Disposition of naproxen, naproxen acyl glucuronide and its rearrangement isomers in the isolated perfused rat liver.

1. An isolated perfused rat liver (IPRL) preparation was used to investigate separately the disposition of the non-steroidal anti-inflammatory drug (NSAID) naproxen (NAP), its reactive acyl glucuronide metabolite (NAG) and a mixture of NAG rearrangement isomers (isoNAG), each at 30 microg NAP equivalents ml perfusate (n = 4 each group). 2. Following administration to the IPRL, NAP was eliminated slowly in a log-linear manner with an apparent elimination half-life (t 1/2) of 13.4 +/- 4.4h. No metabolites were detected in perfusate, while NAG was the only metablolite present in bile in measurable amounts (3.9 +/- 0.8% of the dose). Following their administration to the IPRL, both NAG and isoNAG were rapidly hydrolysed (t 1/2 in perfusate = 57 +/- 3 and 75 +/- 14 min respectively). NAG also rearranged to isoNAG in the perfusate. Both NAG and isoNAG were excreted intact in bile (24.6 and 14.8% of the NAG and isoNAG doses, respectively). 3. Covalent NAP-protein adducts in the liver increased as the dose changed from NAP to NAG to isoNAG (0.20 to 0.34 to 0.48% of the doses, respectively). Similarly, formation of covalent NAP-protein adducts in perfusate were greater in isoNAG-dosed perfusions. The comparative results suggest that isoNAG is a better substrate for adduct formation with liver proteins than NAG.

Evaluation of a pharmacokinetic interaction between remacemide hydrochloride and phenobarbitone in healthy males.

AIMS: To determine whether there is a pharmacokinetic interaction between the antiepileptic drugs remacemide and phenobarbitone. METHODS: In a group of 12 healthy adult male volunteers, the single dose and steady-state kinetics of remacemide were each determined twice, once in the absence and once in the presence of phenobarbitone. The effect of 7 days remacemide intake on initial steady-state plasma phenobarbitone concentrations was also investigated. RESULTS: Apparent remacemide clearance (CL/F) and elimination half-life values were unchanged after 7 days intake of the drug in the absence of phenobarbitone (1.25 +/- 0.32 vs 1.18 +/- 0.22 l kg(-1) h(-1) and 3.29 +/- 0.68 vs 3.62 +/- 0.85 h, respectively). Concomitant administration of remacemide with phenobarbitone resulted in an increase in the estimated CL/F of remacemide (1.25 +/- 0.32 vs 2.09 +/-0.53 l kg-1 h-1), and a decreased remacemide half-life (3.29 +/- 0.68 vs 2.69 +/- 0.33 h). The elimination of the desglycinyl metabolite of remacemide also appeared to be increased after the phenobarbitone intake (half-life 14.72 +/- 2.82 vs 9.61 +/- 5.51 h, AUC 1532 +/- 258 vs 533 +/- 281 ng ml(-1) h). Mean plasma phenobarbitone concentrations rose after 7 days of continuing remacemide intake (12.67 +/- 1.31 vs 13.86 +/- 1.81 microgram ml(-1)). CONCLUSIONS: Phenobarbitone induced the metabolism of remacemide and that of its desglycinyl metabolite. Remacemide did not induce its own metabolism, but had a modest inhibitory effect on the clearance of phenobarbitone.

Population pharmacokinetic modeling of steady state carbamazepine clearance in children, adolescents, and adults.

Carbamazepine (CBZ) clearance decreases from childhood to adulthood and the factors determining this change could include age, size, autoinduction, or maturational changes. This study aims to describe the population pharmacokinetics of CBZ in children and young adults and test the hypothesis that CBZ clearance correlates with weight, surface area, and age. CBZ therapeutic drug monitoring data (sparse data) were collected from child and adult epileptics, and rich data were obtained from a bioequivalence study of CBZ in young adults. Population pharmaco-kinetic analysis was performed using NONMEM V. Forward stepwise, multiple regression was performed on the covariates. Bootstrap validation was performed. A total of 946 observations from 91 subjects, ages 0.7-37 years, were collected and analyzed. A one-compartment, first-order absorption and elimination model, with exponential interindividual error and additive residual error models was developed. The population model was: Clearance (Lhr-1) = ((2.24 x Surface area (m2)) + (0.047 x Dose (mg.kg-1)); Volume of distribution (L) = 0.37 x weight (kg); Absorption rate constant = 0.013 (hr-1). CBZ clearance increased with surface area and dose.

The effect of diflunisal on the elimination of triamterene in human volunteers.

A major metabolic pathway for triamterene (a potassium sparing diuretic) is aromatic hydroxylation followed by sulphate conjugation. Diflunisal (a salicylate anti-inflammatory agent) also undergoes sulphate conjugation of its phenolic group as a major pathway. We investigated the possible effect of diflunisal on the elimination of triamterene (competition for phenolic sulphonation) in six healthy volunteers by studying the disposition of single doses of triamterene (100 mg) taken alone and in the presence of steady-state levels of diflunisal. Diflunisal coadministration (500 mg b.i.d.) had no effect on the pharmacokinetics of triamterene itself. However, plasma AUC of p-hydroxytriamterene sulphate was greater (4.6 times), and its renal clearance lower (0.24 times), in the presence of diflunisal. There was no change in the formation clearance or protein binding of p-hydroxytriamterene sulphate in the presence of diflunisal. The data point to competition for renal excretory pathways rather than sulphonation capacity. This interaction could have clinical relevance since p-hydroxytriamterene sulphate is pharmacologically active.

Valproate metabolism during valproate-associated hepatotoxicity in a surviving adult patient.

The plasma profiles of valproate (VPA), its beta-oxidation metabolites E-2-en-VPA and 3-oxo-VPA and its terminal desaturation metabolite 4-en-VPA, have been measured in a patient receiving NaVPA 1000 mg twice per day from early in the course of serious hepatotoxicity and for 2 weeks after the drug was stopped. Concurrent profiles of liver, renal and haematological function parameters were available. Relative to concurrent plasma VPA concentrations, E-2-en-VPA concentrations were not different to those of the VPA-treated epileptic population at any stage of the illness, whereas 3-oxo-VPA concentrations relative to concurrent VPA concentrations were abnormally high early in the toxicity, abnormally low at its peak (3-5 days later), and comfortably within normal limits for the treated epileptic population late in the recovery phase (9-13 days from the onset). When measurable, plasma 4-en-VPA concentrations were not elevated. The elimination half-life of VPA during the recovery phase was 100 h, which is some 6-12 times greater than values reported for this parameter in normal patients. These data clearly define, in this patient, a link between idiosyncratic VPA-associated hepatotoxicity at its onset and peak and the later stages of VPA beta-oxidation. Whether the beta-oxidation abnormalities are causative or a consequence of an as yet undefined defect is unknown. In this patient, 4-en-VPA was unlikely to have been involved in the pathogenesis of the toxicity.

Phenytoin metabolism by human cytochrome P450: involvement of P450 3A and 2C forms in secondary metabolism and drug-protein adduct formation.

The anticonvulsant phenytoin (5,5-diphenylhydantoin) provokes a skin rash in 5 to 10% of patients, which heralds the start of an idiosyncratic reaction that may result from covalent modification of normal self proteins by reactive drug metabolites. Phenytoin is metabolized by cytochrome P450 (P450) enzymes primarily to 5-(p-hydroxyphenyl-),5-phenylhydantoin (HPPH), which may be further metabolized to a catechol that spontaneously oxidizes to semiquinone and quinone species that covalently modify proteins. The aim of this study was to determine which P450s catalyze HPPH metabolism to the catechol, proposed to be the final enzymatic step in phenytoin bioactivation. Recombinant human P450s were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Novel bicistronic expression vectors were constructed for P450 2C19 and the three major variants of P450 2C9, i.e., 2C9*1, 2C9*2, and 2C9*3. HPPH metabolism and covalent adduct formation were assessed in parallel. P450 2C19 was the most effective catalyst of HPPH oxidation to the catechol metabolite and was also associated with the highest levels of covalent adduct formation. P450 3A4, 3A5, 3A7, 2C9*1, and 2C9*2 also catalyzed bioactivation of HPPH, but to a lesser extent. Fluorographic analysis showed that the major targets of adduct formation in bacterial membranes were the catalytic P450 forms, as suggested from experiments with human liver microsomes. These results suggest that P450 2C19 and other forms from the 2C and 3A subfamilies may be targets as well as catalysts of drug-protein adduct formation from phenytoin.

Apparent autoinduction of valproate beta-oxidation in humans.

AIMS: The study aimed to show whether autoinduction of valproate (VPA) along its beta-oxidation pathway occurred upon chronic dosing in humans. METHODS: Twelve young volunteers without active illness took sodium valproate (NaVPA) 200 mg orally 12 hourly for 3 weeks. On days 7 and 21, serial blood samples and all urine passed over an interdosing interval from 08.00 to 20.00 h were collected for analysis of VPA and certain metabolites. RESULTS: Plasma AUC(0,12 h) of VPA was significantly lower on day 21 than on day 7 (2.40 vs 2.84 micromol ml-1 h, 95% CI for the difference 0.13-0.81 micromol ml-1 h). Significant differences in plasma AUC(0,12 h) of the beta-oxidation metabolites E-2-en-VPA and 3-oxo-VPA were not found. However, formation clearances of plasma VPA to urinary E-2-en-VPA and 3-oxo-VPA were significantly increased from day 7 to day 21 (0. 010 vs 0.024 and 2.57 vs 3.60 ml kg-1 h-1, respectively, 95% CI for the differences -0.025 to -0.004 and -1.72 to -0.34 ml kg-1 h-1, respectively). Formation clearances to VPA-glucuronide (0.534 vs 0. 505 ml kg-1 h-1) and 4-OH-VPA (0.112 vs 0.110 ml kg-1 h-1) were not significantly different. CONCLUSIONS: Regular low dose VPA intake in humans over a period of 3 weeks appears to be associated with a small induction of its metabolism by the beta-oxidation pathway, but not by glucuronidation or 4-hydroxylation.

Steady-state dispositions of valproate and diflunisal alone and coadministered to healthy volunteers.

OBJECTIVE: The effects of coadministration of the non-steroidal anti-inflammatory drug diflunisal (DF) on glucuronidation and beta-oxidation of the antiepileptic agent valproic acid (VPA), and of VPA on DF glucuronidation, were studied in human volunteers. METHODS: Seven healthy male volunteers received sodium valproate (NaVPA, 200 mg) orally twice daily for 7 days, after which all drug intake ceased for 1 month. The volunteers then took DF (250 mg) orally twice daily for 7 days. Both drugs were then taken (at the same doses as previously) twice daily for 7 days. On day 7 of each dosing phase, serial blood samples and all urine passed over the 12-h inter-dosing interval were collected. VPA, DF and selected metabolites were analysed using validated methods. Statistical comparisons of pharmacokinetic parameters were made using paired Student's t-tests. RESULTS: Mean plasma concentrations of total VPA were lower and apparent plasma clearances significantly higher during DF coadministration. This was associated with a significant 20% increase in the unbound fraction of VPA (from 6.6+/-1.3% to 7.9+/-1.8%). The apparent clearance of unbound VPA was not different. There was no evidence of any significant effect of DF coadministration on VPA metabolism: urinary recoveries of and formation clearances to urinary VPA-glucuronide, E-2-en-VPA, 3-oxo-VPA and 4-en-VPA were not significantly altered. However, there was a highly significant 35% increase in the area under the plasma concentration-time curve from 0-12 h (AUC0-12h) of 3-oxo-VPA and its renal clearance was lower, though not significantly so. VPA coadministration had no effect on DF pharmacokinetics or formation clearances of DF to its acyl glucuronide (DAG), phenolic glucuronide (DPG) or sulfate (DS) conjugates. However, plasma AUC0-12h values of the glucuronides were significantly lower and their renal clearances higher (though significantly so only in the case of DPG) during VPA coadministration. CONCLUSIONS: Steady-state coadministration of VPA and DF leads to a significant displacement of VPA from plasma protein binding sites. There was no evidence of competition for glucuronidation capacity or other metabolic interactions. Rather, the interactions detected appeared to be renal in nature, with renal clearance of 3-oxo-VPA being reduced by DF coadministration, and renal clearance of DPG and perhaps DAG being increased by VPA coadministration.

Effect of naproxen co-administration on valproate disposition.

The effects of co-administration of the antiepileptic agent valproic acid (VPA) and the non-steroidal anti-inflammatory drug naproxen (NAP) on their relative dispositions (particularly with respect to glucuronidation) were investigated in human volunteers. Seven healthy males received each drug alone and then in combination (orally twice daily for seven days, 500 mg sodium VPA, 500 mg NAP). On day 7 of each dosing phase, serial plasma and 24 h urine samples were collected for analysis. Co-administration of NAP resulted in significant increases (about 20%, p<0.05) in the apparent plasma clearance of total VPA and in the unbound fraction of VPA in plasma, with the apparent plasma clearance of unbound VPA being unchanged. There were associated increases in the formation clearances to urinary VPA-glucuronide and 3-oxo-VPA, though these were relatively greater for the glucuronidation pathway (and remained significant when formation clearances were calculated using the unbound fraction of drug in plasma). The data thus point to a shift towards glucuronidation as a result of the NAP-induced increase in the unbound fraction of VPA in plasma. By contrast, VPA co-administration caused a decrease (of about 10%, p<0.05) in the apparent plasma clearance of total NAP. Taken in hand with in vitro results showing a VPA-induced displacement (of about 40%) of NAP from plasma protein binding sites, the data strongly support a role for diminished glucuronidation of NAP and its desmethyl metabolite in the presence of co-administered VPA.

Sensitive, high-throughput gas chromatographic-mass spectrometric assay for fluoxetine and norfluoxetine in human plasma and its application to pharmacokinetic studies.

A sensitive, robust gas chromatographic-mass spectrometric assay suitable for use in pharmacokinetic or bioequivalence studies is presented for the selective serotonin reuptake inhibitor, fluoxetine, and its major metabolite, norfluoxetine (N-desmethylfluoxetine). This method employs solid-phase extraction followed by acetylation with trifluoroacetic anhydride and analysis of the derivatives using selected ion monitoring. The lower limit of quantification was 1.0 ng/ml, and the assay was linear for both analytes from 1 to 100 ng/ml. Mean recoveries following solid-phase extraction at concentrations of 5.0, 20 and 100 ng/ml were 91% (fluoxetine) and 87% (norfluoxetine). Assay precision (as mean RSD) and accuracy (as mean relative error) for both analytes were tested at the same three nominal concentrations and were found to be within 10% in all cases. Analysis of fluoxetine concentrations in plasma samples from 18 volunteers following administration of a single 40 mg dose of fluoxetine provided the following pharmacokinetic data (mean+/-SD): Cmax, 32.73+/-9.21 ng/ml; AUC0-infinity, 1627+/-1372 ng/ml h; Tmax, 3.08 h (median); ke, 0.022+/-0.007 h(-1); elimination half-life, 37.69+/-21.70 h.

Improved analytical procedure for the measurement of captopril in human plasma by gas chromatography--mass spectrometry and its application to pharmacokinetic studies.

An enhanced, sensitive GC-MS assay is presented for the highly specific angiotensin-converting enzyme (ACE) inhibitor, captopril. This method improves previously published assays by using solid NEM as stabilizer in the collection tubes, a rapid extraction technique with dichloromethane and back-extraction into base, a commercially available internal standard (thiosalicylic acid) and a capillary GC column. Captopril and the internal standard are measured as their bis-pentafluorobenzyl derivatives. The assay was linear from 10 to 5000 ng/ml with a mean recovery following solvent extraction at 50, 200 and 1000 ng/ml of 77%. At mean values of 45.9, 187 and 980 ng/ml inter-assay precision and accuracy were 4.0, 2.9 and 3.5% and 8.2, 6.5 and 3.1%, respectively. Analysis of captopril concentrations in plasma samples from 20 volunteers following oral administration of 100 mg of captopril provided the following pharmacokinetic data (mean+/-S.D.): Cmax, 1470+/-467 ng/ml; AUC(0-infinity), 1736+/-481 ng/ml.h; Tmax, 0.73 h; k(e), 0.468+/-0.122 h(-1); elimination half life, 1.58/-0.41 h.

Bioactivation of phenytoin by human cytochrome P450: characterization of the mechanism and targets of covalent adduct formation.

The cytochrome P450-dependent covalent binding of radiolabel derived from phenytoin (DPH) and its phenol and catechol metabolites, 5-(4'-hydroxyphenyl)-5-phenylhydantoin (HPPH) and 5-(3',4'-dihydroxyphenyl)-5-phenylhydantoin (CAT), was examined in liver microsomes. Radiolabeled HPPH and CAT and unlabeled CAT were obtained from microsomal incubations and isolated by preparative HPLC. NADPH-dependent covalent binding was demonstrated in incubations of human liver microsomes with HPPH. When CAT was used as substrate, covalent adduct formation was independent of NADPH, was enhanced in the presence of systems generating reactive oxygen species, and was diminished under anaerobic conditions or in the presence of cytoprotective reducing agents. Fluorographic analysis showed that radiolabel derived from DPH and HPPH was selectively associated with proteins migrating with approximate relative molecular weights of 57-59 kDa and at the dye front (molecular weights < 23 kDa) on denaturing gels. Lower levels of radiolabel were distributed throughout the molecular weight range. In contrast, little selectivity was seen in covalent adducts formed from CAT. HPPH was shown to be a mechanism-based inactivator of P450, supporting the contention that a cytochrome P450 is one target of covalent binding. These results suggest that covalent binding of radiolabel derived from DPH in rat and human liver microsomes occurs via initial P450-dependent catechol formation followed by spontaneous oxidation to quinone and semiquinone derivatives that ultimately react with microsomal protein. Targets for covalent binding may include P450s, though the catechol appears to be sufficiently stable to migrate out of the P450 active site to form adducts with other proteins. In conclusion, we have demonstrated that DPH can be bioactivated in human liver to metabolites capable of covalently binding to proteins. The relationship of adduct formation to DPH-induced hypersensitivity reactions remains to be clarified.

The mechanism of the carbamazepine-valproate interaction in humans.

AIMS: The study investigated the mechanism of the interaction between valproate and carbamazepine which causes raised plasma carbamazepine-10,11-epoxide concentrations with unchanged plasma carbamazepine concentrations. This interaction has usually been attributed to valproate inhibiting epoxide hydrolase, the enzyme that catalyses the biotransformation of carbamazepine-10,11-epoxide to carbamazepine-10,11-trans-diol. METHODS: Clearances of plasma carbamazepine, carbamazepine-epoxide and carbamazepine-diol to relevant carbamazepine metabolites present in urine were measured under steady-state conditions in 17 adults receiving carbamazepine as anticonvulsant monotherapy, and in 10 adults taking the drug together with valproate. RESULTS: Plasma carbamazepine-epoxide concentrations were higher, relative to carbamazepine dose, in the co-medicated patients. Plasma apparent clearances of carbamazepine, relative to drug dose, were similar whether or not valproate was taken. Formation clearances of carbamazepine-10,11-trans-diol conjugate, and probably of carbamazepine-10,11-trans-diol, were lower in subjects co-medicated with valproate, and a higher proportion of the carbamazepine dose was excreted in urine as carbamazepine-10,11-epoxide. CONCLUSIONS: Valproate appears to inhibit the glucuronidation of carbamazepine-10,11-trans-diol, and probably also inhibits the conversion of carbamazepine-10,11-epoxide to this trans-diol derivative, rather than simply inhibiting the latter reaction only.

Anticonvulsant therapy in aged patients. Clinical pharmacokinetic considerations.

Alterations in drug disposition that occur with aging are now becoming widely recognised, and there is an increasing number of drugs for which the approach to therapy in elderly patients can be based on pharmacokinetic data. Both healthy aging and comorbid disease can alter the responsiveness of the body to drugs and to their absorption, distribution and elimination. Altered absorption in the elderly has not been documented after oral ingestion of any anticonvulsant drug. Increased adipose tissue in the elderly may raise the apparent volume of distribution (Vd) of lipid-soluble drugs. An increased Vd in the elderly has been shown for diazepam and clobazam, but not midazolam. The data are inconclusive for phenytoin and valproic acid (sodium valproate). The decreased plasma protein binding that often occurs in the elderly has few clinical consequences. The reduced liver function that to occur with aging seems to affect the elimination of drugs that are mainly cleared by oxidative metabolism [e.g. carbamazepine, phenytoin and phenobarbital (phenobarbitone)]. Reduced clearances for methylphenobarbital (methylphenobarbitone), diazepam, midazolam and clobazam occur in elderly men, but not in women. The reduced renal function that is seen in old age affects the disposition of drugs that are eliminated mainly by direct renal excretion. Thus. the clearances of vigabatrin and gabapentin correlate with creatinine clearance. Such considerations may help guide anticonvulsant dosage in the elderly.

Effects of pregnancy on various pathways of human antiepileptic drug metabolism.

Ratios of phenytoin and carbamazepine doses to steady-state plasma concentrations of the drugs (apparent clearances) increase in pregnant women. Mean phenytoin clearance to urinary unconjugated p-hydroxyphenytoin increased from 0.28 +/- SD 0.18 to 0.74 +/- SD 0.37 L/day in 13 pregnant women; mean clearance to p-hydroxyphenytoin glucuronide increased proportionately less (15.25 +/- SD 5.43 to 31.94 +/- SD 16.30 L/day), the proportion of the metabolite that was conjugated falling from 98.4 +/- SD 0.72% to 97.65 +/- SD 0.67%. Mean clearances to urinary phenytoin and phenytoin-dihydrodiol did not increase. In 10 epileptic women, mean clearances of carbamazepine to urinary (a) carbamazepine-10,11-epoxide (1.66 +/- SD 1.2 to 3.70 +/- SD 2.09 L/day), (b) unconjugated carbamazepine-10,11-trans-diol (33.93 +/- SD 10.21 to 47.01 +/- SD 19.58 L/day). (c) unconjugated carbamazepine-acridan (0.24 +/- SD 0.12 to 0.47 +/- SD 0.34 L/day), and (d) unconjugated 2-hydroxy-carbamazepine (0.08 +/- SD 0.09 to 0.66 +/- SD 1.14 L/day) all increased during pregnancy. Mean clearance to unconjugated 3-hydroxy-carbamazepine decreased (0.53 +/- SD 0.25 to 0.18 +/- SD 0.23 L/day). In contrast, mean clearances of carbamazepine to the glucuronides of its first stage metabolites (carbamazepine-diol, 2- and 3-hydroxy-carbamazepine and carbamazepine-acridan, respectively) did not increase in pregnancy. The conversion of carbamazepine to carbamazepine-epoxide increased proportionately more than the conversion of carbamazepine-epoxide to carbamazepine-diol. Pregnancy was thus associated with increased microsomal oxidations of phenytoin and carbamazepine, without proportionate increases in the subsequent hydrolysis of carbamazepine-10,11-epoxide and in the O-glucuronidations of the earlier stage metabolites.

Dose-dependent metabolism of carbamazepine in humans.

48-h steady-state metabolic balance studies were carried out in 17 adults receiving long-term anticonvulsant monotherapy. With increasing carbamazepine dosage (1) carbamazepine overall plasma apparent clearance (CL/F), (2) plasma clearance of carbamazepine to urinary carbamazepine-10,11-epoxide, (3) plasma clearance of carbamazepine-10,11-epoxide to urinary unconjugated carbamazepine-10,11-trans-diol and (4) plasma clearances of carbamazepine to urinary 2- and 3-hydroxy carbamazepine all increased. However, with increasing carbamazepine dose there was no increase in the clearance of carbamazepine to (5) its acridan derivative in urine or of (6) the diol, phenolic or acridan metabolites to their metabolically subsequent conjugates excreted in urine. These findings are consistent with ongoing dose-dependent autoinduction of carbamazepine metabolism along the first two stages, but not the final stage, of the epoxide-diol pathway and, to a lesser extent, along pathways yielding phenolic metabolites. However, conjugations of the various plasma phase I metabolites of carbamazepine are not dose-dependent. Plasma concentration ratios of substances involved in consecutive stages of the epoxide-diol pathway, as in previous published studies, suggested apparent dose dependence of the epoxide-->unconjugated diol stage only. Presumably, increased flux along the first two stages of the full epoxide-diol pathway reduces plasma carbamazepine and carbamazepine-10,11-epoxide concentrations largely in parallel, concealing the dose dependence of the conversion of carbamazepine to its epoxide.

Effect of felbamate on valproic acid disposition in healthy volunteers: inhibition of beta-oxidation.

We assessed the effects of felbamate (FBM) on the disposition of valpr oic acid (VPA) in healthy volunteer men. Eighteen subjects received sodium VPA, 400 mg/day for 21 days. Plasma and urine samples were taken on day 7 to document the steady-state disposition of VPA alone. From day 8 to day 21, subjects received placebo or FBM at the following doses (mg/day): 1,200, 2,400, 3,000, or 3,600 (n = 2-4 per group). Many adverse events (AE) occurred from about day 10; 2 subjects dropped out and 1 continued on a reduced FBM dose. Pharmacokinetic studies were repeated on day 21 for the 16 subjects who completed the study. FBM was measured in plasma and urine by high-performance liquid chromatography (HPLC). VPA and its 2-en, 4-en, and 3-oxo metabolites in plasma, and VPA (nonconjugated and total), and its 3-oxo and 4-hydroxy metabolites in urine were measured by gas chromatography/mass spectrometry (GC/MS). Mean plasma FBM trough concentrations on day 21 ranged from 26.9 mu g/ml (1,200 mg dose) to 76.8 mu g/ml (3,600-mg dose). Mean plasma VPA C max values were 32-42 mu g/ml in the various subgroups when VPA only was administered. Higher plasma VPA levels were observed when FBM was administered concurrently (55.4-63.8 mu g/ml). The excretion of 3-oxo-VPA in urine was significantly lower on day 21 than on day 7, whereas VPA-glucuronide was significantly increased. The effects of FBM on VPA disposition were dose dependent and were maximal at approximately 2400 mg/day. FBM has caused significant inhibition of the beta-oxidation pathway for VPA metabolic clearance, and this had been largely compensated by increased VPA glucuronidation.

Enantioselective analysis of sotalol in plasma by reversed-phase high-performance liquid chromatography using diastereomeric derivatives.

A procedure for the concurrent determination of the (+)- and (-)-enantiomers of sotalol in plasma using high-performance liquid chromatography of diastereomeric derivatives is described. Sotalol is extracted from a 0.5-ml aliquot of plasma at pH 9.3 using ethyl acetate. Atenolol is used as the internal standard. The ethyl acetate is removed under vacuum, and the residue derivatized with R-(-)-1-(1-naphthyl)ethyl isocyanate (NEIC, 0.005% in chloroform) in the presence of trace quantities of carbonate buffer. The chloroform is removed, the residue reconstituted in mobile phase (acetonitrile-water, 39:61, v/v), and an aliquot injected into the HPLC column. A C18 trapping column is used to retain excess derivatizing reagent. While the derivatives are separated on a C18 analytical column with the isocratic mobile phase mentioned above at 1.5 ml/min, the column-switching allows back-flushing of the trapping column to prepare for the next injection. The derivatives were detected using a fluorescence detector with excitation wavelength 280 nm and emission wavelength 320 nm. The method was fully validated, and shown to have excellent linearity, specificity, sensitivity, accuracy and precision. It has been applied to the determination of (+)- and (-)-sotalol in plasma from twelve subjects dosed with racemic sotalol.

Metabolism of carbamazepine and co-administered anticonvulsants during pregnancy.

Urinary excretions of carbamazepine, carbamazepine-10,11-epoxide, carbamazepine-10,11-trans-diol, 9-hydroxyacridan and 2- and 3-hydroxycarbamazepine were measured at various stages of pregnancy, and in the post-natal period, in ten epileptic women, six of whom took no other enzyme-inducing anticonvulsant and four of whom took such co-medication. Mean plasma carbamazepine apparent clearance was increased in pregnancy, but only by virtue of the increased clearance in the anticonvulsant co-medicated women. Alterations in the proportions of the carbamazepine dose cleared via the various excretion pathways studied were quantitatively minor, but there was evidence consistent with impaired conversion of carbamazepine-10,11-epoxide to carbamazepine-10,11-trans-diol during all pregnancies studied. Clearances of carbamazepine to the various excretory products studied were consistent with there being (i) increased urinary excretion of unmetabolised drug in pregnancy, possibly related to the increased glomerular filtration rate, (ii) increased formation of oxidative metabolites of the drug, particularly in women co-medicated with enzyme-inducing anticonvulsants, this effect being offset, in full (in non-co-medicated women) or in part (in co-medicated women) by (iii) inhibition of the epoxide-diol pathway in pregnancy, an inhibition to which folate intake may have contributed.

Expression of cytochrome P450 3A5 in Escherichia coli: effects of 5' modification, purification, spectral characterization, reconstitution conditions, and catalytic activities.

Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Kim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)-1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of approximately 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems [corrected].