The effect of aspirin on valproic acid metabolism.

Urinary valproic acid (VPA) and VPA metabolite profiles were determined before (day 1) and after (day 2) the administration of antipyretic doses of acetylsalicylic acid (ASA) to seven subjects with steady-state levels of VPA. Of the 13 metabolites assayed by GC/MS, levels of (E)-2-ene VPA and 3-keto VPA were significantly decreased on day 2, whereas those of the VPA conjugates (glucuronide) and 4-ene VPA were significantly increased. The beta-oxidation pathway consisting of (E)-2-ene VPA, 3-OH VPA, and 3-keto VPA was decreased from 24.5% +/- 10.3% of total metabolites excreted on day 1 to 8.3% +/- 4.2% on day 2, a decrease of 66% (P less than 0.05). VPA glucuronide content increased from 50.5% +/- 12.6% on day 1 to 65.5% +/- 14% of total excreted on day 2, an increase of 30% (P less than 0.05). The day 2/day 1 ratios of VPA glucuronide correlated significantly with the day 2/day 1 ratios of VPA mean free fraction (r = 0.9424; P = 0.005) in six of the seven subjects. Inhibition of VPA beta-oxidation by salicylate was sufficient to counterbalance the increased elimination of VPA as its conjugates and explains why total clearance of VPA after salicylate remains unchanged even though the free fraction of VPA is increased. Metabolic profiles indicate that salicylate likely inhibits VPA beta-oxidation by reducing valproyl-coenzyme A formation.

Interaction between valproic acid and aspirin in epileptic children: serum protein binding and metabolic effects.

In five of six epileptic children who were taking 18 to 49 mg/kg/day valproic acid (VPA), the steady-state serum free fractions of VPA rose from 12% to 43% when antipyretic doses of aspirin were also taken. Mean total VPA half-life (t1/2) rose from 10.4 +/- 2.7 to 12.9 +/- 1.8 hr and mean free VPA t1/2 rose from 6.7 +/- to 2.1 to 8.9 +2- 3.0 hr when salicylate was present in the serum. The in vitro albumin binding association constant (ka) for VPA was decreased by salicylate, but the in vivo ka value was not affected. The 12-hr (trough) concentrations of both free and total VPA were higher in the presence of serum salicylate in five of six patients. Renal excretion of unchanged VPA decreased in five of six patients, but the VPA carboxyl conjugate metabolite-excretion patterns were not consistently affected. Salicylate appeared to displace VPA from serum albumin in vivo, but the increased VPA t1/2 and changes in VPA elimination patterns suggest that serum salicylate also altered VPA metabolism.

Methadone maintenance: effect of urinary pH on renal clearance in chronic high and low doses.

The subjects were 12 male patients stabilized on methadone for many months or years. A comparison was made of the plasma levels and renal clearance of methadone between patients on "high" doses (80 to 110 mg/day) and those on "low" doses (15 to 40 mg/day). A general trend to higher renal clearance was seen in the "high" -dose group, but on more detailed examination there was a direct correlation only when the patients were categorized by urinary pH. At low pHs, there was nearly a 3-fold increase in renal clearance which was associated with a decreased major metabolite to methadone ratio. No evidence for a difference in rate of metabolism between the two groups was found nor were there differences in hepatic function. It was concluded that urinary pH was a major factor in renal clearance of methadone.

LC/MS analysis of hydroxylation products of salicylate as an indicator of in vivo oxidative stress.

Hydroxyl radical attack upon salicylate leads to the generation of 2,3-dihydroxybenzoic acid (2,3-DHBA) and therefore can be used to assess hydroxyl radical formation both in vitro and in vivo. Evidence is presented for a highly sensitive LC/MS assay for the quantification of 2,3-DHBA. Calibration curves showed linearity within the concentration range tested (0.5-6.5 pmol/microl rat plasma) with a coefficient of determination (r2) greater than 0.99. A detection limit of less than 0.25 pmol for 2,3-DHBA has been achieved. The intra-assay and inter-assay variability were determined to be 4.1% and 12.5%, respectively. This method was evaluated for the determination of drug-induced in vivo generation of oxidative stress by means of 1,1,1-trichloroethane (TCE) a compound that is a pseudosubstrate for cytochrome P450 and is known to induce oxygen reductase activity of this enzyme(s). TCE treated rats had a 6.4-fold increase in the mean maximal plasma 2,3-DHBA concentration as compared to the saline treated rats (p = .009). The developed LC/MS assay requires minimal sample preparation and provides a rapid and sensitive method for quantification of 2,3-DHBA as a specific indicator of hydroxyl radical generation.

Binding of a valproate metabolite to the trifunctional protein of fatty acid oxidation.

The anti-convulsant drug valproate causes hepatic failure in a small percentage of patients. We now report that the valproate metabolite 2,4-dien-valproate binds (IC50 = 42 microM) to the alpha-subunit of the trifunctional protein responsible for the second and third steps in the mitochondrial beta-oxidation of fatty acids. Binding of valproate itself, or of the metabolites 2-envalproate, 4-en-valproate or 3-hydroxy-4-en-valproate, is considerably weaker. We conclude that valproate-induced hepatotoxicity may be due in part to the reversible binding of the valproate metabolite 2,4-dien-valproate or its CoA ester to the alpha-subunit of the trifunctional protein with consequent inhibition of fatty acid oxidation.

Quantitative structure-anticonvulsant activity relationships of valproic acid, related carboxylic acids and tetrazoles.

Valproic acid and several structurally related carboxylic acids and tetrazole analogues have been shown to antagonize seizures induced by pentylenetetrazole in mice. To investigate the influence of the alkyl substituents on the anticonvulsant activity, the octanol-water partition coefficients and relative pKa values were determined. Within the series of active carboxylic acids, there was a good correlation between the anticonvulsant activity and the partition coefficient (r = 0.86). The influence of pKa on the anticonvulsant activity was small but of statistical significance. When the most active compound, 5-heptyltetrazole was added to the carboxylic acid series, a low correlation between the anticonvulsant activity and a linear combination of lipophilicity and pKa resulted. The effect of the polar moieties in alkyl-substituted anticonvulsant compounds was assessed by comparison of the regression equations with either an added pKa or dipole moment term to the term for lipophilicity. It appears that other factors, such as the nature of the alkyl substituent, influence the anticonvulsant activity. The inactivity of the cyclohexylmethyl-substituted compounds, cyclohexylacetic acid and 5-cyclohexylmethyltetrazole may be due to subtle steric effects at a critical step, either involving oxidative metabolism or an interaction at an active site.

Structure-activity relationships of unsaturated analogues of valproic acid.

The principal metabolite of valproic acid (VPA), 2-ene VPA, appears to share most of VPA's pharmacological and therapeutic properties while lacking its hepatotoxicity and teratogenicity, thus making it a useful lead compound for the development of safer antiepileptic drugs. Analogues of 2-ene VPA were evaluated for anticonvulsant activity in mice using the subcutaneous pentylenetetrazole test. Cyclooctylideneacetic acid exhibited a potency markedly exceeding that of VPA itself with only modest levels of sedation. Potency, as either ED50 or brain concentration, was highly correlated (r > 0.85) with volume and lipophilicity rather than with one of the shape parameters calculated by molecular modeling techniques, arguing against the existence of a specific receptor site. Instead, a role for the plasma membrane in mediating the anticonvulsant effect is suggested.

Characterization of thiol-conjugated metabolites of 2-propylpent-4-enoic acid (4-ene VPA), a toxic metabolite of valproic acid, by electrospray tandem mass spectrometry.

The hepatotoxicity of the anticonvulsant drug valproic acid (VPA) is most likely associated with the bioactivation of its metabolite 2-propylpent-4-enoic acid (4-ene VPA), which is known to induce hepatic microvesicular steatosis in rats. This paper presents an on-line liquid chromatographic/tandem mass spectrometric (LC/MS/MS) identification of new glutathione (GSH)-related conjugates of the reactive metabolites of 4-ene VPA. Bile samples collected from male Sprague-Dawley rats dosed intraperitoneally with 4-ene VPA or its [2H7]-analogue (100 mg kg-1) were injected on to an ODS column interfaced to a LC/MS/MS instrument using electrospray ionization. LC was developed such that no overlapping of peaks occurred among those metabolites which may potentially produce common fragment ions of interest. Subsequent comparison of LC retention times and MS/MS full fragment ion spectra generated for putative metabolites with that of authentic reference compounds made available by chemical synthesis confirmed the presence of the GSH, cysteinylglycine, cysteine and N-acetylcysteine (NAC) conjugates of 2-(2'-carboxypentanyl)oxirane (4,5-epoxy VPA) and (E)-2-propylpenta-2,4-dienoic acid ((E)-2,4-diene VPA), respectively. Quantitatively, the biliary thiol conjugates accounted for 5% of the dose. This observation is novel for 4-ene VPA metabolism in terms of the degradation of GSH conjugates to the corresponding mercapturic acids possibly occurring within the liver as opposed to an inter-organ process which involves the kidney. In addition, the GSH- and NAC-glucuronide di-conjugates of (E)-2,4-diene VPA were also identified as the biliary metabolites with the GSH-glucuronide di-conjugate being 10% of the corresponding mono-GSH conjugate. Taken together, these data clearly indicate that reactive metabolites of VPA can react with hepatic GSH via several different metabolic pathways and may subsequently produce depletion of GSH that leads to toxic consequences.

Gas chromatography/negative ion chemical ionization mass spectrometry and liquid chromatography/electrospray ionization tandem mass spectrometry quantitative profiling of N-acetylcysteine conjugates of valproic acid in urine: application in drug metabolism studies in humans.

We report a GC/NICI-MS assay and a LC/ESI-MS/MS assay for the analysis of N-acetylcysteine (NAC) conjugates of (E)-2,4-diene VPA (NAC I and NAC II) identified in humans. The assay also includes the analysis of the NAC conjugate of 4,5-epoxy VPA (NAC III), an identified metabolite in rats treated with 4-ene VPA for its use in metabolic studies in animals. The highly sensitive GC/MS assay was designed to monitor selectively the diagnostic and most abundant [M - 181](-) fragment anion of the di-PFB derivatives of NAC I, NAC II, and NAC IV, the internal standard (IS) and the PFB derivative of NAC III. The higher selectivity of LC/MS/MS methodology was the basis for an assay which could identify and quantitate the underivatized conjugates simultaneously using MRM of the diagnostic ions m/z 130 and 123 arising from the CID of their protonated molecular ions [MH](+). The GC/MS assay employed liquid-liquid extraction whereas the LC/MS/MS assay used a solid-phase extraction procedure. Linearity ranges of the calibration curves were 0.10-5.0microg ml(-1) by GC/MS and 0.10-1.0microg ml(-1) by LC/MS/MS for NAC I, NAC II and NAC III (r(2) = 0.999 or better). Both assays were validated for NAC I and NAC II and provided good inter- and intra-assay precision and accuracy for NAC I and NAC II. The LOQ by LC/MS/MS was 0.1microg ml(-1), representing 1 ng of NAC I and NAC II. The same LOQ (0.1microg ml(-1)) was observed by GC/MS and was equivalent to 100 pg of each metabolite. NAC III was detected at concentrations as low as 0.01 microg ml(-1) by both methods. The total urinary excretion of the NAC conjugates in four patients on VPA therapy was determined to be 0.004-0.088% of a VPA dose by GC/MS and 0.004-0. 109% of a VPA dose by LC/MS/MS.

Cytotoxicity of unsaturated metabolites of valproic acid and protection by vitamins C and E in glutathione-depleted rat hepatocytes.

Valproic acid (VPA) and the unsaturated metabolites, 2-ene VPA and (E)-2,(Z)-3'-diene VPA, demonstrated dose-dependent cytotoxicity in primary cultures of rat hepatocytes, as evaluated by lactate dehydrogenase (LDH) leakage. Cellular glutathione (GSH) was depleted by adding buthionine sulfoximine (BSO) to the culture medium. Induction of cytochrome P450 by pretreatment of rats with phenobarbital or pregnenolone-16 alpha-carbonitrile enhanced the cytotoxicity of parent VPA in BSO-treated hepatocytes. The cytotoxicity of 4-ene VPA was apparent in BSO-treated hepatocytes with detectable loss of cell viability at 1 microM of added 4-ene VPA. Depletion of cellular GSH also increased the cytotoxicities of 2-ene VPA and (E)-2,(Z)-3'-diene VPA. The cytotoxicity of 2-ene VPA was comparable to or higher than that of VPA, producing loss of viability at concentrations > or = 5 mM. Time-course evaluation of hepatocyte response to 4-ene VPA in the GSH-depleted state revealed a delayed cytotoxicity with no effect during the first 12 h of exposure followed by a pronounced toxicity between 12 and 14 h. Two major GSH conjugates of 4-ene VPA metabolites, namely 5-GS-4-hydroxy VPA lactone and 5-GS-3-ene VPA, were detected in 4-ene VPA treated hepatocytes. Consistent with this finding, a 50% decrease in cellular GSH levels was observed following 4-ene VPA treatment. Under similar conditions, neither toxicity nor the GSH conjugated metabolite were detected in cells treated with the alpha-fluorinated 4-ene VPA analogue (alpha-F-4-ene VPA). The antioxidants, vitamin C and vitamin E, demonstrated a cytoprotective effect against 4-ene VPA-induced injury in GSH-depleted hepatocytes. These results are in support of hepatocellular bioactivation of VPA via 4-ene VPA to highly reactive species, which are detoxified by GSH. The susceptibility of hepatocytes to VPA metabolite-mediated cytotoxicity depends on cellular GSH homeostasis.

Metabolism of tocainide in the rat.

The metabolism of tocainide, an oral antiarrhythmic agent, was studied in male Wistar rats following oral administration of 15 mg/kg of tocainide hydrochloride. Qualitative and quantitative identification of the metabolites in urine was carried out by GC-mass spectrometry and electron capture detector gas chromatography. About 15-20% of the dose administered was excreted as intact drug in the urine. An additional 20% of the dose was present as acid hydrolysable conjugates. Enzymatic hydrolysis (beta-glucuronidase) revealed half of the acid hydrolysable conjugates to be a glucuronide. The enzyme mediated hydrolysis was blocked by its specific inhibitor saccharo-1,4-lactone. N-acetyl tocainide, an oxidatively deaminated tocainide, an aldehyde adduct of tocainide, and a cyclic hydantoin derivative of tocainide were also identified as metabolites in the urine samples.

Stereoselective synthesis of the diunsaturated metabolites of valproic acid.

Two diene metabolites of valproic acid (VPA), (E)-2-n-propyl-2,4-pentadienoic acid and (E)-2-(1'-propenyl)-(E)-2-pentenoic acid, were stereoselectively synthesized. Mesylate elimination in the final step to produce the unsaturation at position 2 was stereospecific for the (E)-configuration in the case of 2. Gas chromatography-mass spectroscopy and NMR were used to confirm the configuration of each diene including the minor isomers, (Z)-2-n-propyl-2,4-pentadienoic acid and (Z)-2-(1'-propenyl)-(E)-2-pentenoic acid. Analysis of the dienes, as PFB derivatives by negative chemical ionization GC-MS from a serum extract of a patient on VPA therapy, revealed the presence of four peaks that in order of elution correspond to 9, 18, 1, and 2.

Use of hexadeuterated valproic acid and gas chromatography-mass spectrometry to determine the pharmacokinetics of valproic acid.

Di-[( 3,3,3-2H3]propyl)acetic acid, a hexadeuterated analogue of valproic acid, was synthesized and its pharmacokinetic properties compared with valproic acid. Concentrations of valproic acid and [2H]valproic acid in serum and saliva were determined by GC-MS using selected-ion monitoring. Saliva drug levels were measured with good precision down to 0.1 microgram/mL. Kinetic equivalence of valproic acid and [2H]valproic acid was demonstrated in a single-dose study in a human volunteer. An isotope effect was observed for omega-oxidation, but the difference in metabolism was not sufficient to make [2H]valproic acid biologically nonequivalent. The application of [2H]valproic acid to determine the kinetics of valproic acid under steady-state concentrations was evaluated in the same volunteer. The kinetic data obtained with [2H]valproic acid was consistent with previously reported values for valproic acid including kinetic differences observed between single-dose and steady-state experiments. Saliva levels of valproic acid were found to give a good correlation (r = 0.953) with total serum valproic acid under multiple-dose conditions. A concentration dependence was found for the ratio of saliva valproic acid to free valproic acid in serum, low ratios being observed at high serum concentrations of valproic acid.

GLC analysis of hydrocortisone, triamcinolone acetonide, and desonide in culture media of mouse and human dermal fibroblasts using flame-ionization detection.

A quantitative GLC assay with flame-ionization detection capable of detecting nanogram quantities of hydrocortisone, triamcinolone acetonide, and desonide in biological fluids was developed. This assay consisted of two extractions of the glucocorticoids from 1 N sodium chloride-treated cell culture media into ethyl acetate and subsequent double derivatization with methoxyamine and N-trimethylsilylimidazole. The chemical structures of methoxime-trimethylsilyl derivatives were confirmed by GLC-mass spectrometry. The methoxime-trimethylsilyl derivatives were stable for 24 hr. The applicability of this assay was demonstrated by studies of the glucocorticoid levels in L-929 and human dermal fibroblasts cell culture media over prolonged incubation (0--96 hr).

Free and total serum valproate concentrations: their relationship to seizure control, liver enzymes and plasma ammonia in children.

The relationships between total and free serum valproate (VPA) concentrations and seizure control, serum liver enzyme activity and plasma ammonia concentration were studied in 61 epileptic children. Enzyme-immunoassay (EMIT)R methods gave higher values of total VPA concentration than gas-liquid chromatography (GLC) methods. In over 80% of children with complete seizure control the ranges of total VPA concentration were 140-420 mumol/L with GLC methods and 210-560 mumol/L with EMIT methods. The range of free VPA concentrations in 78% of children with complete seizure control was 8.8-26.4 mumol/L. Increased liver enzyme activity was observed in 6 of the 61 children and raised plasma ammonia concentration in 11 of 50 children. Plasma ammonia concentration was related to total serum VPA but was not related to free serum VPA. Increased serum liver enzyme activity was related to VPA dose per kg but not to free or total serum VPA concentration. Thus free VPA concentrations do not appear to be more useful than total VPA concentrations in predicting seizure control and do not correlate with liver enzyme activity or plasma ammonia concentration.

In vitro investigation of the hepatic extraction of RSD1070, a novel antiarrhythmic compound.

PURPOSE: The hepatic extraction of a novel antiarrhythmic, RSD1070, was investigated to test the hypothesis that the poor bioavailability observed in rats is due to high hepatic metabolism. METHODS: The pharmacokinetics of RSD1070 was examined in rats (n=8) and its metabolism was investigated using pooled rat hepatic microsomes. The free fraction in plasma and microsomal matrices was determined by equilibrium dialysis. Hepatic extraction was predicted by scaling-up of the microsomal kinetic data using the well-stirred liver model. RESULTS: RSD1070 demonstrated tri-exponential decay following single iv bolus administration of a dose of 12 mg/kg. RSD1070 exhibited a rapid elimination, t1/2 of 25 +/- 8 min and a CL(tot) of 71 +/- 9 mL/min/kg. Renal clearance based on 24 h urinary recovery was determined to be insignificant (<< 1% of CL(tot)). A Michaelis-Menten model described the elimination of RSD1070 with a K(m) of 0.45 microg/mL and Vmax of 2.81 microg/min/mg microsomal protein. Taking the V(max)/K(m) ratio (CL(int)) as the basis for scaling, the data from the microsomal kinetic studies (75 mL/min/kg) closely approximated the apparent CL(tot). In the scale-up of the in vitro CL(int), plasma free fraction (1.5%) and microsomal free fraction (15%) were determined and incorporated into the well-stirred liver model. CONCLUSION: RSD1070 is a high hepatic extraction compound (E = 0.94) with a predicted CL(h) value that accounted for the CL(tot) observed in rats.

Monoamine oxidase inhibitors in South American hallucinogenic plants Part 2: Constituents of orally-active Myristicaceous hallucinogens.

Alkaloid constituents in Myristicaceous bark and leaf samples and in purportedly hallucinogenic preparations derived from Myristicaceous sources were qualitatively and quantitatively analyzed using TLC, GC, alkaloid precipitation tests and GC/MS. Fourteen of the 27 bark and leaf samples analyzed contained detectable amounts of alkaloids. The major bases were N,N-dimethyltryptamine (DMT) and/or 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT); much smaller amounts of tryptamine and/or N-methyl-tryptamine (NMT) were also usually present. beta-Carbolines were not detected in the bark or leaf samples. Considerable variation in alkaloid profiles was found, extending to different collections of the same species. Fourteen of the 20 Virola samples contained alkaloids; none of the 6 Iryanthera species had detectable alkaloids. Osteophloem platyspermum contained an indolic base, identified as N-methyl-tryptophan methyl ester. Seven samples of an orally-ingested drug made from Virola spp. were analyzed. All except one contained substantial amounts of tryptamines; the types and proportions of tryptamines present varied greatly between samples. Samples of Yanomama snuff including various admixtures were analyzed and all components but one contained tryptamines. The drug samples having the highest concentrations of alkaloids contained 15-20 mg/g dry wt while the Myristicaceous bark and leaf samples had much lower concentrations ranging from 0.04 to 0.25 mg/g dry wt. beta-Carbolines were detected as trace constituents in only two of the Myristicaceous drug samples. Four Myristicaceous paste samples were bioassayed in self-experiments. Two of the samples were devoid of detectable hallucinogenic or physiological activity, while some degree of oral activity was detected in two other samples. The activity of a number of tryptamine derivatives as monoamine oxidase inhibitors (MAOI) was investigated using an in vitro enzyme assay. Activity was measured using single compounds and mixtures of compounds and the results were compared to the activity of samples of orally-ingested Myristicaceous pastes. Tryptamine derivatives had significantly less MAOI activity than the activity of beta-carboline derivatives measured in a previous study. Some structural correlations for MAOI activity were found for the tryptamine derivatives. Samples of orally-ingested Myristicaceous pastes were assayed for MAOI activity. The inhibition elicited by the paste samples was closely matched by mixtures of tryptamine standards having comparable proportions and concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of valproic acid on organic acid metabolism in children: a metabolic profiling study.

Young children are at increased risk for valproic acid (VPA) hepatotoxicity. Urinary organic acid profiles, as a surrogate of mitochondrial function, were obtained in children 1.9 to 17.3 years of age (n = 52) who were undergoing treatment with VPA for seizure disorders. Age-matched patients receiving treatment with carbamazepine (CBZ; n = 50) and healthy children not undergoing treatment (n = 22) served as controls. Age-related changes in organic acid profiles were observed in all three groups. Although the untreated and CBZ control groups were indistinguishable from each other with respect to the principal-component analysis (PCA) score plots of the subjects, a distinct boundary was apparent between the VPA and each of the control groups. Interindividual variability was observed in the VPA-induced alterations in endogenous pathways corresponding to branched-chain amino acid metabolism and oxidative stress. The data suggest that more detailed metabolomic analysis may provide novel insights into biological mechanisms and predictive biomarkers for children at highest risk for serious toxicity.

Analysis of methadone and metabolites in biological fluids with gas chromatography--mass spectrometry.

The analysis of methadone and its metabolites in biological fluids by gas chromatography--mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 +/- 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography--mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.