Michael A. Zemaitis

Orphan nuclear receptor-mediated xenobiotic regulation in drug metabolism

Abstract The regulation of drug-metabolizing enzymes and transporters has an important role in drug metabolism and many human diseases. The genes that encode these enzymes and transporters are inducible by numerous xenobiotics and endobiotics and the inducibility shows clear species specificity. In the past 4–5 years, orphan nuclear receptors such as PXR and CAR have been established as species-specific xeno-sensors that regulate the expression of many detoxifying enzymes and transporters. Their identification represents a major step forward in understanding the pharmacological and genetic control of the expression of drug-metabolizing enzymes and the implication of this regulation in drug metabolism, drug–drug interactions, and human diseases.

In vivo and in vitro effects of thiuram disulfides and dithiocarbamates on hepatic microsomal drug metabolism in the rat.

Abstract The effects of tetramethylthiuram disulfide (TMTDS) and dimethyldithiocarbamate (DMDTC) on hepatic microsomal drug metabolism were studied after in vivo administration to male rats (1 g/kg, po) and after in vitro addition of the compounds to control microsomal suspensions. Results were compared to the effects of the known inhibitor of drug metabolism, disulfiram (DS, tetraethylthiuram disulfide), its reduced metabolite diethyldithiocarbamate (DDTC), and a common metabolite of all four compounds, carbon disulfide. Twenty-four hours after administration of the disulfides (TMTDS and DS) impairment of microsomal aniline hydroxylase and carboxylesterase activities was observed, while cytochrome P -450 and ethylmorphine N -demethylase activity were unchanged. The reduced thiols (DMDTC and DDTC) caused significant decreases in microsomal cytochrome P -450 and impaired all three microsomal enzymes. In vitro addition of all four compounds to control microsomes at a final concentration of 1 m m impaired aniline hydroxylase and carboxylesterase activity. However, only in vitro addition of TMTDS and DS significantly decreased ethylmorphine N -demethylase activity. This effect may be due to the fact that TMTDS and DS bind to cytochrome P -450 producing a type I spectral change and may, therefore, compete with the type I compound, ethylmorphine, for binding sites on cytochrome P -450. Impairment of aniline hydroxylase activity is the most sensitive indicator of an inhibitory effect of all four compounds on microsomal drug metabolism; this action is not dependent on decreases in cytochrome P -450. In vivo impairment of ethylmorphine N -demethylation by DMDTC and DDTC is related to decreases in microsomal cytochrome P -450 produced by these compounds, which may be due, in part, to their decomposition to CS 2 in the gut. The data indicate that industrial or agricultural exposure to compounds such as TMTDS and DMDTC may impair hepatic metabolism, and thereby enhance pharmacological activity of drugs taken by exposed individuals.

The effects of ethylene oxide (EO) exposure on tissue glutathione levels in rats and mice.

Male Fischer 344 rats and male Swiss-Webster mice were exposed to different atmospheric concentrations of ethylene oxide (EO) for 4 hours. In mice sacrificed immediately after exposure to 100, 450 or 900 ppm EO, there was a concentration related decrease in the GSH levels of all tissues examined. Similar findings were obtained in rats immediately after exposure to 100, 600 or 1200 ppm EO except that blood GSH levels were not affected at any exposure concentration. In both species, lung and liver GSH levels were depressed at all exposure concentrations. Twenty-four hours after exposure to 1200 ppm EO, the GSH concentrations of rat bone marrow and testis had not returned to control levels. Only blood GSH levels remained depressed in mice 48 hours after exposure to 900 ppm EO. The results indicate a marked species difference between rats and mice regarding the effects of EO exposure on blood GSH levels which may have important toxicological implications.

Simultaneous determination of p-aminohippuric acid, acetyl-p-aminohippuric acid and iothalamate in human plasma and urine by high-performance liquid chromatography

A sensitive and specific high-performance liquid chromatographic assay was developed for the simultaneous determination of p-aminohippuric acid (PAH), acetyl-p-aminohippuric acid (aPAH), and iothalamate in human plasma and urine. Plasma samples were prepared by protein precipitation with acetonitrile followed by evaporation, reconstitution in mobile phase, and injection onto a C18 reversed-phase column. Urine samples were diluted with 3 volumes of mobile phase prior to injection. Column effluent was monitored by UV detection at 254 nm. The lower limits of quantification in plasma were 0.5 mg/l for PAH and aPAH, and 1.0 mg/l for iothalamate. The within-day and between-day coefficients of variation in plasma and urine were < or =7.8% for all analytes. This method is well suited for renal function studies using iothalamate and PAH, whether administered as a bolus dose or by continuous infusion, to measure glomerular filtration rate and effective renal plasma flow, respectively.

New high-performance liquid chromatographic method for the determination of alprazolam and its metabolites in serum: instability of 4-hydroxyalprazolam

A high-performance liquid chromatographic method was developed for the determination of alprazolam (ALP) and its active metabolites, alpha-hydroxyalprazolam (AOH) and 4-hydroxyalprazolam (4OH) in human serum. During assay development, the instability of 4OH was revealed. Factors affecting stability of 4OH were then investigated. In this report, the assay methodology for the determination of ALP and AOH, the instability of 4OH, subsequent interference of 4OH breakdown products with AOH quantification, and factors affecting 4OH stability are described. The clinical significance of our findings are reported.

Determination of 20-hydroxyeicosatetraenoic acid in microsomal incubates using high-performance liquid chromatography–mass spectrometry (HPLC–MS)

20-HETE is a potent, vasoconstrictive arachidonic acid metabolite with a limited number of published methods for quantitative assessment of microsomal formation rate. The purpose of this study was to evaluate the utility of HPLC-MS (negative ESI) for quantitation of rat microsomal 20-HETE enzyme kinetics. Calibration curves were linear over 0.75-16 ng on-column (r(2)>0.996). The intra- and inter-assay precision and accuracy were <15%. Microsomal 20-HETE revealed saturable (100 microM) kinetics (brain K(m) and V(max): 39.9+/-6.0 microM and 8.7+/-0.6 pM/min per mg; liver K(m) and V(max): 23.5+/-3.2 microM and 775.5+/-39.8 pmol/min per mg; kidney K(m) and V(max): 47.6+/-8.5 microM and 1933+/-151 pM/min per mg). This paper demonstrates HPLC-MS as an efficient method for quantitating 20-HETE enzyme kinetics in microsomes from rat tissues.

Determination of plasma mexiletine levels with gas chromatography—mass spectrometry and selected-ion monitoring

Mexiletine, 1-(2,6-dimethylphenoxy)-2-aminopropane (Mexitil), is an orally effective agent useful in the treatment of serious ventricular arrhythmias. This paper describes a gas chromatographic-mass spectrophotometric assay with selected-ion monitoring for the measurement of plasma or serum mexiletine levels. The drug and internal standard (p-chlorophenylalanine methyl ester) were extracted from plasma into ethyl acetate-hexane-methanol (60:40:1, v/v). After separation and evaporation of the organic phase, the drug and internal standard were derivatized to their pentafluoropropyl derivatives prior to analysis. The reproducibility of the daily standard curve yielded mean inter- and intra-day coefficients of variability from 0.7 to 11.0%. The coefficients of variability for control plasma samples (0.5 and 1.0 micrograms/ml) ranged from 2.6 to 5.0% and the accuracy of the assay was 106 +/- 6 and 100 +/- 5% for the low and high level controls respectively. The limit of quantitation for the assay was 0.1 micrograms/ml. No interfering peaks were detected in any patient samples. This method can be used as a primary analytical method to measure mexiletine plasma levels or can serve as a convenient back-up method to HPLC procedures when contaminating peaks coelute with mexiletine.

Simplified procedure for measurement of serum dehydroepiandrosterone and its sulfate with gas chromatography–ion trap mass spectrometry and selected reaction monitoring

Dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulfate (DHEAS) are endogenous steroids that have recently been widely publicized as potential treatments for many disorders. This paper describes a gas chromatographic-ion trap mass spectrophotometric assay with selected reaction monitoring for measurement of DHEA and DHEAS levels. The hormones and internal standard (5-androsten-3beta-ol-16-one methyl ester) are extracted from serum with Oasis solid-phase extraction tubes. The extracted steroids are dissolved in methanol and injected into a Finnigan GCQ ion trap mass spectrometer. In the selected reaction mode, both DHEA and DHEAS can be identified and quantified in a single injection. No derivatization or expensive deuterated internal standards are required.

A comparison of the effects of cyclosporine (CsA) on hepatic microsomal drug metabolism in three different strains of rat

1. Male albino Wistar (W), Sprague-Dawley (SD) and Fischer-344 (F344) rats, Rattus norvegicus, were dosed orally once daily with cyclosporine (CsA), 25 mg/kg/day, for 14 days. Control and treated rats were then sacrificed and selected assays were performed to measure differences in hepatic microsomal enzyme activity including: protein (PROT), cytochrome P-450 (CYT-P450) and cytochrome b5 (CYT-b5) levels, NADPH-cytochrome c reductase (CYT-c) activity, ethylmorphine N-demethylase (ETM) and aniline hydroxylase (ANL) activities. 2. Two-way analysis of variance (ANOVA) indicated that the effect of treatment alone showed significant strain-related differences in all parameters examined except CYT-b5 levels, while the effect of strain alone indicated significant differences in PROT, CYT-B5, CYT-c, and ANL. 3. The interaction component of the two-way ANOVA (treatment x strain) indicated strain-related differences in substrate metabolism only (ETM and ANL). 4. F344 rats were found to be significantly different from either Sprague-Dawley or Wistar rats in terms of the effect of CsA on ETM and ANL metabolism.

Barbiturate N-demethylase activity in isolated rat hepatocytes

The isolated hepatocyte preparation has become an increasingly popular system for studying xenobiotic metabolism, and there is a need for methods of determining metabolic activity. Described here is a simple and sensitive radiometric assay for barbiturate N-demethylase activity in the isolated hepatocyte suspension. The demethylation of 1,3-dimethylphenobarbital is determined by measuring the formation of its only significant metabolite, N-methylphenobarbital. The reaction is a typical P-450-mediated dealkylation.