an active metabolite of PB is not involved in the inhibition of GJIC which may, instead, be through an oxidative stress, which is prevented by glutathione.

Epilepsy is one of the most common neurological disorders. Even though existing antiepileptic drugs can render 80% of newly diagnosed patients seizure free, a significant number of patients have chronic intractable epilepsy causing disability with considerable socioeconomic implications. There is, therefore, a need for more potent and effective antiepileptic drugs and drugs with fewer adverse effects, particularly CNS effects. Drugs for the treatment of partial seizures are particularly needed. With major advances in our understanding of the basic neuropathology, neuropharmacology and neurophysiology of epilepsy, numerous candidate novel antiepileptic drugs have been developed in recent years. This review comparatively evaluates the pharmacokinetics, efficacy and adverse effects of 12 new antiepileptic drugs namely vigabatrin, lamotrigine, gabapentin, oxcarbazepine, felbamate, tiagabine, eterobarb, zonisamide, remacemide, stiripentol, topiramate and levetiracetam (ucb-L059). Of the 12 drugs, vigabatrin, lamotrigine and gabapentin have recently been marketed in the UK. Five of these new drugs have known mechanisms of action (vigabatrin, lamotrigine, tiagabine, oxcarbazepine and eterobarb), which may provide for a more rational approach to the treatment of epilepsy. Oxcarbazepine, remacemide and eterobarb are prodrugs. Vigabatrin, gabapentin and topiramate are more promising on the basis of their pharmacokinetic characteristics in that they are excreted mainly unchanged in urine and not susceptible to significant pharmacokinetic interactions. In contrast, lamotrigine, felbamate and stiripentol exhibit significant drug interactions. Essentially, all the drugs are effective in partial or secondarily generalised seizures and are effective to varying degrees in other seizure types. Particularly welcome is the possible effectiveness of zonisamide in myoclonus and felbamate in Lennox-Gastaut syndrome. In relation to adverse effects, CNS effects are observed with all drugs, however, gabapentin, remacemide and levetiracetam appear to exhibit least. There is also the possibility of rational duotherapy, using drugs with known mechanisms of action, as an additional therapeutic approach. The efficacy of these 12 antiepileptic drug occurs despite the fact that candidate antiepileptic drugs are evaluated under highly unfavourable conditions, namely as add-on therapy in patients refractory to drug management and with high seizure frequency. Thus, whilst candidate drugs which do become licensed are an advance in that they are effective and/or are associated with less adverse effects than currently available antiepileptic drugs in these patients, it is possible that these drugs may exhibit even more improved risk-benefit ratios when used in normal clinical practice.

To explore further the structural requirements for ligand interaction with the putative phenobarbital receptor, the pharmacodynamics of CYP2B induction by 5,5-diphenylbarbituric acid, phenytoin (5,5-diphenylhydantoin), barbital (5,5-diethylbarbituric acid) and 5,5-diethylhydantoin were investigated in the male F344/NCr rat. Steady-state total (free plus protein-bound) serum drug concentration, measured after 14 days of administration of the compounds in the diet, was used as an approximation of intrahepatocellular drug concentration. The serum concentrations associated with half-maximal hepatic CYP2B induction (EC50 values) were 6 to 11 microM and 15 to 18 microM for the diphenyl-substituted barbiturate and hydantoin, respectively, based on measurement of pentoxy- or benzyloxyresorufin O-dealkylation activities, or immunoreactive CYP2B1 protein. The corresponding potency values for the diethyl-substituted barbiturate and hydantoin were 16 to 20 microM and > or = 500 microM, respectively. The magnitudes of the maximal CYP2B induction responses elicited by the diphenyl-substituted congeners and by barbital were 94 to 122% of the responses resulting from phenobarbital itself. In contrast, the maximum induction responses elicited by 5,5-diethylhydantoin were only 24% as great as those elicited by phenobarbital. The finding of a CYP2B inducer with a potency value 2 to 3 orders of magnitude lower than those for certain other prototype CYP2B inducers is suggestive but not proof of receptor mediation in the induction process.

The elimination of phenobarbitone (PB) was studied in 14 chronically treated epileptic patients under steady state conditions. PB, [S]-PB-N-glucoside ([S]-PB-N-G) and p-hydroxy-PB (p-OH-PB) were assayed in urine by a HPLC method. Some 57% of the daily dose was recovered in urine, 14% as [S]-PB-N-G, 16% as p-OH-PB (conjugated plus non-conjugated) and 27% as unaltered PB. Thus PB-N-G formation contributed significantly to the elimination of PB during long-term administration of the drug, and there was reason to suspect that some of the PB-N-G formed may have already been degraded to untraced products before excretion from the body.

We report on a 33-year-old female suffering from frequent complex-partial seizures who developed a non-convulsive status epilepticus after one week of antiepileptic therapy with valproate (VPA) which had been added to a basic medication with barbexaclone (BBC) in rapidly increasing dosage. The electroencephalogram (EEG) showed continuous rhythmic generalized sharp and slow wave activity with a frontal maximum. Intravenous administration of 3.0 mg of the benzodiazepine (BZ) receptor antagonist flumazenil under monitoring with video-EEG led to an immediate and marked electroclinical improvement, whereas 6.0 mg of the BZ receptor agonist midazolam was followed by a deterioration both clinically and in the EEG. We discuss the concept of VPA-encephalopathy and the possible mechanisms of the action of flumazenil on VPA-induced as well as on other toxic and metabolic encephalopathies. Flumazenil might antagonize increased benzodiazepine receptor activity with agonistic and even convulsive properties in these encephalopathic syndromes. Further investigations are needed concerning the relation of drug-induced or metabolic encephalopathies and central benzodiazepine receptor activity. We recommend a therapeutic trial with flumazenil, if stupor or decreased seizure control develop in patients treated with valproate.

Conventional analysis of initial uptake and egress rates in isolated hepatocytes is limited in the ability to distinguish between rates of metabolite formation and egress, and to separate basolateral and canalicular transport processes. The present study examined the applicability of kinetic modeling in describing acetaminophen glucuronide (AG) and acetaminophen sulfate (AS) formation and egress in hepatocytes after acute exposure to phenobarbital or p-hydroxyphenobarbital glucuronide (p-OHPBG) in vitro, or in vivo phenobarbital pretreatment. A significant pretreatment effect on AG and AS disposition was seen based on initial rates of egress. In vivo phenobarbital pretreatment decreased the initial egress rate of AG compared to vehicle pretreatment, and the initial egress rate of AS compared to all other treatments. A pharmacokinetic model incorporating AG and AS formation in hepatocytes as well as egress processes (including diffusional and active transport components) was fit to the data. Parameter estimates derived from model fits to the data showed the expected increase in acetaminophen glucuronidation and decrease in sulfation after phenobarbital pretreatment; in addition, an increase in the AG diffusional rate constant and a decrease in the AS diffusional rate constant was apparent. The excretion Vmax for AG was decreased statistically after acute phenobarbital exposure in vitro, and in vivo phenobarbital pretreatment, with a concomitant statistical increase in the Km for AG excretion. In vitro acute p-OHPBG exposure also decreased significantly the excretion Vmax for AG. These data are consistent with the hypothesis that phenobarbital-impaired biliary excretion of AG is a function of impaired canalicular transport due to the presence of p-OHPBG. They further suggest that the mechanism may not be simple competitive inhibition. This work demonstrates the utility of a kinetic modeling approach to differentiate metabolic and transport processes when analyzing data from isolated hepatocyte studies. Additional information may be gained that would not be apparent by conventional methods of analysis.

As part of an investigation of the structural requirements for the induction, by phenobarbital-type inducers, of a coordinate pleiotropic response consisting of increases in hepatic cytochrome P450 2B (P450 2B) activity, increases in other phase I and II enzyme activities, and liver hypertrophy, we have examined a series of analogues of phenobarbital in which the ethyl/phenyl substitution at the sp3 carbon of the parent molecule was kept constant while the heterocyclic portion of the molecule was modified. The induction of hepatic P450 2B protein and ethoxy-, pentoxy-, and (benzyloxy)resorufin O-dealkylation activities, and epoxide hydration activity and liver/body weight ratio increase were examined in male F344/NCr rats fed the various congeners for 14 days at doses equimolar to 500 ppm phenobarbital. Increases in the measured parameters were maximal in rats fed phenobarbital or 5-ethyl-5-phenylhydantoin. The responses to primidone or 2-ethyl-2-phenylsuccinimide were approximately 65% of maximal, while glutethimide yielded a response approximately 50% of maximal. Induction of this response in rats fed the ring-opened and decarboxylated analogues, (ethylphenylacetyl)urea and 2-ethyl-2-phenylmalonamide, were < 25% of maximal. 5-Ethyl-5-phenyloxazolidinedione caused minimal increases in the measured end points when administered at a dose equimolar to 500 ppm phenobarbital. The profound differences among the congeners in ability to induce P450 2B protein and associated catalytic activities were not due to differences in food consumption by the various groups of rats.(ABSTRACT TRUNCATED AT 250 WORDS)

We describe here a new approach for multilabel immunoassays. The starting point of this approach is a new nonradioisotopic immunoassay (carbonyl metallo immunoassay) based on the use of metal carbonyl complexes as tracers and Fourier transform infrared (FT-ir) spectroscopy as the detection method. We show here that the judicious choice of the organometallic tracers associated with multicomponent analysis of FT-ir spectroscopic data provides the simultaneous rapid and reliable quantitation of pmol of the organometallic tracers. The feasibility of this approach is demonstrated in the case of the two major antiepileptic drugs phenobarbital and carbamazepine.

Valproate has been shown to interact with all major antiepileptic drugs. The interaction with phenobarbital is the most clinically significant. The mechanism of the interaction was evaluated in the in vivo rat and in vitro liver perfusion system. Phenobarbital and parahydroxyphenobarbital (PbOH) were administered with and without valproate treatment. In vivo, after administration of PbOH, valproate caused a significant inhibition of both the renal clearance of unchanged PbOH (40%) and the formation clearance (ClF) of its glucuronide conjugate (44%). When coadministered with phenobarbital, valproate caused a significant decrease in the total plasma clearance of phenobarbital (95.4 +/- 29.0 to 65.8 +/- 20.2 ml/hr/kg), with no apparent effect on the phenobarbital renal clearance or the ClF of PbOH. Valproate did cause a significant inhibition (50%) of formation of a minor metabolite, metahydroxyphenobarbital. The largest effect of valproate appears to be on unknown pathways of phenobarbital elimination. In the isolated perfused rat liver, the ClF of PbOH and its glucuronide conjugate were determined. Valproate caused a small (10%) but significant decrease in the ClF of PbOH. As seen in vivo, the most significant effect of valproate was on the ClF of the PbOH glucuronide (66% decrease). In conclusion, inhibition of PbOH formation by valproate cannot account entirely for the increased plasma concentrations of phenobarbital that occur when valproate is added to therapy. A complete understanding of the mechanism will require a complete accounting of the phenobarbital dose in rat or in humans.

The purpose of this study was to determine the influence of age on the pharmacokinetic/pharmacodynamic relationship of phenobarbital in male BN/BiRij rats aged 4, 15, 26, 31 and 36 months. The pharmacokinetics were studied on basis of the plasma concentration vs. time profile and the excretion of phenobarbital and parahydroxyphenobarbital in urine and feces after an i.v. dose of 20 mg/kg. The pharmacodynamics were determined as the threshold dose and cerebrospinal fluid threshold concentration of phenobarbital for the onset of loss of righting reflex (as a measure of the anesthetic effect) during an i.v. infusion at a rate of 3 mg/min. The dose requirement for the anesthetic effect decreased with increasing age from 263 +/- 4 mg/kg (mean +/- S.E.M.) for the 4-month-old rats to 202 +/- 6 mg/kg for the 31-month-old rats. Only minor changes in the pharmacokinetic parameters, the metabolite profile and the distribution of phenobarbital between plasma (total and free), cerebrospinal fluid and brain tissue were observed. With respect to the pharmacodynamics, however, evidence for an increase in brain sensitivity was observed, as reflected in a decrease in the threshold cerebrospinal fluid concentration from 181 +/- 4 mg/l (mean +/- S.E.M.) at 4 months to 134 +/- 4 mg/l at 31 months. It is concluded that the decreased dose requirement of phenobarbital in elderly BN/BiRij rats is due to an increased brain sensitivity as a result of the aging process per se rather than detectable pathology.

1. Phenobarbitone (1) and three of its N-alkyl derivatives, and primidone (10) and four of its N-alkyl derivatives, were orally administered separately to two human volunteers. Total urine was collected for approximately 2 weeks following each dose, and the drugs and their metabolites were assayed by g.l.c.-mass spectrometry. 2. Recoveries in the phenobarbitone series increased from approximately 40% to approximately 50% as alkylation of (1) increased. There was a linear relationship between the extent of p-hydroxylation and the lipophilicity (log P) of the substrates. The increased total recovery was largely attributable to increased p-hydroxylation. 3. Urinary recoveries in the primidone series decreased from approximately 80% for (10) to approximately 30% for its diakyl derivatives, despite a slight increase in p-hydroxylation with increasing alkylation (and increasing lipophilicity). The decreased recovery was mainly the result of decreased urinary excretion of the drug.

The effect of the phenobarbital-type monooxygenase inducers is accomplished via the native molecule. This made it possible to transform the typical substrate of cytochrome P-450b aminopyrine into an inducer of this isozyme by blocking the substrate molecule position undergoing monooxygenation. Substitution of two methyl groups in the aminopyrine -N(CH3)2 position by an isopropyl group gave rise to a clear-cut inducive effect. This was registered by spectral, kinetic, radiological and immunochemical methods.

Previously, the N-glucosylation of phenobarbital had been observed only in humans. The results of a species screen (mouse, rat, guinea pig, rabbit, cat, dog, pig, and monkey) found that only mice excreted the N-glucosides of phenobarbital in urine after ip administration of sodium phenobarbital. The major diastereomer excreted by the mouse had the R configuration at the C-5 position of the barbiturate ring. The N-glucoside metabolites accounted for a small percentage of the dose (approximately 0.5%). Following ip dosing of the mouse with the phenobarbital N-glucosides, free phenobarbital could be detected in the urine. Upon ip or intercerebroventricular (icv) injection of the phenobarbital N-glucosides, minimal CNS activity was observed in the mouse.