Purification and characterization of akr1b10 from human liver: role in carbonyl reduction of xenobiotics.

Members of the aldo-keto reductase (AKR) superfamily have a broad substrate specificity in catalyzing the reduction of carbonyl group-containing xenobiotics. In the present investigation, a member of the aldose reductase subfamily, AKR1B10, was purified from human liver cytosol. This is the first time AKR1B10 has been purified in its native form.

Carbonyl reduction of naltrexone and dolasetron by oxidoreductases isolated from human liver cytosol.

The opioid receptor antagonist naltrexone and the antiemetic 5-HT(3) receptor antagonist dolasetron are ketonic drugs that are efficiently reduced to their corresponding alcohols in-vivo. These experiments aimed at characterizing the role in these reactions of individual oxidoreductases present in human liver cytosol. Aldo-keto reductases (AKRs) and carbonyl reductase (CR, EC 1.

The metabolic fate of amitriptyline, nortriptyline and amitriptylinoxide in man.

Amitriptyline (AT), the most widely used tricyclic antidepressant, undergoes oxidative metabolism in the side chain with production of the secondary amine nortriptyline (NT), a primary amine, and the N-oxide amitriptylinoxide (AT-NO); in addition, direct conjugation leads to a quaternary ammonium-linked glucuronide. Hydroxylation of AT or NT at the ethylene bridge of the central seven-membered ring results in four isomeric alcohols and occurs with high stereo- and enantioselectivity, the (-)-(E)-10-hydroxy compounds usually being the major products. The disposition of the alcohols is also partially enantioselective, for instance with regard to glucuronidation and reversible oxidation to ketones.

Enantioselectivity of carbonyl reduction of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone by tissue fractions from human and rat and by enzymes isolated from human liver.

Detoxication of the tobacco-specific carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in humans is mainly due to carbonyl reduction to the chiral alcohol 4-methylnitrosamino-1-(3-pyridyl)-1-butanol (NNAL), which undergoes glucuronidation and excretion. NNAL has a carcinogenic potential with (S)-NNAL being more tumorigenic in the mouse. Therefore, the enantioselectivity of NNK reductases seems toxicologically relevant.

Tertiary N-glucuronides of clozapine and its metabolite desmethylclozapine in patient urine.

In experiments with expressed human UDP-glucuronosyltransferase 1A4 (UGT1A4), the antipsychotic clozapine proved to be conjugated to two different glucuronides, one of which was identified as the quaternary ammonium glucuronide derivatized at the N-methylpiperazine group; this compound had previously been isolated from patient urine. An additional glucuronide produced in larger quantity was assumed to be conjugated at the secondary nitrogen of the central ring to form 5-N-glucuronide, but this was not proven. The analogous olanzapine 10-N-glucuronide was found to make a major contribution to urinary metabolites in human volunteers.

Isolation and identification of clozapine metabolites in patient urine.

Biotransformation products of the atypical neuroleptic clozapine were isolated from urine samples of three schizophrenic patients by solid-phase extraction, liquid-liquid extraction for the separation of unpolar and polar metabolites, and thin-layer chromatography followed by final purification by high-performance liquid chromatography. Their structures were elucidated by mass spectrometry and (1)H NMR spectroscopy and in some cases by enzymatic deconjugation. Besides the known metabolites desmethylclozapine, clozapine N-oxide, 8-deschloro-8-hydroxyclozapine, and 8-deschloro-8-hydroxydesmethylclozapine, the unpolar fraction contained 7-hydroxyclozapine and a compound in which the piperazine ring of clozapine was partially degraded to an ethylenediamine derivative.

Purification and characterization of oxidoreductases-catalyzing carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) in human liver cytosol.

1. Four enzymes were purified to homogeneity from human liver cytosol and were demonstrated to be responsible for carbonyl reduction of the tobacco-specific nitrosamine 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK). 2.

Comparative N-glucuronidation kinetics of ketotifen and amitriptyline by expressed human UDP-glucuronosyltransferases and liver microsomes.

Like other basic amphiphilic drugs, the (S)-enantiomer of the antiallergic drug ketotifen exhibited biphasic kinetics when it was converted to two isomeric quaternary ammonium-linked glucuronides in human liver microsomes. For (R)-ketotifen this applied when incubations were carried out in the absence of a detergent. Two UDP-glucuronosyltransferases (UGTs) present in human liver, UGT1A4 and UGT1A3, were previously shown to catalyze tertiary amine N-glucuronidation when expressed in HK293 cells.

High-affinity stereoselective reduction of the enantiomers of ketotifen and of ketonic nortriptyline metabolites by aldo-keto reductases from human liver.

Aldo-keto reductases (AKR) form an enzyme superfamily catalyzing the reduction of carbonyl compounds and in some cases the reverse oxidation of alcohols as well. In particular, a role in drug metabolism has been considered for the AKR1C family, but published data failed to reveal low Km drug substrates. Moreover, structure activity relationships using chemically related substrates have not been established.

Conjugation of the enantiomers of ketotifen to four isomeric quaternary ammonium glucuronides in humans in vivo and in liver microsomes.

The antiallergic drug ketotifen is chiral due to a nonplanar seven-membered ring containing a keto group. Earlier studies have revealed glucuronidation at the tertiary amino group as a major metabolic pathway in humans. Chemical synthesis of glucuronides from racemic ketotifen now led to four isomers separable by HPLC of which two each could be ascribed to (R)-(+)- and (S)-(-)-ketotifen by synthesis from the enantiomers.

Pharmacokinetics of clozapine and its metabolites in psychiatric patients: plasma protein binding and renal clearance.

Aims: N-Desmethylclozapine and clozapine N-oxide are major metabolites of the atypical neuroleptic clozapine in humans and undergo renal excretion. The aim of this study was to investigate to what extent the elimination of these metabolites in urine contributes to the total fate of clozapine in patients and how they are handled by the kidney.

Methods: From 15 psychiatric patients on continuous clozapine monotherapy, blood and urine samples were obtained during four 2 h intervals, and clozapine and its metabolites were assayed in serum and urine by solid-phase extraction and h.

Variability of diphenhydramine N-glucuronidation in healthy subjects.

The H1-antagonist diphenhydramine can undergo direct glucuronidation at its tertiary amino group with formation of a quaternary ammonium glucuronide. The intraindividual variability in the amount of N-glucuronide excretion in urine was investigated in two female volunteers who repeatedly took single doses of 25 mg diphenhydramine hydrochloride without and with concomitant administration of ascorbic acid or ammonium chloride for urine acidification. Another two female and four male subjects underwent single tests without and with additional ascorbic acid.

Biphasic kinetics of quaternary ammonium glucuronide formation from amitriptyline and diphenhydramine in human liver microsomes.

The tricyclic antidepressant amitriptyline and the H1-receptor antagonist diphenhydramine are conjugated in human liver microsomes fortified with UDP-glucuronic acid at their tertiary amino groups with the formation of quaternary ammonium glucuronides. The kinetics of the reactions were found to be biphasic with apparent KM1 and KM2 values of 1.4 microM and 311 microM for amitriptyline and 2.

[Amalgam--"etiology" of psychiatric disorders?].

In a liability lawsuit an expertise had to answer the question whether a mania in the course of an affective psychosis could have been caused by chronic mercury intoxication resulting from dental amalgam fillings. On the basis of current psychiatric and toxicological knowledge, such an association can be disproved. Mercury intake from amalgam fillings does not lead to toxic concentrations in organs or body fluids.

Elevated norharman plasma levels in alcoholic patients and controls resulting from tobacco smoking.

Plasma norharman and harman levels were measured by solvent extraction and HPLC with fluorescence detection in alcohol-dependent patients undergoing in-patient abstinence treatment and in control subjects. In both groups, randomly collected samples from smokers contained higher mean norharman levels than those from non-smokers. In three volunteers norharman concentrations rose sharply after smoking of one or two cigarettes and declined to near-basal levels within one hour after one cigarette.

Stereoselective reversible ketone formation from 10-hydroxylated nortriptyline metabolites in human liver.

1. E- and Z-10-hydroxynortriptyline are major metabolites of amitriptyline and nortriptyline in man. Upon incubation with human liver microsomes or cytosol, these metabolites were oxidized to the corresponding ketones, E- and Z-10-oxonortriptyline.

Comparison of procedures for measuring the quaternary N-glucuronides of amitriptyline and diphenhydramine in human urine with and without hydrolysis.

The activities of beta-glucuronidases from Helix pomatia, Escherichia coli and rat towards the N-glucuronides of amitriptyline and diphenhydramine were considerably lower than those towards standard substrates. The two N-glucuronides were analysed in urine samples by the following procedures: HPLC of the intact conjugate after solid-phase extraction on a cation exchanger cartridge or after direct injection of urine; HPLC of the aglycone after hydrolysis with beta-glucuronidase from H. pomatia or E.

Renal clearance of pyridostigmine in myasthenic patients and volunteers under the influence of ranitidine and pirenzepine.

1. To assess the contribution of tubular secretion to the renal excretion of pyridostigmine, and its modification by other cationic drugs, six volunteers were given single oral doses of 60-mg pyridostigmine bromide with and without co-administration of ranitidine or pirenzepine. Renal clearances were determined by h.

Enantioselective amitriptyline metabolism in patients phenotyped for two cytochrome P450 isozymes.

In 26 hospitalized patients with depression, a combined pharmacogenetic test with dextromethorphan, a substrate of cytochrome P450IID6, and mephenytoin, the S-form of which is hydroxylated by a P450IIC isozyme, was carried out before amitriptyline therapy. Metabolites were determined in 24-hour urine samples collected on treatment day 8, and the contributions of individual compounds, including the four isomers of 10-hydroxyamitriptyline and 10-hydroxynortriptyline to total excretion were calculated. Formation of (-)-E-10-hydroxyamitriptyline and (-)-E-10-hydroxynortriptyline apparently depends on the activity of cytochrome P450IID6 because negative correlations existed between the log metabolic ratio of dextromethorphan and the relative quantities of these enantiomers.

Stereoselective inhibition of nortriptyline hydroxylation in man by quinidine.

1. Four volunteers phenotyped as extensive metabolizers of sparteine took 25 mg nortriptyline hydrochloride and collected urine for 72-80 h. Total free and conjugated 10-hydroxynortriptyline (10-OH-NT) accounted for 54-58% of the dose and it was reduced to 25-40% when 50 mg quinidine sulphate was ingested on the first and second day.

Urinary metabolites of amitriptylinoxide and amitriptyline in single-dose experiments and during continuous therapy.

In a cross-over design, six healthy volunteers received 50 mg amitriptylinoxide (AT-NO) IV and orally and 50 mg amitriptyline (AT) IV. Urine was collected completely for 8 h and occasionally up to 48 h. In addition, five patients each under treatment with AT-NO or AT for tension headache collected 24-h urine samples.