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Die Pharmakogenetik untersucht, inwieweit genetische Polymorphismen der Proteine, die die pharmakokinetischen und pharmakodynamischen Prozesse eines Arzneimittels kontrollieren, fur die interindividuellen Unterschiede in Wirkung und Nebenwirkung verantwortlich sind. Im Gegensatz zur Pharmakogenetik nutzt die Pharmakogenomik einen genomweiten Ansatz zur Identifizierung von Genen bzw. Gennetzwerken, die an der Entstehung von Erkrankungen beteiligt sind bzw. als therapeutische Ziele fur neue Arzneistoffe dienen konnen. Die zurzeit am besten charakterisierten pharmakogenetischen Polymorphismen betreffen die Arzneimittel metabolisierenden Enzyme Zytochrom-P450-2C9, -2C19 und -2D6 bzw. die Thiopurinmethyltransferase, fur die in klinischen Studien relevante Konsequenzen fur die Arzneimitteltherapie gezeigt werden konnten. Das ultimative Ziel pharmakogenetischer/-genomischer Forschung ist es, unter Verwendung einer neuen Krankheits- und Therapieklassifikation auf molekularer Ebene eine spezifische Arzneimitteltherapie bei genetisch definierten Untergruppen von Patienten durchzufuhren.

Die Kenntnis, wie ein Arzneistoff auf den Organismus einwirkt (Pharmakodynamik) und wie der Organismus auf das Arzneimittel einwirkt (Pharmakokinetik), stellt die Basis fur eine individualisierte Therapie dar. Eine individualisierte Therapie ist deswegen fur die Wirksamkeit und Sicherheit so wichtig, weil sowohl in der Dosis-Konzentrations-Beziehung als auch in der Konzentrations-Wirkungs-Beziehung betrachtliche Unterschiede von Patient zu Patient bestehen konnen. Denn die Praxis zeigt, dass die unterschiedlichen Dosis-Wirkungs-Beziehungen ein wichtiges Problem – wohl das wichtigste uberhaupt – in der Arzneimitteltherapie darstellen, weil sie sich unmittelbar auf die Wirksamkeit (Risiko der Unterdosierung) und Sicherheit (Risiko der Uberdosierung) auswirken. Besonders ausgepragte interindividuelle Unterschiede werden vor allem in der Konzentrations-Beziehung beobachtet. Daraus resultiert die therapeutische Relevanz der Pharmakokinetik. Ziel der vorliegenden Ubersicht ist, die einzelnen pharmakokinetischen Prozesse unter diesem Gesichtspunkt zu beleuchten. Pharmakodynamische Unterschiede, die z.B. auf den Ebenen der Rezeptordichte, Rezeptorpolymorphismen oder der Signaltransduktion bestehen konnen, sind nicht Gegenstand dieser Ubersicht.

The effect of verapamil and its enantiomers and metabolites on cardiac action potential repolarizing potassium channels was tested. For this purpose, the potassium channels Kv1.1, Kv1.5, Kir2.1, and HERG, and the IsK subunit of the IKs-channel complex were expressed in Xenopus oocytes and two-electrode voltage-clamp experiments were performed. Verapamil induced a concentration-dependent block of Kv1. 1-, Kv1.5-, IKs-, and HERG-induced currents with IC50 values of 14.0 +/- 2.7 microM (n = 4), 5.1 +/- 0.5 microM (n = 6), 161.0 +/- 26.3 microM (n = 4), and 3.8 +/- 0.2 microM (n = 5), respectively. The same potency of HERG channel inhibition was observed for the optical enantiomers (+)-verapamil (IC50 = 3.5 +/- 0.4 microM, n = 5) and (-)-verapamil (IC50 = 4.0 +/- 0.7 microM, n = 4), as well as the derivatives norverapamil (D591; IC50 = 3.8 +/- 0.3 microM, n = 4) and D703 (IC50 = 2.2 +/- 0.4 microM, n = 4). The verapamil metabolites D620 and D617 did not block HERG-induced currents at concentrations of up to 30 microM (n = 3). These results demonstrate that cardiac delayed rectifier potassium currents are sensitive targets to calcium channel blockers.

The majority of synthetic drugs with chiral centers are administered as racemates. Thus chemically, and to an even greater extent biologically, a racemic drug is not a single compound, but a 50:50 mixture of two enantiomeric drugs. No generalization can be made concerning whether and to what extent the activity, in either qualitative or quantitative terms, differs between enantiomers. It is not unusual for the enantiomers of a drug to have a high degree of enantioselectivity for one action but no enantioselectivity for another action. For instance S-propranolol is at least two orders of magnitude more potent than R-propranolol with regard to beta-adrenoceptor antagonism. However, the two enantiomers are equipotent with regard to their membrane stabilizing effect. It is often overlooked that enantioselectivity in the activity of enantiomers as determined in vitro cannot be extrapolated to the in vivo situation since enantioselective drug disposition can lead to an enantiomer ratio in vivo which differs substantially from that in the dosage form administered. Enantioselectivity in drug disposition seems to be the rule rather than the exception and, depending on whether the active or less active enantiomer is preferentially affected, there may be amplification or attenuation of in vivo as compared to the in vitro drug potency.

Microsomal epoxide hydrolase is a biotransformation enzyme which is involved in the hydrolysis of various epoxides and epoxide intermediates. In the present study, its distribution was investigated in both normal human tissues and human tumours of different histogenetic origin using immunohistochemical techniques. In normal tissue, epithelial cells were more often and more intensely immunostained than mesenchymal cells. The main epithelial cell types expressing microsomal epoxide hydrolase were hepatocytes, acinus cells of the pancreas, and cells of salivary and adrenal glands. Immunostained cells of mesenchymal origin included monocytes, fibrocytes, fibroblasts, vessel endothelium, muscle cells, and cells of the reproductive system. Three patterns of expression were observed in tumour tissues: (1) moderate or strong in hepatocellular carcinomas, tumours of the adrenal gland, and theca-fibromas of the ovary; (2) inhomogeneous staining pattern of variable intensity in breast cancer, lung cancer, colorectal carcinomas, carcinoid tumours, and some tumours of mesenchymal origin; and (3) no expression in malignant melanomas, malignant lymphomas, and renal carcinomas. These data indicate that microsomal epoxide hydrolase expression is not restricted to tissue of any particular histogenetic origin. Nonetheless, immunohistochemical identification of microsomal epoxide hydrolase may be helpful in some well-defined histological settings, for example, confirmation of hepatocellular carcinoma.

Between 45 and 60% of all drugs currently used are metabolized by the CYP3A4 protein. CYP3A4 expression in liver varies up to 60-fold in the general population, which can lead to ineffective drug therapy (high CYP3A4) or, on the other hand, to harmful drug reactions (low CYP3A4). Most of this variability has been attributed to genetic factors, but to date their identity remains unknown. Recently, it was shown that CYP3A expression is largely controlled by the pregnane X receptor (PXR). We, therefore, hypothesized that polymorphisms in PXR may contribute to CYP3A4 variability. The presence of PXR variants was investigated in two ethnic groups, Caucasians and Africans. Six missense mutations leading to variant PXR proteins were identified, and their consequences on CYP3A4 expression were analyzed. Expressed in LS174T cells, three protein variants, V140M, D163G, and A370T, exhibited altered basal and/or induced transactivation of CYP3A promoter reporter genes. Thus, these natural PXR protein variants may play a role in the observed interindividual variability of CYP3A4 expression and may be involved in rare, atypical responses to drugs or altered sensitivities to carcinogens.

The human cytochrome P450, CYP2B6, is involved in the metabolism of several therapeutically important drugs and environmental or abused toxicants. In this study, we present the first systematic investigation of genetic polymorphism in the CYP2B6 gene on chromosome 19. A specific direct sequencing strategy was developed based on CYP2B6 and CYP2B7 genomic sequence information and DNA from 35 subjects was completely analysed for mutations throughout all nine exons and their exon-intron boundaries. A total of nine novel point mutations were identified, of which five result in amino acid substitutions in exon 1 (C64T, Arg22Cys), exon 4 (G516T, Gln172His), exon 5 (C777A, Ser259Arg and A785G, Lys262Arg) and exon 9 (C1459T, Arg487Cys) and four are silent mutations (C78T, G216C, G714A and C732T). Polymerase chain reaction-restriction fragment length polymorphism tests were developed to detect each of the five nonsynonymous mutations in genomic DNA. By screening a population of 215 subjects the C64T, G516T, C777A, A785G and C1459T mutations were found at frequencies of 5.3%, 28.6%, 0.5%, 32.6% and 14.0%, respectively. Haplotype analysis revealed six different mutant alleles termed CYP2B6*2 (C64T), *3 (C777A), *4 (A785G), *5 (C1459T), *6 (G516T and A785G) and *7 (G516T, A785G and C1459T). By analysing a large number of human liver samples, significantly reduced CYP2B6 protein expression and S-mephenytoin N-demethylase activity were found in carriers of the C1459T (R487C) mutation (alleles *5 and *7). These data demonstrate that the extensive interindividual variability of CYP2B6 expression and function is not only due to regulatory phenomena, but also caused by a common genetic polymorphism.

Using a stable isotope technique we investigated the pharmacokinetics and pharmacodynamics of gallopamil after administration of 50 mg pseudoracemic gallopamil every 12 h for 7 doses (72 h).Six male healthy volunteers were studied. After the seventh dose the pharmacokinetics and pharmacodynamics were assessed. Serum levels of gallopamil were measured by gas chromatography/mass spectrometry. Effects of gallopamil were measured by ECG recording.The apparent oral clearances (R: 4.8 l min-1 (95% CI: 2.9-6.8); S: 5.5 l min-1 (95% CI: 2.5-8.5)) and half-lives (R: 6.2 h; S: 7.2 h) of R- and S-gallopamil were similar (P0.05). The serum protein binding (fu R: 0.035 (95% CI: 0.026-0. 045); S: 0.051 (95% CI: 0.033-0.069)) and the renal elimination (% of dose R: 0.49%; S: 0.71%) were enantioselective. Gallopamil had a potent effect on the PR interval (% prolongation 35.7% (95% CI: 14. 0-57.3)). No changes in other electrocardiographic or cardiovascular parameters were observed.The pharmacokinetics and bioavailability of the racemic drug gallopamil are not stereoselective at steady-state and are therefore not substantially altered compared with the single dose administration of gallopamil.

The effect of verapamil and its enantiomers and metabolites on cardiac action potential repolarizing potassium channels was tested. For this purpose, the potassium channels Kv1.1, Kv1.5, Kir2.1, and HERG, and the IsK subunit of the IKs-channel complex were expressed in Xenopus oocytes and two-electrode voltage-clamp experiments were performed. Verapamil induced a concentration-dependent block of Kv1. 1-, Kv1.5-, IKs-, and HERG-induced currents with IC50 values of 14.0 +/- 2.7 microM (n = 4), 5.1 +/- 0.5 microM (n = 6), 161.0 +/- 26.3 microM (n = 4), and 3.8 +/- 0.2 microM (n = 5), respectively. The same potency of HERG channel inhibition was observed for the optical enantiomers (+)-verapamil (IC50 = 3.5 +/- 0.4 microM, n = 5) and (-)-verapamil (IC50 = 4.0 +/- 0.7 microM, n = 4), as well as the derivatives norverapamil (D591; IC50 = 3.8 +/- 0.3 microM, n = 4) and D703 (IC50 = 2.2 +/- 0.4 microM, n = 4). The verapamil metabolites D620 and D617 did not block HERG-induced currents at concentrations of up to 30 microM (n = 3). These results demonstrate that cardiac delayed rectifier potassium currents are sensitive targets to calcium channel blockers.

The development of CYP2D6 has been attributed to the need of earth-dwelling animals to detoxify toxic xenobiotics (phytoalexins) present in plants. This hypothesis has been extrapolated to humans, but is yet unconfirmed. Therefore, we studied two Amerindian populations as the best available model to test the effect of selection through diet on human CYP2D6 evolution. The frequency of sparteine poor metabolizers in Ngawbe was 4.4% (n = 344), while the frequency in Embera was 2.2% (n = 153). Among Ngawbe and Embera, CYP2D6*4 (allelic frequencies for each tribe, respectively: 0.171; 0.14), CYP2D6*6 (0.005; 0.011) and CYP2D6*10 (0.175; 0.069) were detected, while CYP2D6*3, CYP2D6*5, CYP2D6*9 and CYP2D6*16 were absent. All poor metabolizers possessed either CYP2D6*4 or CYP2D6*6 and there were no disagreements between genotypic and phenotypic data. The total frequency of mutant alleles showed no difference among Amerindians or when compared to Caucasians. It was higher than in Chinese, since the frequency of CYP2D6*4 was higher in Amerindians. XbaI restriction fragment length polymorphisms haplotypes were very homogeneous in Amerindians, because the only fragment that hybridized with the CYP2D6 cDNA probe was the 29 kb (not 42/44 kb or 11.5/13 kb). This indicated no gene cluster recombinations that generate insertions or deletions. We propose that in earlier hominids and humans, CYP2D6 had increasingly become a vestigial characteristic unconstrained by dietary stressors, as a result of cultural survival strategies. Human CYP2D6 evolution was preferentially affected by random genetic drift, and not by adaptive or purifying selection.

Using a stable isotope technique which allows simultaneous and differential measuring of orally and intravenously administered drugs we compared the pharmacokinetics and pharmacodynamics of unlabelled modified release verapamil p.o. (steady state) and deuterated verapamil i.v. (single dose) following morning and evening administration.Twelve female and 12 male healthy volunteers were studied in a randomized, crossover design. During the last day of each treatment period (day 6 and day 10) pharmacokinetics and pharmacodynamics (PR interval) of verapamil were assessed; 1 h before ingestion of a new R/S-verapamil 240 mg modified release formulation (08.00 h vs 20.00 h) a single dose of 10 mg d7-R/S-verapamil was administered intravenously. Serum levels of unlabelled and labelled R/S-verapamil were measured by gas chromatography/mass spectrometry. In selected samples of serum which were chosen at tmin,po and tmax,po the enantiomers were separated by chiral high-performance liquid chromatography in order to calculate R- to S-verapamil serum concentration ratios.We observed no significant differences in pharmacokinetics (AUCpo, Cmax, tmax, CLo, F and R/S enantiomer ratio) between morning and evening treatment with modified release verapamil and there was no influence of time of dosing on mean prolongation of PR interval. AUCiv, CL, Vss and d7-R/d7-S enantiomer ratio following verapamil i.v. did not show circadian variation. t1/2 was slightly but statistically significantly increased after the morning infusion. PR-prolongation was significantly greater after verapamil i.v. in the morning than in the evening. The 90% confidence intervals of the differences between morning and evening administration in AUCpo, Cmax and AUCiv were within the equivalence range of 0.8-1.25.Time of dosing has no significant influence on pharmacokinetics and pharmacodynamics of this new modified release formulation of verapamil. Circadian variation in presystemic metabolism of verapamil was not observed.

The calcium channel blocker verapamil [2,8-bis-(3,4-dimethoxyphenyl)-6-methyl-2-isopropyl-6-azaoctanitrile+ ++] undergoes extensive biotransformation in man. We have previously demonstrated cytochrome P450 (CYP) 3A4 and 1A2 to be the enzymes responsible for verapamil N-dealkylation (formation of D-617 [2-(3,4-dimethoxyphenyl)-5-methylamino-2-isopropylvaleronitrile], and verapamil N-demethylation (formation of norverapamil [2,8-bis-(3,4-dimethoxyphenyl)-2-isopropyl-6-azaoctanitrile]), while there was no involvement of CYP3A4 and CYP1A2 in the third initial metabolic step of verapamil, which is verapamil O-demethylation. This pathway yields formation of D-703 [2-(4-hydroxy-3-methoxyphenyl)-8-(3,4-dimethoxyphenyl)-6-methyl-2-isopro pyl-6-azaoctanitrile] and D-702 [2-(3,4-dimethoxyphenyl)-8-(4-hydroxy-3-methoxyphenyl)-6-methyl-2-isopro pyl-6-azaoctanitrile]. The enzymes catalyzing verapamil O-demethylation have not been characterized so far. We have therefore identified and characterized the enzymes involved in verapamil O-demethylation in humans by using the following in vitro approaches: (I) characterization of O-demethylation kinetics in the presence of the microsomal fraction of human liver, (II) inhibition of verapamil O-demethylation by specific antibodies and selective inhibitors and (III) investigation of metabolite formation in microsomes obtained from yeast strain Saccharomyces cerevisiae W(R), that was genetically engineered for stable expression of human CYP2C8, 2C9 and 2C18. In human liver microsomes (n=4), the intrinsic clearance (CLint), as derived from the ratio of Vmax/Km, was significantly higher for O-demethylation to D-703 compared to formation of D-702 following incubation with racemic verapamil (13.9 +/- 1.0 vs 2.4 +/- 0.6 ml*min-1*g-1, mean+/-SD; p0.05), S-verapamil (16.8 +/- 3.3 vs 2.2 +/- 1.2 ml* min-1*g-1, p0.05) and R-verapamil (12.1 +/- 2.9 vs 3.6 +/- 1.3 ml*min-1*g-1; p0.05), thus indicating regioselectivity of verapamil O-demethylation process. The CLint of D-703 formation in human liver microsomes showed a modest but significant degree of stereoselectivity (p0.05) with a S/R-ratio of 1.41 +/- 0.17. Anti-LKM2 (anti-liver/kidney microsome) autoantibodies (which inhibit CYP2C9 and 2C19) and sulfaphenazole (a specific CYP2C9 inhibitor) reduced the maximum rate of formation of D-703 by 81.5 +/- 4.5% and 45%, that of D-702 by 52.7 +/- 7.5% and 72.5%, respectively. Both D-703 and D-702 were formed by stably expressed CYP2C9 and CYP2C18, whereas incubation with CYP2C8 selectively yielded D-703. In conclusion, our results show that enzymes of the CYP2C subfamily are mainly involved in verapamil O-demethylation. Verapamil therefore has the potential to interact with other drugs which inhibit or induce these enzymes.

Two different reaction mechanisms for the formation of the two human enamine-structured sparteine metabolites by cytochrome P450 2D6 have been discussed in the literature. These mechanisms are either initial one-electron oxidation of N1 of sparteine followed by deprotonation of the aminium radical cation, resulting in the formation of different carbon radicals and oxygen rebound of the carbon radicals, or oxidation of the carbon atoms adjacent to N1 by the enzyme, directly producing the respective carbon radicals. With a spectrum of deuterium-labeled isotopomers of sparteine, stereoselectivity and kinetic isotope effects of human sparteine metabolism were investigated by in vitro and in vivo experiments and were compared with chemical oxidation of 17-oxosparteine. These experiments revealed that the major human sparteine metabolite 2,3-didehydrosparteine is formed via highly stereoselective abstraction of the 2 beta-hydrogen atom; the deuterium label was completely retained during metabolism when 2R-[2H]sparteine was used as substrate. Chemical oxidation of 17-oxosparteine by Ce4+, as a model for one-electron oxidation of N1 of a sparteine-like structure, resulted in the sole formation of the 5,6-unsaturated enamine, and no 2,3-unsaturated enamine, structurally equivalent to the human major metabolite, was found. An unequivocal discrimination between the two possible reaction mechanisms was not possible by simple interpretation of the magnitude of the kinetic deuterium isotope effects. However, results of competitive and noncompetitive experiments revealed the presence of a nondissociative enzymatic mechanism for the formation of the two sparteine metabolites, i.e., the sparteine molecule that is bound to the substrate binding site of cytochrome P450 2D6 performs orientational changes without dissociating from the activated enzyme/substrate complex before the product-determining first irreversible reaction step. These results agree with the hypothesis that sparteine metabolism proceeds by direct carbon oxidation. Because electron transfer from amines to P450 may occur over some distance, the possibility of a sequential electron-proton transfer reaction during sparteine metabolism cannot be ruled out completely as an alternative reaction mechanism for sparteine metabolism.

Eine Vielzahl der heute therapeutisch eingesetzen Arzneimittel besitzt ein oder mehrere Chiralitatszentren und weist somit das Phanomen der Stereoisomerie auf. Eine Ubersicht von 866 regelmasig in Deutschland verschriebenen Arzneimitteln zeigt, das 449 chiral sind. Von diesen 449 duralen Verbindungen sind 186 naturlichen Ursprungs oder sind Modifikationen naturlich vorkommender Verbindungen. Aufgrund der Stereospezifitat der biologischen Synthese liegen 183 (98%) dieser Arzneimittel in stereochemisch reiner Form vor. Im Gegensatz dazu entstehen bei der chemischen Synthese von Arzneimitteln mit einem Chiralitatszentrum Razemate, wenn nicht eine stereospezifische Synthese eingesetzt wird. 225 (85%) der 266 Arzneimittel mit einem Chiralitatszentrum, die durch chemische Synthese gewonnen werden, liegen als Razemate, lediglich 41 als stereochemisch reine Verbindungen vor (Roth & Kleemann, 1982). Chemisch und in einem noch viel groseren Umfang biologisch sind Razemate keine Einzelverbindungen, sondern eine 50:50- Mischung von 2 Enanuomeren. In einem achiralen Milieu haben Enantiomere identische chemische und physikalische Eigenschaften mit Ausnahme der Tatsache, das sie polarisiertes Licht in die entgegengesetzte Richtung drehen. Interagieren Enantiomere jedoch mit anderen duralen Verbindungen, wie z.B. den Markomolekulen eines Rezeptors oder Enzyms, so beobachtet man haufig eine hohes Mas an Stereoselektivitat.

The activity of metabolizing enzymes determines plasma concentrations and hence effects of drugs. Identification of these enzymes may allow the prediction of both the interaction potential of drugs and the variability deriving from certain pathways. The antiarrhythmic propafenone is extensively biotransformed to the active metabolites 5-hydroxypropafenone and N-desalkylpropafenone. Whereas 5-hydroxylation is catalyzed by CYP2D6, the enzyme involved in N-dealkylation has not been identified. We, therefore, characterized the enzyme involved in the formation of N-desalkylpropafenone by using both in vitro [human liver microsomes, specific antibodies or inhibitors, and stably expressed cytochrome P450 (P450) enzymes] and in vivo (formation of N-desalkylpropafenone in patients under conditions of chronic therapy) approaches. Formation of N-desalkylpropafenone can be described by Michaelis-Menten kinetics. A strong correlation was observed between maximum rate of formation (Vmax) of N-desalkylpropafenone and the amount of CYP1A2 (r = 0.83, p0.001) and CYP3A (r = 0.54, p0.05) in the microsomal fraction of 20 human livers. In vitro intrinsic clearances (derived from Vmax/Km) indicated a wide interindividual variability in seven human livers (from 0.01 to 0.1 ml/hr/mg of protein). Antibodies directed against CYP3A and CYP1A2 inhibited formation of N-desalkylpropafenone by 54 +/- 10% and 24 +/- 16%, respectively. The CYP2D6-mediated formation of 5-hydroxypropafenone was unaffected by these antibodies. Verapamil (substrate of CYP3A4 and CYP1A2) and midazolam (substrate of CYP3A4) were competitive inhibitors of N-desalkylpropafenone formation (Ki = 70 microM and 25 microM for verapamil and midazolam, respectively). Coding sequences for CYP1A2 and CYP3A4 were inserted in a yeast expression vector and introduced into Saccharomyces cerevisiae strain W(R). Both CYP1A2 and CYP3A4 catalyzed N-dealkylation of propafenone, with specific activities of 0.32 pmol/min/pmol of P450 and 0.16 pmol/min/pmol of P450, respectively. Our data indicate that N-dealkylation of propafenone is mediated via CYP3A4 and CYP1A2. From experiments on the molecular level interactions of propafenone with other drugs that are metabolized by CYP3A4 and CYP1A2 can be predicted. Such interactions have been reported for cyclosporin, rifampicin, warfarin, and theophylline. Moreover, in vitro intrinsic clearances showed a wide interindividual variability. Therefore, variable plasma concentrations of the active metabolite N-desalkylpropafenone are expected in vivo. We tested this hypothesis in 14 patients (dose of 150 mg of propafenone three times per day) during chronic oral therapy and observed steady state plasma concentrations of N-desalkylpropafenone ranging from 4 to 293 ng/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Mexiletine is a low clearance drug which undergoes extensive metabolism in man. In vitro studies with human liver microsomes have suggested that major oxidation pathways of mexiletine are predominantly catalyzed by the genetically determined debrisoquine 4-hydroxylase (cytochrome P450IID6) activity. In this study, we investigated the role of debrisoquine polymorphism and the effects of low dose quinidine, a selective inhibitor of cytochrome P450IID6, on the disposition of mexiletine. Fourteen healthy volunteers, 10 with the extensive metabolizer (EM) and 4 with the poor metabolizer (PM) phenotype, received a single 200-mg dose of mexiletine hydrochloride orally on two occasions (1 week apart), once alone and once under steady-state conditions for quinidine (50 mg QID). During the phase mexiletine alone, total clearance, nonrenal clearance and partial metabolic clearance of mexiletine to hydroxymethylmexiletine, to m-hydroxymexiletine and to p-hydroxymexiletine were decreased in PM compared to EM (all P less than .05). In EM, quinidine decreased mexiletine total clearance from 621 +/- 298 to 471 +/- 214 ml/min (mean +/- S.D.; P less than .05) and mexiletine nonrenal clearance from 583 +/- 292 to 404 +/- 188 ml/min (P less than .05). Moreover, quinidine increased mexiletine elimination half-life in EM from 9 +/- 1 to 11 +/- 2 h (P less than .05). In these subjects, partial metabolic clearance to hydroxymethylmexiletine, m-hydroxymexiletine and p-hydroxymexiletine were decreased by quinidine coadministration 5-, 4- and 7-fold, respectively, whereas partial metabolic clearance to N-hydroxymexiletine was unaffected. Changes induced by quinidine in EM were correlated to their debrisoquine metabolic ratio. Thus, genetically determined or pharmacologically induced modulation of cytochrome P450IID6 activity represents a major determinant of mexiletine disposition.

Four different mutations of the cytochrome P450 CYP2D6 gene associated with the poor metabolizer phenotype (PM) of the debrisoquine/sparteine polymorphism were analyzed by Xba I restriction fragment length polymorphism (RFLP) analysis and a polymerase chain reaction (PCR)-based DNA amplification method in DNA of 394 healthy European subjects; 341 of these were phenotyped by sparteine or debrisoquine administration and urinary metabolic ratios (MR). Our study demonstrates the efficiency of the PCR-test for phenotype prediction; 96.4% of individuals were correctly predicted, i.e., 100% of the extensive metabolizers (EMs) and 86.0% of the poor metabolizers (PMs). In contrast, Xba I RFLP analysis was far less informative, predicting the phenotype in only 26.8% of PMs. By combining both DNA tests, the prediction rate of the PM phenotype increased to 90.6%. A point mutation at a splice-site consensus sequence termed D6-B represented the most common mutant CYP2D6 gene and accounted for more than 75% of mutant alleles. In addition, other known mutations such as D6-D (14%), D6-A (5%), and the rare D6-C mutation bring the identified mutant alleles to greater than 95% of all mutant PM-alleles. Most of Xba I 44-kb alleles were confirmed as mutant alleles carrying the D6-B mutation. However, 9.7% did not have this mutation and may express a functional CYP2D6 gene. Moreover, all Xba I 16 + 9-kb alleles contained the D6-B mutation. Heterozygous EM individuals had a significantly higher MR when compared to homozygous EMs. Genotyping provides an important advantage for investigations of the influence of CYP2D6 activity on drug therapy and its association with certain diseases.

Many drugs are used as racemates, and the enantiomers may differ in terms of pharmacological properties and disposition. Stereoselective disposition of the enantiomers can arise from metabolism of the enantiomers via different routes catalyzed by different enzymes. In contrast, the enantiomers may be metabolized by the same enzyme at different rates. In the latter case, the enantiomers can compete for this metabolic step, giving rise to the possibility of an enantiomer/enantiomer interaction. We have chosen the antiarrhythmic propafenone, for which in vivo data indicated an interaction between (S)- and (R)-propafenone, as a model substance to study the mechanism underlying that interaction in human liver microsomes. We used the cytochrome P450IID6-mediated 5-hydroxylation of propafenone as a model pathway, because this metabolic step constitutes the major route of biotransformation of propafenone. The Michaelis-Menten kinetics for 5-hydroxylation were determined after incubation of (R)- and (S)-propafenone and a pseudoracemate consisting of (S)-[2H4]propafenone and (R)-propafenone. Inhibition experiments were performed using (S)-[2H4]propafenone as an inhibitor of the 5-hydroxylation of (R)-propafenone, and vice versa. The kinetic model of mixed alternative substrates was used to simulate inhibition experiments. Experimental data were compared with those predicted by this model. We observed a substantial stereoselectivity after incubation of the individual enantiomers [(S)-propafenone: Vmax, 10.2 pmol/micrograms/hr, and Km, 5.3 microM; (R)-propafenone: Vmax, 5.5 pmol/micrograms/hr, and Km, 3.0 microM]. In contrast, no substrate stereoselectivity was observed after incubation of the pseudoracemate [3.1 pmol/micrograms/hr for (S)-[2H4]propafenone and 3.3 pmol/micrograms/hr for (R)-propafenone]. Application of the model revealed Ki values of 2.9 and 5.2 microM for the inhibition of 5-hydroxylation of (S)-[2H4]-propafenone by (R)-propafenone and for inhibition of 5-hydroxylation of (R)-propafenone by (S)-[2H4]-propafenone, respectively. The predicted and the experimental data were in good agreement, and both indicated the mode of inhibition to be competitive. In conclusion, the enantiomers of propafenone interact with respect to 5-hydroxylation, with (R)-propafenone being a more potent inhibitor than the S-enantiomer with respect to cytochrome P450IID6-mediated 5-hydroxylation. Because beta-blocking properties of propafenone reside in the S-enantiomer, inhibition of metabolism of this enantiomer by (R)-propafenone may have therapeutic consequences.

The oxidative N-dealkylation of verapamil has been studied in a family of five members with two propositi with an inherited deficiency of trimethylamine N-oxidation (fish-odour syndrome). The results were assessed for possible co-segregation of the trimethylamine N-oxidation phenotype and any observed deficiency in oxidative N-dealkylation. The general pattern of metabolism of verapamil in the five subjects studied was similar to that reported in earlier investigations. Moreover, there were no differences between the two affected subjects and other family members with respect to the metabolic pattern. It is concluded that there is no functional segregation with respect to the mechanisms controlling trimethylamine N-oxidation and verapamil N-dealkylation.

Approximately 50% of all drugs in therapeutic use have a chiral center and hence exhibit stereoisomerism. Chiral drugs derived from natural sources are stereochemical pure due to the stereospecificity of biological synthesis. By contrast, most of the drugs with a chiral center produced by chemical synthesis are only available as racemates, since chemical synthesis usually leads to a racemate unless stereospecific synthesis is employed [Roth & Kleemann, 1982; Simonyi, 1984].

Basically, the behaviour of all drugs is determined, at least to some extent, by genetic factors. It is thus possible to state that “pharmacogenetics” is a basic component of the interindividual variability of all drugs. It is, however, customary to restrict the term “pharmacogenetics” when one (or more) distinct subpopulations can be identified as far as their metabolic capacities towards drugs or group of drugs are concerned.

The biochemical basis underlying the genetic polymorphism of drug N-acetylation was investigated using a combination of in vivo and in vitro assays for arylamine N-acetyltransferase (NAT) activity and content in human liver. The acetylator phenotype of 26 surgical patients was determined using caffeine as an innocuous probe drug by measurement of the 5-acetyl-amino-6-formylamino-3-methyluracil to 1-methylxanthine molar ratio in urine. Liver wedge biopsies from these patients and livers from 24 organ donors were then used for measurement of N-acetyltransferase activity with the substrate sulfamethazine and for quantitation of immunoreactive N-acetyl-transferase protein. In vivo (caffeine metabolites in urine) and in vitro (sulfamethazine acetylation) measures of N-acetyl-transferase activity correlated very highly (r = 0.98). Moreover, in all subjects tested, slow acetylation both in vivo and in vitro was associated with a decrease in the quantity of immunodetectable N-acetyltransferase protein in liver cytosol relative to that seen in cytosols from rapid acetylator livers. Two kinetically distinct enzyme activities, designated NAT-1 and NAT-2, were partially purified from low- and high-activity livers and their relationship to acetylator status was determined. Low acetylation capacity was related to decreases in the liver content of both of these immunologically related proteins. The results demonstrate that genetically defective arylamine N-acetylation is due to a parallel decrease in the quantity of two structurally and functionally similar acetylating enzymes.

A World Health Organisation (European Regional Office) working party has been established to review the progress of clinical pharmacology in European countries. As part of this review a questionnaire on the teaching of chlinical pharmacology was sent to the Deans of all 350 medical schools in the region. Very few replies were received from U.S.S.R., Greece and Portugal and these countries' returns were not analysed further. The overall compliance rate (excluding these countries) was 82% with a figure of 84% from Western Europe and 74% from Eastern Europe. An average time of 96 h (range 0–320) was devoted to pharmacology teaching in the medical curriculum in Western Europe with 124 (0–240) h in Eastern Europe. In contrast 28 h (0–210) was devoted to clinical pharmacology teaching in Western Europe and 27 h (0–90) in Eastern Europe. On average in Western Europe each medical school had 2 individuals trained in clinical pharmacology with 1.3 posts in the subject and the figures for Eastern Europe were 2.3 and 1.1 respectively. However these figures hide a wide variance in the teaching of clinical pharmacology. Particularly in Western Europe there are a number of medical schools in Italy, Spain and the Federal Republic of Germany (FRG) where clinical pharmacology is not taught and there is a dearth of individuals trained in the subject. Every effort to encourage clinical pharmacology and its teaching should be made, particularly in these countries.

Previous investigations in animals and one retrospective study in man suggest that verapamil can prevent anthracycline-induced cardiomyopathy. In the following study, patients with acute myeloid leukemia (AML) treated with double induction and consolidation chemotherapy (AML COOP study 1986, [3]) were randomized in a group with and without accompanying low-dose oral verapamil treatment. Since July 1986, 64 patients have been included. Thirty patients have been evaluated for pre- and posttreatment cardiological investigations. So far, no significant difference in cardiotoxicity has been observed either between the verapamil and nonverapamil group or between the two induction chemotherapy regimens (TAD/TAD - TAD/HAM).

The use of anthracyclines and anthrachinones in antineoplastic treatment is limited by the possible induction of irreversible cardiomyopathy [1, 2, 6, 7, 9]. In 1976, Daniels et al. observed a protective effect of subcutaneously administered verapamil in white rabbits [4]. Mullerleile et al. reported a beneficial effect of low-dose oral verapamil in 22 patients with different tumors treated with doxorubicin [10]. The aim of this study was to determine whether verapamil can protect against anthracycline cardiomyopathy without altering the clinical efficiency of the antineoplastic regimen in patients with acute myeloid leukemia (AML) [8, 10–12].

LKM-1 ANTIBODY POSITIVE AUTOIMMUNE HEPATITIS IN IDENTICAL TWINS: IN VITRO INHIBITION OF THE TARGET ANTIGEN CYTOCHROME P45O db1 AND IN VIVO PHENOTYPE

A high-performance liquid chromatography method is described which allows for the simultaneous determination of antipyrine, 4-hydroxyantipyrine, norantipyrine and 3-hydroxyantipyrine. Hydrolysis conditions with respect to source of glucuronidase, amount of glucuronidase, pH of incubation and addition of antioxidant to the incubation medium proved to be very critical for norantipyrine and to a lesser extent 4-hydroxyantipyrine. The method is of good sensitivity, specificity, accuracy and reproducibility. Thus, the method is well suited for detailed metabolic studies.

The diminished sympathomimetic pressor activity of monohydroxylated phenylalkylamines after oral administration has been attributed to incomplete enteric absorption. Therefore, urinary excretion of the unchanged drug and its metabolites has been compared after intravenous and oral administration of 3H-m-octopamine to eight patients. Identical amounts of 3H-activity (80% of the dose) were excreted after the two routes of dosing, so enteric absorption has been assumed to be complete. Significant differences were found in the fraction of free urinary m-octopamine, which amounted to 10.5% of the dose after infusion and 0.58% after oral administration. The only metabolic pathways for m-octopamine are deamination and conjugation. Following oral administration the percentage of conjugates was considerably higher than after intravenous infusion. This metabolic pattern appears typical of all phenylalkylamines with a hydroxyl group in the meta position. Ring hydroxylation to catecholamines was not observed. The enzymes mainly responsible for conjugation after oral administration are located in the gut wall. The resulting ""first pass effect'', i.e. metabolism prior to the access to the central compartment, can account for the diminished pharmacodynamic effect after dosing by this route.

Rifampicin clearance increases during multiple dose therapy due to its known induction of hepatic enzymes, which leads to autoinduction of its own metabolism. Therefore, we investigated the pharmacokinetics of rifampicin (RMP) and its major metabolites, 25-desacetyl- (D-RMP) and 3-formyl-RMP (F-RMP), in two groups of six patients with active pulmonary tuberculosis who received either chronic oral or intravenous RMP therapy in combination with isoniazid and ethambutol. Serum concentrations of RMP were determined by HPLC at the beginning and after one and three weeks of chronic dosing. It was found that while the volume of distribution of RMP remained constant over the study period, the systematic clearance of RMP increased during multiple dosing. The bioavailability of oral RMP decreased from 93% after a single oral dose to 68% after three weeks. The decrease could not be attributed solely to an increase in hepatic clearance. This suggests that gut wall metabolism may also be induced during chronic therapy with RMP, leading to presystemic clearance of RMP. The metabolites were also eliminated more rapidly after repeated RMP administration (induction of phase II transformations).