Human Liver Microsomal Incubations Research Articles

Roles of human hepatic cytochrome P450s 2C9 and 3A4 in the metabolic activation of diclofenac.

Recently, it was shown that diclofenac was metabolized in rats to reactive benzoquinone imines via cytochrome P450-catalyzed oxidation. These metabolites also were detected in human hepatocyte cultures in the form of glutathione (GSH) adducts. This report describes the results of further studies aimed at characterizing the human hepatic P450-mediated bioactivation of diclofenac. The reactive metabolites formed in vitro were trapped by GSH and analyzed by LC/MS/MS. Thus, three GSH adducts, namely, 5-hydroxy-4-(glutathion-S-yl)diclofenac (M1), 4'-hydroxy-3'-(glutathion-S-yl)diclofenac (M2), and 5-hydroxy-6-(glutathion-S-yl)diclofenac (M3), were identified in incubations of diclofenac with human liver microsomes in the presence of NADPH and GSH. The formation of the adducts was taken to reflect the intermediacy of the corresponding putative benzoquinone imines. While M2 was the dominant metabolite over a substrate concentration range of 10-50 microM, M1 and M3 became equally important products at >/=100 microM diclofenac. The formation of M2 was inhibited by sulfaphenazole or an anti-P450 2C9 antibody (5-10% of control values). The formation of M1 and M3 was inhibited by troleandomycin, ketoconazole, or an anti-P450 3A4 antibody (30-50% of control values). In studies in which recombinant P450 isoforms were used, M2 was generated only by P450 2C9-catalyzed reaction, while M1 and M3 were produced by P450 3A4-catalyzed reaction. Good correlations were established between the extent of formation of M2 and P450 2C9 activities (r = 0.93, n = 10) and between the extent of formation of M1 and M3 and P450 3A4 activities (r = 0.98, n = 10) in human liver microsomal incubations. Taken together, the data suggest that the biotransformation of diclofenac to M2 is P450 2C9-dependent, whereas metabolism of the drug to M1 and M3 involves mainly P450 3A4. Although P450s 2C9 and 3A4 both catalyze the bioactivation of diclofenac, P450 2C9 is capable of producing the benzoquinone imine intermediate at lower drug concentrations which may be more clinically relevant.

Quantification of flunitrazepam's oxidative metabolites, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in hepatic microsomal incubations by high-performance liquid chromatography

A high-performance liquid chromatographic assay for the quantification of the oxidative metabolites of flunitrazepam, 3-hydroxyflunitrazepam and desmethylflunitrazepam, in human liver microsomal incubations was developed. Both metabolites were quantifiable in a single assay following a solvent extraction and reversed-phase high-performance liquid chromatography with UV detection. Standard curve concentrations for both metabolites ranged from 0.2 to 10 μ M. Assay performance was determined using quality control samples and the intra- and inter-day accuracy and precision as determined by the coefficient of variations which were less than 15% (0.5–6 μ M) for both metabolites. This method provides good precision and accuracy for use in kinetic studies of the oxidative metabolism of flunitrazepam in human liver microsomes.

The quantitative determination of cilostazol and its four metabolites in human liver microsomal incubation mixtures by high-performance liquid chromatography

A high-performance liquid chromatography-ultraviolet (HPLC-UV) method for the quantitation of cilostazol and four of its principal metabolites (i.e. OPC-13015, OPC-13213, OPC-13217 and OPC-13326) in human liver microsomal solutions was developed and validated. Cilostazol, its metabolites, and the internal standard (OPC-3930), were analyzed by protein precipitation followed by reverse-phase HPLC separation on a TSK-Gel ODS-80TM (150×4.6 mm, 5 μm) column and a Cosmil C-18 column (150×4.6 mm, 5 μm) in tandem and UV detection at 254 nm. An 80 min gradient elution of mobile phase acetonitrile in acetate buffer (pH=6.50) was used to obtain quality chromatography and peak resolution. All the analytes were separated from each other, with the resolution being 2.43–17.59. The components of liver microsomal incubation mixture and five metabolic inhibitor probes (quinidine sulfate, diethyl dithiocarbamate (DEDTC), omeprazole, ketoconazole and furafylline) did not interfere with this analytical method. The LOQ was 1000 ng ml −1 for cilostazol and 100 ng ml −1 for each of the metabolites. This method has been validated for linear ranges of 100–4000 ng ml −1 for OPC-13213, OPC-13217 and OPC-13326; 100–2000 ng ml −1 for OPC-13015; and 1000–20 000 ng ml −1 for cilostazol. The percent relative recovery of this method was established to be 81.2–101.0% for analytes, with the precision (% coefficient of variation (CV)) being 2.8–7.7%. The autosampler stability of the analytes was evaluated and it was found that all analytes were stable at room temperature for a period of at least 17 h. This assay has been shown to be precise, accurate and reproducible.

Labelling Studies for Structure Elucidation of a New Hydroxymetabolite of Tramadol

Tramadol, racemic 1-(3-methoxyphenyl)-2-(dimethylaminomethyl)cyclohexane-1-ol, is an effective analgesic drug. Metabolites of tramadol described so far originate from O- and N-demethylation and are excreted in urine directly or after conjugation. A further metabolite was found in human liver microsome incubations and in the urine of volunteers after ingestion of tramadol. To elucidate the structure of the new metabolite, seven deuterated isotopomers of tramadol have been synthesized and ingested by volunteers. The mass spectra of the metabolites derived showed (i) that it was a hydroxy metabolite, (ii) that the hydroxy group was not located on the aromatic ring, the side chain, or the positions 2 and 6 of the cyclohexane ring, (iii) that the hydroxy-group was introduced to one of the the positions 3, 4 or 5 of the cyclohexane ring. The hydroxy metabolite was formed preferentially from the (-)-enantiomer, (1S,2S)-tramadol.

Determination of the dihydrocodeine metabolites, dihydromorphine and nordihydrocodeine, in hepatic microsomal incubations by high-performance liquid chromatography

A high-performance liquid chromatography assay for the oxidative metabolites of dihydrocodeine, nordihydrocodeine and dihydromorphine, formed in human liver microsomal incubations, is described. A simple solvent extraction followed by reversed-phase high-performance liquid chromatography with UV detection allows quantification of both metabolites in a single assay. Standard curve concentration ranges for dihydromorphine and nordihydrocodeine were 0.05–5 and 0.2–20 μM, respectively. Assay performance was assessed by intra- and inter-day accuracy and precision of quality control (QC) samples. The difference between the calculated and the actual concentration and the relative standard deviation were less than 15% at low QC concentrations and less than 10% at medium and high QC concentrations for both analytes. The method provides good precision, accuracy and sensitivity for use in kinetic studies of the oxidative metabolism of dihydrocodeine in human liver microsomes.

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.

In vitro metabolism of dexamethasone (DEX) in human liver and kidney: The involvement of CYP3a4 and CYP17 (17,20 LYASE) and molecular modelling studies

Dexamethasone (DEX) has previously been shown to be extensively metabolised to 6-hydroxylated and side-chain cleaved metabolites in human liver in vitro. CYP3A4 is responsible for 6α- and 6β-hydroxylation of DEX and CYP17 is thought to mediate side-chain cleavage to generate 9αfluoro-androsta-1,4-diene'11β'hydroxy'16α-methyl-3,17-dione (9αF-A). Although 9αF-A has not previously been isolated as a metabolite in its unhydroxylated form in human liver incubations, it is formed as an intermediate metabolite, which is subsequently rapidly hydroxylated to OH-9αF-A. A main part of this study has been to conclusively show that DEX undergoes extensive side-chain cleavage to form 9αF-A in human kidney fractions, which is in contrast to profiles obtained for DEX metabolism in parallel human liver microsomal incubations where 6-hydroxylation is the predominant pathway. Furthermore, molecular models of CYP3A4 and CYP17 (17,20 lyase) have been used to model the enzyme fits of DEX. From these modelling studies it has been shown that DEX complements both putative enzyme active sites in orientations likely to lead to the formation of the metabolites identified in vitro. We have also been able to rationalise the preferential formation of the 6βOH-DEX isomer.

Identification of three cytochrome P450 isozymes involved in N-demethylation of citalopram enantiomers in human liver microsomes.

Using in vitro techniques, the present study demonstrates that CYP2D6, and 3A4 are involved in N-demethylation of citalopram (CIT) enantiomers. Human liver microsome incubations performed with specific inhibitors of these three CYP isozymes have shown up to 60% inhibition of demethylcitalopram production. cDNA expressed human cytochrome P-450 3A4, 2C19 and 2D6 isozymes, but not CYP1A2, were identified to be involved in N-demethylation of CIT enantiomers. Kinetics using cDNA expressed CYP2C19 and CYP3A4 show K(m) values in the same range: 198 microM, 211 microM for CYP2C19 and 169 microM, 163 microM for CYP3A4 for S- and R-CIT demethylation, respectively. In contrast, kinetics using cDNA expressed CYP 2D6 show a K(m) of 18 microM and 22 microM for S- and R-CIT demethylation, respectively. Nevertheless, kinetics using cDNA expressed CYP2C19 and 3A4 have a range of Vmax values ten times higher than that of CYP2D6. For this reason, intrinsic clearance values (Vmax/K(m)) for S- and R-CIT were within a small range for these three isozymes: 0.25 to 0.39 microliter h-1 x pmol-1 of CYP. CYP2D6 has an opposite stereoselectivity in the biotransformation of CIT enantiomers than CYP2C19 and 3A4; the S/R ratios of the intrinsic clearance were 0.71, 1.57 and 1.37, respectively. Taking into account that CYP isozymes are expressed at various levels, CYP2D6, which is expressed at lower levels than CYP2C19 and CYP3A4, plays a minor role in the biotransformation of CIT enantiomers. These results confirm that the use of cDNA expressed CYP isozymes is a potent tool for the measurement of kinetic constants and help to predict clearance modifications of CIT enantiomers, especially in poor metabolizers of mephenytoin (with a CYP2C19 deficiency) or patients comedicated with potent CYP2C19 or 3A4 inhibitor(s). For instance, fluvoxamine (100 microM) inhibits CIT N-demethylation by 64% in microsomes.

Oxidation of Butadiene Monoxide tomeso-and (±)-Diepoxybutane by cDNA-Expressed Human Cytochrome P450s and by Mouse, Rat, and Human Liver Microsomes: Evidence for Preferential Hydration ofmeso-Diepoxybutane in Rat and Human Liver Microsomes

Butadiene monoxide (BM) can be oxidized by cDNA-expressed human cytochrome P450 enzymes and by mouse, rat, and human liver microsomes to yieldmeso- and (±)-diepoxybutane (DEB). The DEB diastereomers were separated by gas chromatography (GC) and characterized by GC–mass spectrometry. Of eight cDNA-expressed human P450 enzymes examined, only incubations with 2E1, 2A6, and 2C9 led to DEB detection; total DEB formation rate by 2E1 was nearly four- and sixfold higher than the rates observed with 2C9 and 2A6, respectively, while incubations with 1A1, 1A2, 2B6, 2D6, or 3A4 did not lead to DEB detection.meso-DEB was detected preferentially in 2A6 and 2E1 incubations (ratios nearly 2:1), whereas incubations with 2C9 led to detection of both isomers in approximately equal amounts. Incubations of mouse, rat, or human liver microsomes with BM and chlorzoxazone provided further evidence for the involvement of 2E1 in BM oxidation;meso-DEB was the major DEB form detected at high BM concentrations. TheVmax/Kmratio for total DEB formation obtained with mouse liver microsomes was higher than the ratios obtained with rat and human liver microsomes. However, DEB hydrolysis in human liver microsomes was greater than that observed with rat liver microsomes, while no hydrolysis was detectable in mouse liver microsomes. When the DEB diastereomers were added in a 1:1 ratio to human or rat liver microsomes, selective hydrolysis ofmeso-DEB was observed. These results, which characterize new metabolic reactions for BM and DEB, may be of toxicological importance, as significant species differences reflecting both the stereoselective oxidation of BM by P450 enzymes and the subsequent preferentialmeso-DEB hydration by epoxide hydrolase have been demonstrated.

Identification of CYP4A11 as the Major Lauric Acid ω-Hydroxylase in Human Liver Microsomes

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the ω- and ω-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate ω-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing ω-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate ω-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (Mr= 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the humanCYP4A11cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate ω-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the ω-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate ω-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a loneKmof 48.9 μMwith aVMAXof 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate ω-hydroxylase activity while ω-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r= 0.89;P< 0.001) was found between immunochemically determined CYP4A11 content and laurate ω-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate ω-hydroxylating enzyme expressed in human liver.

Cytochromes P450, 1A2, and 2C9 are responsible for the human hepatic O-demethylation of R- and S-naproxen

A preliminary report implicated cytochrome P450 (CYP) 2C9 in the human liver microsomal O-demethylation of S-naproxen, suggesting that this pathway may be suitable for investigation of human hepatic CYP2C9 in vitro. Kinetic and inhibitor studies with human liver microsomes and confirmatory investigations with cDNA-expressed enzymes were undertaken here to define the role of CYP2C9 and other isoforms in the O-demethylation of R- and S-naproxen. All studies utilised a newly developed sensitive and specific HPLC assay that measured the respective O-desmethyl metabolites of R- and S-naproxen in incubations of human liver microsomes and in COS cell lysates. Microsomal R- and S-naproxen O-demethylation kinetics followed Michaelis-Menten kinetics, with respective mean apparent K m values of 123 μM and 143 μM. Sulfaphenazole, a specific inhibitor of CYP2C9, reduced the microsomal O-demethylation of R-and S-naproxen by 43% and 47%, respectively, and the CYP1A2 inhibitor furafylline decreased R- and S-naproxen O-demethylation by 38% and 28%, respectively. R,S-Mephenytoin was a weak inhibitor of R- and S-naproxen O-demethylation, but other CYP isoform specific inhibitors (e.g., coumarin, diethyldithiocarbamate, quinidine, troleandomycin) had little or no effect on these reactions. cDNA-expressed CYP2C9 and CYP1A2 were both shown to O-demethylate R- and S-naproxen. Apparent K m values (92–156 μM) for the reactions catalysed by the recombinant enzymes were similar to those observed for human liver microsomal R- and S-naproxen O-demethylation. The data demonstrate that CYP2C9 and CYP1A2 together account for the majority of human liver R- and S-naproxen O-demethylation, precluding the use of either R- or S-naproxen as a CYP isoform-specific substrate in vitro and in vivo.

High-performance liquid chromatographic assay for human liver microsomal omeprazole metabolism

Assays for the measurement of omeprazole metabolites in plasma and urine have been reported, but when applied to the determination of omeprazole metabolites formed by human liver microsomal incubations there were obvious limitations in sensitivity. The present high-performance liquid chromatographic (HPLC) assay, which comprises extraction, evaporation and reconstitution, is several-fold more sensitive with a limit of detection of approximately 2 pmol (2 n M in incubate) for omeprazole sulphone and 25 pmol (25 n M in incubate) for hydroxyomeprazole. Extraction efficiency is essentially quantitative and is highly reproducible (coefficient of variation = 2.1% for both metabolites). The assay is linear over a wide range of concentrations and the formation of the metabolites is linear with respect to both time (to 15 min) and protein concentration (to 1.5 mg/ml). Two minor metabolites, one of which was identified tentatively as 5-O-desmethylomeprazole, were also formed by human liver microsomes and could be determined by this method. Preliminary studies of the formation of omeprazole sulphone and hydroxyomeprazole showed that the formation kinetics in human liver microsomes were biphasic for both metabolites, suggesting that at least two different cytochrome P450 isoforms are involved in their formation.

Metabolism of gestodene in human liver cytosol and microsomes in vitro

The metabolism of the progesteron gestodene has been studied in human liver cytosol and microsomal incubations. Extraction with diethyl ether was followed by radiometric HPLC analysis. Metabolites were identified by co-chromatography with authentic standards and mass spectrometry (electron impact and chemical ionization). All the cytosolic incubations (n = 4 livers) produced dihydrogestodene as the major metabolite, with lesser amounts of a tetrahydro derivative. It was not possible to separate the 5α- and 5β- isomers of dihydrogestodene on the chromatographic system used. Values of Km and Vmax for the Δ4 reductase were determined. Androstenedione (Ki = 2.85 ± 1.5 μM; n = 4) and cortisol (Ki = 24.1 ± 8.9 μM; n = 4) both inhibited the Δ4-reductase. In contrast desogestrel showed virtually no inhibition at concentrations up to 200 μM. The major microsomal metabolite of gestodene was a hydroxylated derivative although mass spectral analysis was unable to determine the position of insertion of the hydroxyl moiety. The hydroxylation of gestodene (1 μM) was markedly inhibited by ketoconazole (IC50 < 0.1 μM), and also by cyclosporin. This suggests that the cytochrome P450 isozyme CYP3A4 is important in gestodene metabolism. Theophylline and tolbutamide (substrates of CYPIA and CYP2C, respectively) did not affect gestodene metabolism at concentrations up to 100 μM. In conclusion, the major biotransformation of gestodene (A-ring reduction) occurs in the cytosolic fraction of human liver. Microsomal hydroxylation appears to be catalysed by CYP3A4.

Oxidation of cyclopenta[cd]pyrene by human and mouse liver microsomes and selected cytochrome P450 enzymes.

The metabolism of the environmental pollutant and suspected human carcinogen, cyclopenta[cd]pyrene (CPP), was investigated. Human liver microsomes from three individuals were examined, as well as CD-1 mouse liver microsomes. Five new metabolites recently identified in our lab, 4-hydroxy-3,4-dihydroCPP, 3,4-dihydroCPP-cis-3,4-diol, 4-oxo-3,4-dihydroCPP, 3,4,9,10-tetrahydroCPP-trans-3,4-trans-9,10-tetrol, and trans-3,4-dihydroCPP-3, 4,x-triols, as well as the known major metabolite, 3,4-dihydroCPP-trans-3,4-diol, were all observed from the incubations of human liver microsomes and CPP. Even though all three human samples were capable of producing all the metabolites identified from the mouse liver microsomal incubations of CPP, the quantity of each metabolite varied among the microsomal samples. In an attempt to explain the variation among human liver samples, the microsomes derived from genetically engineered cells containing specific cytochrome P450 isozyme cDNAs were employed. It was found that the 3,4-cyclopenta double bond can be oxidized by the cytochrome P450 enzymes 1A1, 1A2, and 3A4. The 9,10 K-region double bond was not efficiently oxidized by cytochrome P450 1A1, but by P450 1A2 either from CPP or from the t-3,4-dihydrodiol. The lack of catalytic activity of 3A4 toward the t-3,4-dihydrodiol, despite its high activity toward CPP oxidation to tetrahydrotetrols, suggests the possibility of two dihydrodiol epoxides, 3,4-dihydrodiol 9,10-epoxide and 9,10-dihydrodiol 3,4-epoxide, of CPP.

Suicidal inactivation of human cytochrome P-450 by carbon tetrachloride and halothane in vitro.

A significant loss of human cytochrome P-450 was observed during the anaerobic incubation of NADPH-reduced human liver microsomes obtained from surgical samples, in presence of carbon tetrachloride or halothane. In order to prevent any interference in the classical spectrum of cytochrome P-450 with CO, the method of Johannesen & DePierre (1978) was modified to obtain cytochrome P-450 determination. The enzyme inactivation reaction showed pseudo-first order kinetics and was accompanied by destruction of the haem tetrapyrrolic structure, as indicated by a significant loss of its porphyrin fluorescence. Values of about 200 and 700 were calculated for the partition ratio between metabolic turnover of the substrate and enzyme inactivation during reductive incubation of one of these microsomal preparations with limiting concentrations of CCl4 and halothane, respectively. The results indicate that human liver cytochrome P-450 can be inactivated reductively in vitro by CCl4 and halothane reactive metabolites and suggest that a suicide type of mechanism, similar to that which was recently demonstrated to occur, for both substrates, with rat liver microsomes (Manno et al. 1988a & 1991), may also be involved in the inactivation of the human enzyme(s).

Identification of potentially neurotoxic pyridinium metabolite in the urine of schizophrenic patients treated with haloperidol

Evidence that the parkinsonian inducing agent MPTP is biotransformed to a pyridinium species that selectively destroys nigrostriatal neurons in humans and subhuman primates has prompted studies to evaluate the metabolic fate of the structurally related neuroleptic agent haloperidol. With the aid of a highly sophisticated atmospheric pressure ionspray HPLC/MS/MS assay, unambiguous evidence has been obtained for the presence of the haloperidol pyridinium species in extracts of urine obtained from haloperidol-treated patients and in extracts of NADPH-supplemented human liver microsomal incubation mixtures containing haloperidol. The potential significance of the formation of this putative neurotoxic pyridinium species is considered.

Elucidation of the structure and receptor binding studies of the major primary, metabolite of dihydroergotamine in man.

The plasma concentrations and urinary excretion of dihydroergotamine and its metabolites have been measured after a single oral administration of 3 mg tritium-labelled drug to 6 male volunteers. The plasma level of non-volatile radioactivity declined biphasically with alpha- and beta-phase half-lives of 2.1 h and 32.3 h, respectively. The peak plasma concentration was reached within 3.2 h. Urinary excretion of total non-volatile radioactivity was low, amounting to 1.0% of the dose. The parent drug and four metabolites could be quantitated in urine and plasma samples. Metabolite 4 (8'-hydroxy-dihydroergotamine) was isolated from incubates of rat and monkey liver microsomal preparations. In human liver microsomal incubates, metabolite 4 was shown to be the primary metabolite of dihydroergotamine. In receptor binding studies performed with mammalian brain preparations, metabolite 4 had IC50-values at 6 monoaminergic binding sites similar to those of dihydroergotamine. Thus, it appears that the active principle consists at least of dihydroergotamine and its 8'-hydroxy derivative. As the concentration of metabolite 4 exceeded 5-7 times that of dihydroergotamine in urine and plasma, the bioavailability of dihydroergotamine should be reevaluated, taking into account the plasma concentrations of the parent drug and of its active metabolite, 8'-hydroxy-dihydroergotamine.

Molecular aspects of debrisoquine metabolism studied by gas chromatography mass spectrometry with electron capture negative ion chemical ionisation

A gas chromatographic mass spectrometric assay using electron capture negative ion chemical ionisation has been developed for the analysis of 4-hydroxydebrisoquine in human liver microsomal incubations. The assay has been used to show that sparteine, guanoxan and phenformin are competitive inhibitors of human liver debrisoquine 4-hydroxylase activity in vitro.

Unilateral paralysis of the diaphragm and larynx associated with inflammatory lung disease.

The benzyloxy-substituted lactone cyclooxygenase-2 inhibitor DFB [3-[(3,4-difluorobenzyl)oxy]-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5<i>H</i>)-one] is metabolized in human and rat liver microsomal incubations and hepatocytes to a fluorescent metabolite, DFH [3-hydroxy-5,5-dimethyl-4-[4-(methylsulfonyl)phenyl]furan-2(5<i>H</i>)-one]. This process is CYP3A-mediated in both species, as demonstrated by incubations with recombinant CYP3A enzymes and experiments with inhibitory antibodies. Measurement of DFH fluorescence can be used as a rapid readout of CYP3A activity following microsomal or cultured hepatocyte incubations. In rat and human hepatocytes treated with prototypical inducers, the formation of DFH was linear for the first 30 min, with no secondary metabolism of DFH, such as phase II glucuronidation, observed at early time points. Using a panel of four prototypical inducers (phenobarbital, dexamethasone, phenytoin, and rifampicin), the correlation between testosterone 6β-hydroxylation in cultured human hepatocytes and CYP3A enzyme level in cell lysate was confirmed. DFB debenzylation was then shown to correlate well with testosterone 6β-hydroxylation in hepatocytes treated with these four inducers. Primary cultured rat and human hepatocyte induction assays were optimized for 24- and 96-well plates, respectively. Controls were established to evaluate whether test compounds demonstrate time-dependent CYP3A inhibition to avoid false negative results. Thus, the use of DFB, a fluorogenic CYP3A-selective probe substrate, affords a fast, efficient, and robust assay for the measurement of CYP3A induction in both rat and human cultured primary hepatocytes.