Cytochrome P450 enzyme system: genetic polymorphisms and impact on clinical pharmacology

Transcription

1 Review Article Ann Clin Biochem 1999; 36: Cytochrome P450 enzyme system: genetic polymorphisms and impact on clinical pharmacology Jan van der Weide and Linda S W Steijns From the Department of Clinical Chemistry, Psychiatric Hospital Veldwijk, PO Box BA Ermelo, the Netherlands SUMMARY. The cytochrome P450 (CYP) enzyme system is involved in the metabolism and elimination of numerous widely used drugs. The capacity of this system varies from one person to another, leading to variable drug excretion rates and intersubject differences in the final serum drug concentrations. For this reason, therapeutic response and side-effects vary widely between patients treated with the same dose ofdrug. The intersubject variability in metabolic rate is largely determined by genetic factors. Some CYP enzymes, including CYP2D6 and CYP2CI9, are genetically polymorphic. Several mutant alleles have been described. Environmental factors such as smoking, diet and co-administration of medications might also influence the CYP enzyme activity. By the use of genotyping or phenotyping methods every individual can be classified as either a poor, an intermediate, an extensive or an ultrarapid metabolizer. If this could be performed prior to drug therapy, the knowledge could be applied to drug selection and dose adjustment in order to reach therapeutic serum drug levels. Additional key phrases: drug metabolism; genotyping NOMENCLATURE The most important elimination pathway for lipophilic drugs is cytochrome P450 (CYP) (EC )-dependent oxidation. CYP mediates biotransformation to polar metabolites, which can be excreted by the kidneys. The human hepatic CYP system consists of over 30 related isoenzymes with different, sometimes overlapping, substrate specificities. The CYP enzymes have been classified in a systematic way on the basis of their amino acid sequence. A standard nomenclature system, categorizing enzymes in gene families and subfamilies, has been developed.' Families are indicated by the abbreviation for cytochrome P450 (CYP), followed by an arabic number. Members of a family are at least 40% identical. Within a family, enzymes with greater than 55% sequence homology are included in the same subfamily. Subfamilies are indicated by a letter following the family number. Individual genes, coding for one specific isoenzyme, have a second arabic number Correspondence: Dr J van der Weide. jvdweide@worldonline.nl after the letter.i-' The nomenclature system is depicted in Table I. At least 74 CYP gene families, of which 14 are ubiquitous in all mammals, have been described so far. 1 The enzymes belonging to the families CYPI, CYP2 and CYP3 catalyse the oxidative biotransformation of exogenous compounds, including many drugs, (pro)carcinogens, (pro) mutagens and alcohols. The other CYP families are involved in the metabolism of endogenous substances, such as fatty acids, prostaglandins and steroid and thyroid hormones." Table 2 shows some CYP enzymes that are relevant for TABLE 1. Cytochrome P450 (CYP) nomenclature system Family CYPI CYP2---> CYP3 Subfamily lcyp2c---> CYP2D Individual gene [ CYP2C9 CYP2Cl9 722

2 Cytochrome P450 enzyme system 723 drug metabolism and some clinically important substrates. POLYMORPHIC DRUG METABOLISM Genetic polymorphism The metabolic capacity of the CYP enzyme system is not equal in all members of a population. As a result, the metabolic conversion and excretion rate of drugs vary between individuals, from extremely slow to ultrafast. For many drugs, four major phenotypes can be distinguished: poor metabolizers (PMs), intermediate metabolizers (IMs), extensive metabolizers (EMs) and ultrarapid metabolizers (UMs). The intersubject variability in metabolic rate is largely determined by genetic factors.v' A number of CYP enzymes are known to be genetically polymorphic. Mutant alleles carrying certain nucleotide substitutions, deletions, insertions or gene conversions are known, which may result in CYP enzymes with abnormal activity. Some mutants, the so-called null alleles, lead to enzyme deficiency or total absence of enzyme activity. This genetically determined variance in enzyme activity results in the different drug metabolism phenotypes. CYP2D6 Of all CYP enzymes, the highly genetically polymorphic enzyme debrisoquine-4-hydroxylase, or CYP2D6, has been the most extensively studied. CYP2D6 is involved in the oxidative metabolism of more than 40 widely prescribed drugs (Table 2). The gene that codes for the CYP2D6 enzyme has been localized to chromosome 22, where it forms part of the CYP2D gene cluster with two non-functional pseudogenes, termed CYP2D7P and CYP2D8P. 7 The CYP2D6 gene contains nine exons within a total of 4378 base pairs. More than 50 different CYP2D6 alleles have so far been identified.f-? Their expanding number has led to agreements on a common nomen- clature, which has recently been updated.!? The allelic variants are divided into subgroups, sharing the same characteristic mutation(s). The CYP2D6 allele subgroups, associated with absent, decreased, normal or increased enzyme activity, are listed in Table 3. 9,11-27 Five to 10% of Caucasians lack CYP2D6 activity completely because of the inheritance of two mutant CYP2D6 null alleles. These subjects are classified as PMs, with an impaired metabolism of CYP2D6 substrates.p The majority of defective allelic variants of the CYP2D6 gene that give rise to the PM phenotype have now been identified. Up to 7% of Caucasians are UMs of CYP2D6 substrates, owing to the inheritance of alleles with duplication or amplification of functional CYP2D6 genes, causing an excessive amount ofenzyme to be expressed.u-" The metabolic capacity of the rest of the population lies somewhere between the extremes. IMs have mutations on the CYP2D6 gene, which cause only a partial decrease in enzyme activity. Subjects who are either homozygous for the normal-functioning alleles, or heterozygous with one active and one defect allele, are classified as EMs. Among EMs, metabolic rate can range considerably. In subjects homozygous for the active alleles, CYP2D6 drugs are metabolized more efficiently than the heterozygous genotypes. The latter are more at risk for drug-drug interaction if two or more CYP2D6 drugs have to be administered concomitantly. In general, the drug metabolism phenotype is determined by the number of functional CYP2D6 genes present.s!' In non-caucasian populations, PM and UM phenotypes of CYP2D6 substrates may occur with different prevalences. In Orientals, for example, there are hardly any PMs and UMs «I %), but the frequency of the 1M phenotype is very high" In some African populations, prevalences ofup to 29% for the UM phenotype have been reported.'! TABLE 2. Enzyme CYPIA2 CYP2CI9 CYP2D6 CYP2C8-9 CYP3A4 CYP2El Cytochrome P450 (CYP) enzymes and substrates Substrates Caffeine, clozapine, fluvoxarnine, haloperidol, paracetamol, theophylline Amitriptyline, barbiturates (hexobarbital), clomipramine, diazepam, imipramine, rnephenytoin, omeprazole, proguanil Antiarrhythrnics, antihypertensives, {J-blockers, tricyclic antidepressants, antipsychotics, selective serotonin reuptake inhibitors, morphine derivatives Ibuprofen, phenytoin, S-warfarin Carbamazepine, quinidine Acetone, ethanol, paracetarnol

3 724 Van der Weide and Steijns TABLE 3. Cytochrome P450 (CYP) allele subgroups, characteristic mutationts), enzyme activity and frequency among Caucasians Enzyme Allelic frequency Designation Characteristic mutation(s) activity (%) Reference CYP2D6*1 Wild type Normal CYP2D6*2 G 1749C, C 2938 T, G 4268C substitutions Normal 30 II CYP2D6*3 A 2637 deletion Deficient 2 12 CYP2D6*4 G I934A substitution Deficient CYP2D6*j Gene deletion Deficient 2 14 CYP2D6*6 T 1795 deletion Deficient 2 15 CYP2D6*7 A 3023C substitution Deficient CYP2D6*8 G I846 T substitution Deficient CYP2D6*9 (A2701-A2703) or (G2702-A2704) deletion Decreased CYP2D6*10 C 188 T, G 1749C, G 426SC substitutions Decreased CYP2D6*11 G 971C substitution Deficient CYP2D6*12 G 212A substitution Deficient CYP2D6*13 Hybrid: 2D7 exon I, 2D6 exons 2-9 Deficient CYP2D6*14 G 1s46A substitution Deficient CYP2D6*15 T 226 insertion Deficient CYP2D6*16 Hybrid: 2D7 exons 1-7, 2D6 exons 8-9 Deficient CYP2D6*1 x2 Gene duplication Increased I 23 CYP2D6*2x2 Gene duplication Increased 1 5 II CYP2D6*4x2 Gene duplication Deficient CYP2C19*1 Wild type Normal CYP2C19*2 G 6SIA substitution exon 5 Deficient CYP2CI9*3 G6]6A substitution Deficient CYP2C19*4 A1G substitution Deficient CYP2C19 A second CYP enzyme which exhibits genetic polymorphism is S-mephenytoin-hydroxylase, or CYP2C19. The enzyme catalyses the metabolism of some tricyclic antidepressants and barbiturates (see Table 2). In addition to the wild-type gene, three allelic variants are known, which are associated with the complete absence of CYP2Cl9 activity (see Table 3) Occurring homozygously or heterozygously together, these null alleles give rise to the PM phenotype, with an impaired metabolism of CYP2Cl9 substrates. In contrast to the CYP2D6 polymorphism, no UM phenotype has been demonstrated for CYP2C19. The prevalence of PM caused by CYP2C19 deficiency is 2-6% in Caucasians and as high as 18-23% in Oriental populations.p Other CYP enzymes The enzyme CYP2C9 is also known to be polymorphic. Several mutant alleles have been characterized, probably associated with decreased enzyme activity.v For other CYP enzymes involved in the metabolism and elimination of drugs, such as CYP1A2 and CYP2El, there is some evidence of genetic heterogeneity in the population. For la2, about 12% PMs have been demonstrated.w" However, the exact molecular basis for these polymorphisms and the clinical significance have not been characterized.pv" Non-genetic factors Although individual metabolic capacity IS determined mainly by genetic background, several internal and environmental factors might influence the activity of CYP enzymes: for example, age, gender, certain diseases with hepatic involvement, smoking, nutrition and a1cohop 34.35,37-40 In addition, the use of comedication can change the metabolic capacity of the CYP enzyme system. Many drugs are known to interfere with certain enzymes by inhibition or induction, or by utilizing those enzymes in their metabolism. For example, there is competition for CYP2D6 between antipsychotics and tricyclic antidepressants. If they are adjusted concomitantly, the metabolism of the latter will be slowed down because ofthe higher affinity ofthe former for CYP2D6. 35 In general, any substrate for a specific CYP enzyme is potentially capable of inhibiting the metabolism of another substrate." Drugs such as carbamazepine and

4 Cytochrome P450 enzyme system 72S barbiturates act as inducers of several CYP enzymes, thereby speeding up the metabolism of co-administered CYP substrates. Many selective serotonin reuptake inhibitors, as well as quinidine which is not a CYP2D6 substrate, are known to be inhibitors of CYP2D6. When administered with drugs that are metabolized by this enzyme, EMs will, in effect, be converted to the PM phenotype. 6,41.42 CLINICAL CONSEQUENCES OF POLYMORPHIC DRUG METABOLISM There are numerous case reports demonstrating that variations in CYP enzyme activity can lead to intersubject differences in therapeutic efficacy of drugs. 9,35,36,43-45 In general, PM subjects, either genetically determined or drug-induced, will develop higher serum drug concentrations in comparison with EMs, causing an increased risk of suffering from concentration-dependent sideeffects when subjected to standard recommended doses. UM subjects, on the other hand, will not reach therapeutic serum concentrations upon treatment with standard doses. They may fail to respond to treatment, leading to false accusations of non-compliance. When the parent compound is a prodrug, which requires bioactivation by the enzyme to the active drug, the opposite effects can be observed in PMs and UMs. As individual differences in drug disposition could be compensated for in part by dose adjustment according to the metabolic capacity," determination of drug metabolism phenotypes prior to drug therapy seems useful. PM and UM subjects can thus be identified and drug selection and dosage could be tailored to these individual patients from the beginning, which will help avoid adverse reactions or therapeutic failure. 36,43,45 However, the clinical significance of a specific enzyme polymorphism for a specific drug is not always clear and depends on several factors. A substantial amount of the drug should be metabolized by the enzyme. When the affected pathway plays only a minor part in the overall elimination of the drug, or alternative excretion pathways are available, the effect of the polymorphism on metabolic rate, and therefore on clinical outcome, will be negligible. In addition, the resulting variability in elimination rate should have some clinical impact. This is the case with drugs having a narrow therapeutic range, i.e. when the toxic serum concentration is just above the therapeutic concentration. On the basis of these criteria, the frequency of use and the usually (life)long period ofpharmacotherapy, the polymorphism of CYP2D6 in particular seems to be of great clinical importance for tricyclic antidepressants, certain antipsychotics and cardiovascular cornpounds.l'v" Individual metabolic capacity could be estimated by either phenotyping or genotyping approaches. Phenotyping is accomplished by administration of a probe drug, known to be selectively metabolized by the CYP enzyme under study, followed by measurement of the metabolic ratio (the ratio of drug dosage or unchanged drug to metabolite in serum or urine). Phenotyping takes into account all internal and external factors influencing the activity of the specific enzyme, and it reveals drug-drug interactions or defects in the overall process of drug metabolism. However, the method has several drawbacks. The protocols of testing are rather complicated and, especially in the case of psychiatric patients, difficult to perform correctly. In addition, there are risks of adverse drug reactions. Genotyping involves identification of defined genetic mutations on the CYP genes that give rise to the specific drug metabolism phenotype. By screening for genetic variants, an individual's drug metabolism phenotype can be characterized. Genotyping is simple to perform and provides results within 24 h, allowing for rapid intervention. The method just requires a small blood, tissue or urine sample, which can be taken at any time, and it is not affected by underlying diseases or by co-administration of drugs. Genotyping needs to be done only once in a lifetime. GENOTYPING METHODS FOR IDENTIFICATION OF PM AND UM SUBJECTS CYP2D6 Poor metabolization caused by CYP2D6 enzyme deficiency may be detectable with close to 100% accuracy by screening for all known nonfunctional CYP2D6 alleles. 8,43,45 The CYP2D6 null alleles and their inactivating mutation(s) are listed in Table 3. Most of these deficiencycausing mutations can be detected by fast and easy assays based on the well-known polymerase chain reaction (PCR), followed by restrictiondigestion.pr" When there is no suitable restriction endonuclease available for detection of a specific mutation, as is the case with CYP2D6*6

5 726 Van der Weide and Steijns for example, PCR products could be used also for dot-blotting, followed by hybridization with labelled allele-specific oligonucleotide probes." Nearlyevery CYP2D6 null allele listed in Table 3 can be detected by one of these methods. However, it is unnecessary and impractical to carry out routine screening for so many mutations. In our hospital we perform genotyping for the three most common defect gene variants, i.e. CYP2D6*3, CYP2D6*4 and CYP2D6*5, in every patient before starting pharmacotherapy. This allows identification of at least 95% of PMs in the Caucasian population.t" Screening for the remaining null alleles is performed when poor metabolism appears to be present on the basis of the dose/concentration ratio and is not found in the three allelic variants mentioned above. UM caused by CYP2D6 gene duplication can be detected by PCR-based methods as well. 49,5o The genomic organization of an allele with duplicate CYP2D6 genes is shown in Fig. I. With primer pair cyp17/cyp32, a 3'6-kb PCR fragment amplified from the 2D6-2D6 region is observed in subjects having duplicate genes. In addition, a 5 2-kb fragment from a 2D7-2D6 intergenic region should be obtained from every sample as an internal control of the PCR reaction. Amplification of a 3 2-kb fragment indicative of a 2D6-2D6 intergenic sequence by primer combination cyp207/cyp32 is performed as a control reaction. This assay yields product only in subjects with duplicate genes. The resulting PCR fragments are separated and detected in ethidium bromide-containing agarose gels. In the case of homozygosity, it is clear which CYP2D6 allelic variant has been duplicated. In heterozygotes, additional PCR assays, 1kb as depicted in Fig. I for genotype *1/*4, should be performed in order to identify the duplicated gene.? CYP2C19 Poor metabolization caused by CYP2CI9 deficiency can be detected by genotyping only with a sensitivity of about 80% in Caucasians." By usinga PCR assay followed by restriction-digestion the non-functional *2 variant can be identified, accounting for 75% of CYP2C19 alleles in PMs genotyped." In the Oriental population, a second mutation, CYP2C19*3, was found in conjunction with *2, accounting for 100% of alleles in Oriental PMS. 26 In Caucasians this *3 allele is rare, just like the recently characterized allele CYP2C19*4.n Other mutations associated with CYP2CI9 deficiency are yet to be discovered. CYP2C9 The allelic variants CYP2C9*2 and CYP2C9*3, probably leading to impaired metabolism of CYP2C9-depcndent drugs, can be detected by PCR and restriction-digestion. In Caucasians, allele frequencies of 12 5% and 8 5% are reported, respectively. 32 Further studies are required to assess to what extent CYP2C9 genetic polymorphism affects enzyme activity and metabolic rate. The specificity of both CYP2D6 and CYP2C19 genotyping for determination of drug metabolism phenotypes is 100% when appropriate control reactions are built in." The presence of an allele with duplicate functional CYP2D6 genes, in addition to a normal CYP2D7 ~ IICYP2DS-dUp1trrJC----- CYP2D > <32 A1 17> <32 A2 <32 207> <32 B <32~"'F :'~" c 2x2F~ <92 17> 207> 2x2F> c 42~O 42,7 "I allele: CCTGGTGAGCCCAT + Banll (GPuGCPy'C) -> 231 bp + 33 bp "4 allele: CCTGGTGACCCCAT + Banll (GPuGCPy'C) -> 264 bp FIGURE 1. Genomic organization ofan allele with duplicate CYP2D6 genes. The shaded area indicates the 'extra' sequence compared with a 'normal' allele. The polymerase chain reaction-based methodfor identification ofcyp2d6 gene duplication is depicted schematically: A 1 = internal control product; A2 = product indicative for gene duplication; B= positive control product; C and c= identification 0/*4 allele duplication (only in the case ofthe * 1/*4 genotype). Reprinted in part from Lovlie et al.,50 with permission from Elsevier Science. Ann CU" Biochem 1999: 36

6 Cytochrome P450 enzyme system 727 functioning allele, always results in ultrarapid metabolism of CYP2D6 drugs. Genotyping is not very laborious. Screening for the most common defective allelic variants of CYP2D6, CYP2Cl9 and CYP2C9 and for the CYP2D6 gene duplication could be done within 24 h at a cost of 18 each when performed as a routine assay. CONCLUSION It is the opinion of some physicians and medical scientists that genotyping should be performed routinely in each instance when the drug of choice is substrate for a polymorphic enzyme, especially when a close relationship between serum drug concentration and effect is demonstrated. 6 36,43.44 Alternatively, genotyping should be indicated when individuals demonstrate suboptimal response to drugs that are substrates for polymorphic enzymes. Genetic analysis of CYP2D6 and CYP2Cl9 gives a fairly good prediction of PM and UM metabolic phenotypes, and this information can be used for individualization and optimization of drug therapy. Dose adjustment or the selection of an alternative drug that is not substrate for the polymorphic enzyme will help to avoid therapeutic failure and wrong suspicion of noncompliance in VMs, and the development of side-effects in PMs. In the case of 1M and heterozygous EM phenotypes, the clinician should be aware of a higher risk of drug-drug interactions when taking co-medication. There would be a considerable cost associated with screening all patients before starting pharmacotherapy. On the other hand, there will be a reduction in costs associated with toxic episodes or therapeutic failure and subsequent intervention. Overall, the cost could be neutral. Some hospitals have implemented routine genotyping techniques, but the relevance of this practice has been the subject of much debate. For many drugs the clinical effect of polymorphic drug metabolism is not clear. Serious side-effects do not invariably occur in persons with elevated serum drug levels. Moreover, administration of drugs based on genotyping is no guarantee that appropriate serum drug levels are attained. Other CYP enzymes may be involved in the elimination of a drug, and nongenetic factors may also influence enzyme activity. To evaluate the contribution of genetic polymorphism to adverse drug reactions and to define the true relevance of genotyping in clinical practice, controlled prospective studies using a multidisciplinary approach are necessary. Based on the evidence at hand, it is suggested that an appropriate practice would be genotyping, forewarning the physician that a patient is susceptible to an increased risk of side-effects, non-response or drug-drug interaction, in combination with monitoring the patient's progress regularly and determining serum levels of drugs at appropriate time intervals. REFERENCES Nelson DR, Koymans L, Kamataki T, Stegeman 11, Feyereisen R, Waxman DJ, et al. P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 1996; 6: Nebert DW, Nelson DR, Coon MJ, Estabrook RW, Feyereisen R, Fujii-Kuriyama Y, et at. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature. DNA Cell Bioi 1991; 10: Murray M. P450-enzymes: inhibition mechanisms, genetic regulation and effect of liver disease. C/in Pharmacokinet 1992; 23: Gonzalez FJ, Idle JR. Pharmacogenetic phenotyping and genotyping - present status and future potential. C/in Pharmacokinet 1994; 26: May DG. Genetic differences in drug disposition. J Clin Pharmacol 1994; 34: Meyer VA, Amrein R, Balant LP, Bertilsson L, Eichelbaum M, Guentert TW, et al. Antidepressants and drug-metabolizing enzymes - expert group report. Acta Psychiatr Scand 1996; 93: Kimura S, Vmeno M, Skoda RC, Meyer VA, Gonzalez FJ. The human debrisoquine 4-hydroxylase (CYP2D6) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene, Am J Hum Genet 1989; 45: Marez D, Legrand M, Sabbagh N, Lo-Guidice JM, Spire C, Lafitte 11, et al. Polymorphism of the cytochrome P450 CYP2D6 gene in a European population: characterization of 48 mutations and 53 alleles, their frequencies and evolution. Pharmacogenetics 1997; 7: Sachse C, Brockmoller J, Bauer S, Roots I. Cytochrome P450 2D6 variants in a Caucasian population: allele frequencies and phenotypic consequences. Am J Hum Genet 1997; 60: Daly AK, Brockmoller J, Broly F, Eichelbaum M, Evans WE, Gonzalez FJ, et al. Nomenclature for human CYP2D6 alleles. Pharmacogenetics 1996; 6: II Johansson I, Lundqvist E, Bertilsson L, Dahl ML, Sjoqvist F, Ingelman-Sundberg M. Inherited amplification of an active gene in the cytochrome P450 CYP2D6 locus as a cause of ultrarapid metabolism of debrisoquine. Proc Natl Acad Sci USA 1993; 90: Kagimoto M, Heim M, Kagimoto K, Zeugin T, Meyer VA. Multiple mutations of the hyman

7 728 Van der Weide and Steijns cytochrome P450IID6 gene (CYP2D6) in poor metabolizers of debrisoquine. J Bioi Chem 1990; 265: Gough AC, Miles JS, Spurr NK, Moss JE, Gaedigk A, Eichelbaum M, et al. Identification of the primary gene defect at the cytochrome P450 CYP2D locus. Nature 1990; 347: Gaedigk A, Blum M, Gaedigk R, Eichelbaum M, Meyer VA. Deletion of the entire cytochrome P450 CYP2D6 gene as a cause of impaired drug metabolism in poor metabolizers of the debrisoquine/sparteine polymorphism. Am J Hum Genet 1991; 48: IS Saxena R, Shaw GL, Reiling MV, Frame IN, Moir DT, Evans WE, et at. Identification of a new variant CYP2D6 allele with a single base deletion in exon 3 and its association with the poor metabolizer phenotype. Hum Mol Genet 1994; 3: 923--{j 16 Evert B, Griese EV, Eichelbaum M. A missense mutation in exon 6 of the CYP2D6 gene leading to a histidine 324 to proline exchange is associated with the poor metabolizer phenotype of sparteine. Naunyn-Schmiedeberg's Arch Pharmaco/1994; 350: Tyndale R, Aoyama T, Broly F, Matsunanga T, Inaba T, Kalow W, et al. Identification of a new variant CYP2D6 allele lacking the codon encoding Lys-281: possible association with the poor metabolizer phenotype. Pharmacogenetics 1991; 1: Yokota H, Tamura A, Furuya H, Kimura S, Watanabe M, Kanazawa I, et al. Evidence for a new variant CYP2D6 allele CYP2D6J in a Japanese population associated with lower in vivo rates of sparteine metabolism. Pharmacogenetics 1993; 3: Marez D, Sabbagh N, Legrand M, Lo-Guidice JM, Boone P, Broly F. A novel CYP2D6 allele with an abolished splice recognition site associated with the poor metabolizer phenotype. Pharmacogenetics 1995; 5: Marez D, Legrand M, Sabbagh N, Lo-Guidice JM, Boone P, Broly F. An additional allelic variant of the CYP2D6 gene causing impaired metabolism of sparteine. Hum Genet 1996; 97: Daly AK, Fairbrother KS, Andreassen OA, London SJ, Idle JR, Steen VM. Characterization and PCR-based detection of two different hybrid CYP2D7P/CYP2D6 alleles associated with the poor metabolizer phenotype. Pharmacogenetics 1996; 6: Sachse C, Brockrnoller J, Bauer S, Reum T, Roots I. A rare insertion of T 226 in exon I of CYP2D6 causes a frameshift and is associated with the poor metabolizer phenotype: CYP2D6* 15. Pharmacogenetics 1996; 6: Dahl ML, Johansson I, Bertilsson L, Ingelman Sundberg M, Sjoqvist F. Vltrarapid hydroxylation of debrisoquine in a Swedish population. Analysis of the molecular genetic basis. J Pharmacol Exp Ther 1995; 274: Masimirembwa CW, Johansson I, Hasler JA, Ingelman-Sundberg M. Genetic polymorphism of cytochrome P450 CYP2D6 in a Zimbabwean population. Pharmacogenetics 1993; 3: DeMorais SM, Wilkinson GR, Blaisdell J, Nakamura K, Meyer UA, Goldstein JA. The major genetic defect responsible for the polymorphism of S-mephenytoin metabolism in humans. J Biol Chem 1994; 269: I DeMorais SM, Wilkinson GR, Blaisdell J, Meyer VA, Nakamura K, Goldstein JA. Identification of a new genetic defect responsible for the polymorphism of S-mephenytoin metabolism in Japanese. Mol Pharmacol 1994; 46: Ferguson RJ, DeMorais SM, Benhamou S, Bouchardy C, Blaisdell J, Ibeanu G, et al. A new genetic defect in human CYP2C19: mutation of the initiation codon is responsible for poor metabolism of S-mephenytoin. J Pharmacol Exp Ther 1998; 284: Alvan G, Bechtel P, Iselius L, Gundert-Remy U. Hydroxylation polymorphisms of debrisoquine and mephenytoin in European populations. Eur J cu«pharmacol 1990; 39: Agundez JAG, Ledesma MC, Ladero JM, Benitez J. Prevalence of CYP2D6 gene duplication and its repercussion on the oxidative phenotype in a white population. Clin Pharmacol Ther 1995; 57: Bertilsson L, Dahl ML, Ingelman-Sundberg M, Johansson I, Sjoqvist F. Interindividual and interethnic differences in polymorphic drug oxidation - implications for drug therapy with focus on psychoactive drugs. In: Pacifici G, Fracchia GN, eds. Advances in Drug Metabolism in Man. Luxembourg: Office for Official Publications of the European Communities, 1995: Alklillu E, Persson I, Bertilsson L, Johansson I, Rodrigues F, Inghelman-Sundberg M. Frequent distribution of ultrarapid metabolizers of debrisoquine in an Ethiopian population carrying duplicated and multiduplicated functional CYP2D6 alleles. J Pharmacol Exp Ther 1996; 278: 441--{j 32 Kimura M, Ieiri I, Mamiya K, Urae A, Higuchi S. Genetic polymorphism of cytochrome P450s, CYP2CI9 and CYP2C9 in a Japanese population. Ther Drug Manit 1998; 20: Butler MA, Lang NP, Young JF. Determination of CYPIA2 and NAT2 phenotype in human populations by analysis of caffeine urinary metabolites. Pharmacogenetics 1992; 2: Batt AM, Magdalou J, Vincent-Viry M, Ouzzine M, Fournel-Gigleux S, Galteau MM, et al. Drug metabolizing enzymes related to laboratory medicine: cytochromes P-450 and VDP-glucuronosyltransferases. Clin Chim Acta 1994; 226: Glue P, Banfield C. Psychiatry, psychopharmacology and P450s. Hum Psychopharmacol 1996; 11: Linder MW, Prough RA, Valdes R. Pharmacogenetics: a laboratory tool for optimizing therapeutic efficiency. Clin Chem 1997; 43: 254--{i6 37 Sonne J. Drug metabolism in liver disease: implication for therapeutic drug monitoring. Ther Drug Monit 1996; 18: I 38 Kall M, Yang 0, Andersen 0, Clausen J. In vivo induction of human phase I and phase II enzymes by diet. J Cell Biochcm Suppl 1995; 19A: 197

8 Cytochrome P450 enzyme system Vainer JL, Chouinard G. Interaction between caffeine and clozapine. J Clin Psychopharmacol 1994; 14: Carillo JA, Jerling M, Bertilsson L. Comments to 'Interaction between caffeine and clozapine'. J Clin Psychopharmacol 1995; 15: Nemeroff CB, DeVane CL, Pollock BG. Newer antidepressants and the cytochrome P450 system. Am J Psychiatry 1996; 153: Michalets EL. Clinically significant cytochrome P 450 drug interactions. Pharmacotherapy 1998; 18: Chen S, Wen-Hwei C, Blouin RA, Mao Z, Humphries LL, Craig Meek Q, et al. The cytochrome P450 2D6 (CYP2D6) enzyme polymorphism: screening costs and influence on clinical outcomes in psychiatry. Clin Pharmacol Ther 1996; 60: Kroemer HK, Eichelbaum M. Molecular bases and clinical consequences of genetic cytochrome P450 2D6 polymorphism. Life Sci 1995; 56: Griese EU, Zanger UM, Brudermanns U, Gaedigk A, Mikus G, Morike T, et al. Assessment of the predictive power of genotypes for the in-vivo catalytic function of CYP2D6 in a German population. Pharmacogenetics 1998; 8: Wolf CR, Moss JE, Miles JS, Gough AC, Spurr NK. Detection of debrisoquine hydroxylation phenotypes. Lancet 1990; 336: Ezquieta B, Varela JM, Jariego C, Oliver A, Gracia R. Nonisotopic detection of point mutations in CYP21B gene in steroid 2I-hydroxylase deficiency. cu«chem 1996; 42: Dahl ML, Johansson I, Porsmyr Palmertz M, Ingelman-Sundberg M, Sjiiqvist F. Analysis of the CYP2D6 gene in relation to debrisoquine and desipramine hydroxylation in a Swedish population. cu«pharmacol Ther 1992; 51: Steijns LSW, Van der Weide J. Ultrarapid drug metabolism: PCR-based detection of CYP2D6 gene duplication. Clin Chem 1998; 44: Lovlie R, Daly AK, Molven A, Idle JR, Steen VM. Ultrarapid metabolizers of debrisoquine: characterization and PCR-based detection of alleles with duplication of the CYP2D6 gene. FEBS Lett 1996; 392: 3Q-4 51 Brockmiiller J, Roots 1. Assessment of liver metabolic function: clinical implications. Clin Pharmacokinet 1994; 27: Acceptedfor publication 9 April/999 Ann C/in Biochem 1999: 36