Ursula Breyer-Pfaff

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. AKR1B10 showed a molecular mass of 35 kDa upon gel filtration and SDS-polyacrylamide gel electrophoresis. Kinetic parameters for the NADPH-dependent reduction of the antiemetic 5-HT3 receptor antagonist dolasetron, the antitumor drugs daunorubicin and oracin, and the carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) to the corresponding alcohols have been determined by HPLC. Km values ranged between 0.06 mM for dolasetron and 1.1 mM for daunorubicin. Enzymatic efficiencies calculated as kcat/Km were more than 100 mM–1 min–1 for dolasetron and 1.3, 0.43, and 0.47 mM–1 min–1 for daunorubicin, oracin, and NNK, respectively. Thus, AKR1B10 is one of the most significant reductases in the activation of dolasetron. In addition to its reducing activity, AKR1B10 catalyzed the NADP+-dependent oxidation of the secondary alcohol (S)-1-indanol to 1-indanone with high enzymatic efficiency (kcat/Km = 112 mM–1 min–1). The gene encoding AKR1B10 was cloned from a human liver cDNA library and the recombinant enzyme was purified. Kinetic studies revealed lower activity of the recombinant compared with the native form. Immunoblot studies indicated large interindividual variations in the expression of AKR1B10 in human liver. Since carbonyl reduction of xenobiotics often leads to their inactivation, AKR1B10 may play a role in the occurrence of chemoresistance of tumors toward carbonyl group-bearing cytostatic drugs.

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. In contrast, correlations were positive for nortriptyline, (+)‐E‐10‐hydroxynortriptyline, (−)‐Z‐10‐hydroxynortriptyline, and (+)‐Z‐10‐hydroxynortriptyline. The mephenytoin hydroxylase seems to participate in side‐chain demethylation to the secondary and primary amines, because the log metabolic ratio of mephenytoin correlated negatively with the relative quantity of E‐10‐hydroxydidesmethylamitriptyline and positively with that of amitriptyline and its N‐glucuronide.

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. When 12 patients kept a smoking-free interval of at least 6 h, they had similarly low plasma norharman concentrations (20 +/- 8 pg/ml) as 18 non-smoking control subjects (17 +/- 8 pg/ml) or as 13 smoking controls who had abstained from smoking (20 +/- 6 pg/ml). Ten of the patients smoked one cigarette and within 5-10 min attained norharman levels of 177 +/- 147 pg/ml plasma. The high prevalence of smokers among chronic alcoholics probably explains the previous finding of elevated norharman plasma levels in these patients.

Pyridostigmine kinetics in healthy subjects and patients with myasthenia gravis

Comparative pyridostigmine kinetics in plasma were measured in 10 healthy subjects given 4 mg iv and 60 mg oral pyridostigmine bromide. As determined from the AUC ratio, oral availability was 11.5% to 18.9% (X̄ = 14.3%). Mean t½ of the plasma level decline after oral dosing was 200 minutes, twice as long as the terminal elimination t½ after intravenous infusion (97 minutes). Thus absorption may proceed at a slower rate than elimination. Comparison of intraindividual data revealed strict dependence of the AUC on the infused dose (2, 4, and 8 mg) in one subject and variability in AUC up to a factor of two when two subjects took oral pyridostigmine three times. Patients with myasthenia who were receiving continuous therapy with oral pyridostigmine had AUC values per unit dose corresponding to those in healthy subjects. Storage stability of pyridostigmine in plasma required acidification of samples and storage at −75° C. When native plasma was kept at −20° C, there was appreciable loss of pyridostigmine within 1 to 2 months, the extent of which depended on the initial concentration.

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. In the present investigation, a modified procedure was developed for the isolation of AKR1C1, 1C2, and 1C4 (dihydrodiol dehydrogenases 1, 2, and 4) from human liver cytosol along with carbonyl reductase (EC 1.1.1.184), a member of the short-chain alcohol dehydrogenase superfamily. The kinetics of NADPH-dependent reduction by the closely related enzymes AKR1C1 and 1C2 were studied with the structurally similar substrates (R)- and (S)-ketotifen and E- and Z-10-oxonortriptyline by HPLC measurement of the products. Km values varied between 2.6 and 53 microM and Vmax values between 5 and 313 mU/mg protein; substrate inhibition with Ki around 30 microM occurred in the reduction of E- and Z-10-oxonortriptyline by AKR1C1. The reactions were strictly stereospecific with production of one enantiomeric alcohol from each ketotifen enantiomer and of the (+)-enantiomers of E- and Z-10-hydroxynortriptyline. Enzymatic NADP+ -dependent oxidation of the alcohols mirrored the reduction with regard to stereochemical specificity. All four ketones were no or poor substrates of carbonyl reductase, whereas haloperidol was reduced by this enzyme with low affinity, but high efficiency.

Red cell ouabain binding sites, Na+K+-ATPase, and intracellular Na+ as individual characteristics

Red cells were used as model cells to assess the relationship between the number of ouabain binding sites and the Na+K+-ATPase activity in humans. (3H) ouabain binding to intact red cells was found to be a reversible reaction and rectilinear Scatchard plots were obtained. Interindividual comparison revealed identical slopes, whereas the abscissa intercepts differed, indicating variations in the number of ouabain binding sites. The number of sites ranged from 330 to 890 (x = 453 ± 104 SD) per red cell in 73 individuals and was intraindividually stable during the course of 3–12 months. The number of sites was significantly lower in the middle-age females compared to younger ones. A linear relationship (r=0.96) was found between the total number of ouabain binding sites of intact red cells and the Na+K+-ATPase activity of red cell ghosts. The catalytic activity varied little among individuals; a turnover number of 5500 ± 250 (SD) molecules ATP hydrolyzed per site and per min was calculated. A highly significant correlation was established between [Na+]i of red cells in vivo and the number of ouabain binding sites.

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. Introduction of a second hydroxy group results in isomeric glycols. Oxidative attack at an aromatic ring is a minor pathway leading to dihydrodiols and phenols. Numerous metabolites originate by combinations of reactions in the ring system and the side chain. AT‐NO is by about one‐third excreted in unchanged form or as 10‐hydroxy derivative; the major part is reduced to AT and metabolized further. The review covers current knowledge on the enzymes participating in the individual pathways. Their quantitative importance is inferred from kinetic studies in volunteers and patients and from experiments in vitro. Clinical consequences of biochemical findings mainly derive from the impact of the polymorphic CYP2D6 mediating (−)‐(E)‐10‐hydroxylation and from its potential inhibition by other psychoactive drugs.

The contribution of α1-acid glycoprotein, lipoproteins, and albumin to the plasma binding of perazine, amitriptyline, and nortriptyline in healthy man

SummaryParameters for the binding of perazine (PER), amitriptyline (AT) and nortriptyline (NT) to plasma and to single plasma proteins were determined by equilibrium dialysis. The highest affinity (K at least 105 M−1) and lowest capacity (first site 1 mol/mol) towards all three drugs was exhibited by α1-acid glycoprotein (α1-AGP). From the parameters, α1-AGP was estimated to contribute 43% to total binding of PER and 49 and 31%, respectively, to AT and NT binding in samples with normal protein concentrations. Fractions bound to total lipoproteins would amount to 32% (PER), 40% (AT) and 52% (NT), respectively, while the contribution of albumin would range from 11% (AT) to 25% (PER). The extent of the binding to plasma was compared with that to single proteins and their mixtures. Binding to combinations of α1-AGP, lipoproteins and albumin exceeded that to plasma with PER, but not with AT and NT. This leads to the assumption that additional plasma constitutents interfere with PER binding.

Antidepressive effect and pharmacokinetics of amitriptyline with consideration of unbound drug and 10-hydroxynortriptyline plasma levels

In 27 inpatients with primary affective disorder the urinary excretion of 3-methoxy-4-hydroxyphenylglycol (MHPG) was measured prior to a 4-week treatment with 150 mg amitriptyline (AT)/day. Ratings according to the Hamilton depression scale were performed before therapy and repeated after 2 and 4 weeks. Plasma levels of AT, nortriptyline (NT), and E-10-hydroxynortriptyline (OHNT) were assayed weekly, and binding of AT to plasma proteins was determined in one sample. Better therapeutic results were obtained at intermediate, as compared to low and high concentrations of AT or AT plus NT. Independent evaluation of AT and metabolite levels revealed that patients with AT of 50–125 mg/ml responded particularly well when NT did not exceed 95 ng/ml or when NT plus OHNT was below 150 ng/ml. Outside this ‘therapeutic window’ the outcome was markedly poorer. Interindividual variation of AT binding was much smaller than variation of total concentrations. Evaluation of free, instead of total levels did not help to clarify the relationship between clinical and pharmacokinetic variables. Plasma levels within the optimal ranges were found in more patients with high than with low MHPG excretion. The frec fraction of OHNT in plasma of healthy subjects was about 35%.

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

The opioid receptor antagonist naltrexone and the antiemetic 5‐HT3 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 1.1.1.184) purified from human liver cytosol were incubated with varying substrate concentrations and 6β‐naltrexol or reduced dolasetron were analysed by HPLC. AKR1C1, AKR1C2, and AKR1C4 were able to reduce both substrates. On the basis of kcat/Km values, AKR1C4 was nearly 1000‐fold more efficient in reducing naltrexone than was AKR1C1, while AKR1C2 was of intermediate efficiency. Substrate inhibition was observed on incubating AKR1C2 or AKR1C4 with naltrexone. In contrast, dolasetron was also a substrate of CR. AKR1C1 and AKR1C4 were the most efficient enzymes in producing reduced dolasetron. We concluded that the efficient reduction of naltrexone by AKR1C4 probably causes the high 6β‐naltrexol/naltrexone ratio in man. The rapid disappearance from human plasma of dolasetron given intravenously and its virtual absence after oral dosage are explained by its liability to reduction by several enzymes, including CR which shows widespread expression in human tissues.