Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques.

The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound.

Identification of human liver cytochrome P450 isoforms mediating omeprazole metabolism.

1 The in vitro metabolism of omeprazole was studied in human liver microsomes in order to define the metabolic pathways and identify the cytochrome P450 (CYP) isoforms responsible for the formation of the major omeprazole metabolites. 2 The four major metabolites identified in vitro, in tentative order of importance, were hydroxyomeprazole, omeprazole sulphone, 5-O-desmethylomeprazole, and an unidentified compound termed metabolite X. Omeprazole pyridone was also detected but could not be quantitated.

The glucuronidation of hydroxylated metabolites of benzo[a]pyrene and 2-acetylaminofluorene by cDNA-expressed human UDP-glucuronosyltransferases.

The capacity of four cDNA-expressed human liver UDP-glucuronosyltransferases (UGT), UGT1*6, UGT2B7, UGT2B10 and UGT2B11, to glucuronidate hydroxylated metabolites of benzo[a]pyrene (B[a]P) and 2-acetylaminofluorene (AAF) has been investigated. UGT1*6 and UGT2B7 glucuronidated a range of B[a]P and AAF metabolites with a degree of regiospecificity, although UGTs 2B10 and 2B11 were inactive towards all compounds screened. UGT2B7 glucuronidated the B[a]P trans 4,5- and 7,8-dihydrodiols and the 1-,2-,4-,5-,6-,8-,9- and 10-monophenols.

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 nM in incubate) for omeprazole sulphone and 25 pmol (25 nM in incubate) for hydroxyomeprazole. Extraction efficiency is essentially quantitative and is highly reproducible (coefficient of variation = 2.

cDNA cloning and expression of two new members of the human liver UDP-glucuronosyltransferase 2B subfamily.

Two new UDP-glucuronosyltransferase cDNAs, designated UGT2B10 and UGT2B11, encoding 528 amino acid proteins were isolated from a human liver cDNA library. The deduced amino acid sequences of UGTs 2B10 and 2B11 share > 76% sequence similarity with other known human liver UGT2B subfamily isoforms and < 48% sequence similarity with UGT1 family proteins. COS-7 cells transfected with UGT 2B10 and 2B11 synthesized proteins with respective molecular masses of 49kDa and 51kDa.

High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes.

A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both > 90%.

Tolbutamide hydroxylation in humans: lack of bimodality in 106 healthy subjects.

The tolbutamide hydroxylation capacity was studied in 106 healthy unrelated volunteers from the Australian population. Following a 500 mg oral dose of tolbutamide, the ratio of metabolites (hydroxytolbutamide plus carboxytolbutamide) to unchanged tolbutamide excreted in urine from 6 to 12 h post-dose (urinary metabolic ratio, MR) was determined. Metabolic ratio values did not appear bimodally distributed, even following various transformations of the data (i.

Specificity of substrate and inhibitor probes for human cytochromes P450 1A1 and 1A2.

Kinetic and inhibitor studies using cDNA-expressed enzymes and human liver microsomes have characterized the specificity of a range of cytochrome P450 (CYP) 1A substrate and inhibitor probes towards the two isoforms comprising this subfamily. Expressed CYP1A1 and CYP1A2 both catalyzed the O-deethylation of phenacetin, although the apparent Km was about 4-fold lower for CYP1A2 (25 vs. 108 microM).

Site-directed mutation studies of human liver cytochrome P-450 isoenzymes in the CYP2C subfamily.

Evidence from human studies in vivo and in vitro strongly suggests that the methylhydroxylation of tolbutamide and the 4-hydroxylation of phenytoin, the major pathways in the elimination of these two drugs, are catalysed by the same cytochrome P-450 isoenzyme(s). In the present study we used site-directed mutagenesis and cDNA expression in COS cells to characterize in detail the kinetics of tolbutamide and phenytoin hydroxylations by seven CYP2C proteins (2C8, 2C9 and variants, and 2C10) in order to define the effects of small changes in amino acid sequences and the likely proteins responsible in the metabolism of these two drugs in man. Tolbutamide was hydroxylated to varying extents by all expressed cytochrome P-450 isoenzymes, although activity was much lower for the expressed 2C8 protein.

Complementary deoxyribonucleic acid cloning and expression of a human liver uridine diphosphate-glucuronosyltransferase glucuronidating carboxylic acid-containing drugs.

A cDNA clone, designated UGT2B7 variant, encoding a 529-amino acid human liver microsomal uridine diphosphate-glucuronosyltransferase (UGT) was isolated from a lambda gt11 human liver cDNA library. UGT2B7 variant synthesized in COS-7 cells was screened for activity toward a range of clinically used drugs and other xenobiotics. The expressed enzyme glucuronidated several carboxylic acid-containing nonsteroidal antiinflammatory agents including, in order of relative substrate activity, naproxen, ketoprofen, ibuprofen, fenoprofen, tiaprofenic acid, benoxprofen, zomepirac, diflunisal and indomethacin.

Co-regulation of phenytoin and tolbutamide metabolism in humans.

1. The disposition of phenytoin and tolbutamide was compared in eighteen healthy young adults separately administered single therapeutic doses (sodium phenytoin 300 mg, tolbutamide 500 mg) of the two drugs. 2.

Perturbation of paracetamol urinary metabolic ratios by urine flow rate.

The effects of high and low urine flow rates on the urinary metabolic ratios for paracetamol glucuronidation, sulphation and oxidation were determined at steady-state in seven healthy young adult volunteers. Metabolic partial clearances were unaffected by urine flow rate, but individual paracetamol metabolic ratios varied 2.5- to 3.

Caffeine as a probe for human cytochromes P450: validation using cDNA-expression, immunoinhibition and microsomal kinetic and inhibitor techniques.

The molecular basis for the use of caffeine (CA; 1,3,7-trimethylxanthine) as a probe for specific human cytochromes P450 has been investigated. The CA 1-, 3- and 7-demethylations (to form theobromine, paraxanthine and theophylline, respectively) all followed biphasic kinetics in human liver microsomes. Mean apparent Km values for the high- and low-affinity components of the demethylations ranged from 0.

Interethnic differences in drug glucuronidation: a comparison of paracetamol metabolism in Caucasians and Chinese.

Paracetamol disposition following a single oral 1 g dose of the drug was compared in groups (n = 12) of healthy young adult male Caucasians and Chinese. There was no difference between the groups in terms of paracetamol oral clearance, elimination half-life, or partial metabolic (glucuronidation, sulphation, oxidation) and renal clearances. The results demonstrate that drug glucuronidation is not universally impaired in Chinese and, together with previously published data, that paracetamol glucuronidation is minimally affected by race.

1-Methylxanthine derived from theophylline as an in vivo biochemical probe of allopurinol effect.

The urinary 1-methyluric acid (1MU) to 1-methylxanthine (1MX) ratio has been assessed as a biochemical index of oxipurinol effect in vivo in man. Dosing with theophylline was used to produce 1MX as an intermediate metabolite in six healthy volunteers. A sigmoid Emax model was fitted to the data and gave a mean plasma oxipurinol IC50 of 3.

Stereospecific high-performance liquid chromatographic assay for the enantiomers of phenylpropanolamine in human plasma.

A stereospecific high-performance liquid chromatographic assay for the enantiomers of phenylpropanolamine (PPA) in human plasma has been developed. The method is based on reaction of extracted PPA with the chiral derivatizing agent 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate to form diastereomeric thiourea derivatives. These thiourea derivatives are stable for at least 24 h, readily separable by reversed-phase chromatography, and amenable to ultraviolet detection at 254 nm.

Caffeine renal clearance and urine caffeine concentrations during steady state dosing. Implications for monitoring caffeine intake during sports events.

1. Relationships between the plasma and urine concentrations and clearances of caffeine over successive dosage intervals at steady-state were investigated in six healthy volunteers administered caffeine, 150 mg 8 hourly for 6 days. 2.

Tolbutamide and phenytoin hydroxylations by cDNA-expressed human liver cytochrome P4502C9.

A human cytochrome P4502C9 cDNA clone has been isolated from a human liver bacteriophage Lambda gt11 library using oligonucleotide probes. Expression of the 1762 base pair cDNA in COS cells demonstrated that the encoded enzyme has a molecular mass of 55 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The expressed enzyme catalysed the methylhydroxylation of tolbutamide with an apparent Km of 131.

Assessment of the drug inhibitor specificity of the human liver 4-methylumbelliferone UDP-glucuronosyltransferase activity.

The data suggest that the 4MU-UDPGT activity of human liver microsomes probably contributes to the glucuronidation of a limited number of clinically used drugs. However, confirmation of this ultimately requires studies to be performed with purified isozymes, cDNAs expressed in cell culture, or specific inhibitory antibodies.

Relationship between phenytoin and tolbutamide hydroxylations in human liver microsomes.

1. The metabolic interaction of phenytoin and tolbutamide in human liver microsomes was investigated. 2.

Drug glucuronidation in humans.

Glucuronidation is a major metabolic pathway for a large number of drugs in humans. Conjugation of drugs and other chemicals with glucuronic acid is catalyzed by the multigene UDP-glucuronosyltransferase family. It is believed that a number (unspecified at present) of glucuronosyltransferase isozymes, which probably differ in terms of substrate specificity and regulation, contribute to drug glucuronidation.

Mechanism of formation of 6-amino-5-(N-methylformylamino)-1-methyluracil and 3,7-dimethyluric acid from theobromine in the rat in vitro: involvement of cytochrome P-450 and a cellular thiol.

1. The involvement of glutathione (GSH) and cytochrome P-450 in the conversion of theobromine to 6-amino-5-(N-methylformylamino)-1-methyluracil (3,7-DAU) and 3,7-dimethyluric acid (3,7-DMU) has been investigated in rat liver microsomal incubations. 2.

Characterization of paracetamol UDP-glucuronosyltransferase activity in human liver microsomes.

A specific high performance liquid chromatographic assay has been developed for the measurement of paracetamol glucuronide formation by the microsomal fraction of human liver. The procedure has been used to characterize paracetamol glucuronidation kinetics in human livers microsomes and to assess the substrate specificity of the paracetamol UDP-glucuronosyltransferase (UDPGT) activity. Paracetamol glucuronidation followed Michaelis-Menten kinetics, suggesting the involvement of a single form of UDPGT, or possibly two or more forms of UDPGT with similar affinities for paracetamol, in this reaction.