Magnus Ingelman-Sundberg

Genetic polymorphisms of cytochrome P450 2D6 (CYP2D6): clinical consequences, evolutionary aspects and functional diversity

CYP2D6 is of great importance for the metabolism of clinically used drugs and about 20–25% of those are metabolised by this enzyme. In addition, the enzyme utilises hydroxytryptamines as endogenous substrates. The polymorphism of the enzyme results in poor, intermediate, efficient or ultrarapid metabolisers (UMs) of CYP2D6 drugs. It is plausible that the UM genotype, where more than one active gene on one allele occurs, is the outcome of selective dietary selection in certain populations in North East Africa. The UM phenotype affects 5.5% of the population in Western Europe. A hypothesis for the evolutionary basis behind selection for CYP2D6 gene duplications is presented in relation to selection for Cyp6 variants in insecticide resistant Drosophila strains. The polymorphism of CYP2D6 significantly affects the pharmacokinetics of about 50% of the drugs in clinical use, which are CYP2D6 substrates. The consequences of the polymorphism at ordinary drug doses can be either adverse drug reactions or no drug response. Examples are presented where CYP2D6 polymorphism affects the efficacy and costs of drug treatment. Predictive CYP2D6 genotyping is estimated by the author to be beneficial for treatment of about 30–40% of CYP2D6 drug substrates, that is, for about 7–10% of all drugs clinically used, although prospective clinical studies are necessary to evaluate the exact benefit of drug selection and dosage based on the CYP2D6 genotype.

A common novel CYP2C19 gene variant causes ultrarapid drug metabolism relevant for the drug response to proton pump inhibitors and antidepressants

Many drugs, including proton pump inhibitors and certain antidepressants, are metabolized by the polymorphic cytochrome P450 (CYP) 2C19 enzyme. A significant portion of extensive metabolizers do not reach appropriate drug levels, and our objective was to investigate any genetic background.

Polymorphic human cytochrome P450 enzymes: an opportunity for individualized drug treatment

Approximately 40% of human P450-dependent drug metabolism is carried out by polymorphic enzymes, which can cause abolished, quantitatively or qualitatively altered or enhanced drug metabolism. The latter situation is due to stable duplication, multiduplication or amplification of active genes, most likely in response to dietary components that have resulted in a selection of alleles with multiple non-inducible genes. Several examples exist where subjects carrying certain alleles suffer from a lack of drug efficacy due to ultrarapid metabolism or, alternatively, adverse effects from the drug treatment due to the presence of defective alleles. Knowledge in this field has grown rapidly and can now be applied to both drug development and clinical practice. This is facilitated by the recent development of high-throughput methods for mutation detection and oligonucleotide chips array technology for the identification of a multitude of mutations in the genes encoding drug-metabolizing enzymes. The outcome will allow for safer and more efficient drug therapies.

Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1).

The liver microsomal ethanol-inducible cytochrome P-450 (P-450IIE1) form is known to exhibit a high rate of oxidase activity in the absence of substrate and it was therefore of interest to evaluate whether this form of P-450 could contribute to microsomal and liposomal NADPH-dependent oxidase activity and lipid peroxidation. The rate of microsomal NADPH-consumption, O2--formation, H2O2-production and generation of thiobarbituric acid (TBA) reactive substances correlated to the amount of P-450IIE1 in 28 microsomal samples from variously treated rats. Anti-P-450IIE1 IgG inhibited, compared to control IgG, microsomal H2O2-formation by 45% in microsomes from acetone-treated rats and by 22% in control microsomes. NADPH-dependent generation of TBA-reactive products was completely inhibited by these antibodies, whereas preimmune IgG was essentially without effect. Liposomes containing reductase and P-450IIE1 were peroxidized in a superoxide dismutase (SOD) sensitive reaction at a 5-10-fold higher rate than membranes containing 3 other forms of cytochrome P-450. Lipid peroxidation in reconstituted vesicles dependent on the presence of P-450IIB1 was by contrast not inhibited by SOD. Microsomal peroxidase activities, using 15-(S)-hydroperoxy-5-cis-8,11,13-trans-eicosatetraenoic acid as a substrate were high in microsomes from phenobarbital- or ethanol-treated rats but low in membranes from isoniazid-treated rats, having the highest relative level of P-450IIE1. It is suggested that the oxidase activity of P-450IIE1 contributes to microsomal NADPH-dependent lipid peroxidation. The combined action of the oxidase activity by P-450IIE1 and the peroxidase activities by P-450IIB1 and other forms of P-450 may be important for the high rate of lipid peroxidation observed in e.g. microsomes from ethanol- or acetone-treated rats. The possible importance of cytochrome P-450IIE1-dependent lipid peroxidation in vivo after ethanol abuse is discussed.

Nomenclature for human CYP2D6 alleles.

To standardize CYP2D6 allele nomenclature, and to conform with international human gene nomenclature guidelines, an alternative to the current arbitrary system is described. Based on recommendations for human genome nomenclature, we propose that alleles be designated by CYP2D6 followed by an asterisk and a combination of roman letters and arabic numerals distinct for each allele with the number specifying the key mutation and, where appropriate, a letter specifying additional mutations. Criteria for classification as a separate allele and protein nomenclature are also presented.

Cytochrome P450 pharmacogenetics and cancer

The cytochromes P450 (CYPs) are key enzymes in cancer formation and cancer treatment. They mediate the metabolic activation of numerous precarcinogens and participate in the inactivation and activation of anticancer drugs. Since all CYPs that metabolize xenobiotics are polymorphic, much emphasis has been put on the investigation of a relationship between the distribution of specific variant CYP alleles and risk for different types of cancer, but a consistent view does not yet exist. This is to a great extent explained by the fact that the CYPs involved in activation of precarcinogens are in general not functionally polymorphic. This is in contrast to CYPs that are active in drug biotransformation where large interindividual differences in the capacity to metabolize therapeutic drugs are seen as a consequence of polymorphic alleles with altered function. This includes also some anticancer drugs like tamoxifen and cyclophosphamide metabolized by CYP2D6, CYP2C19 and CYP2B6. Some P450 forms are also selectively expressed in tumours, and this could provide a mechanism for drug resistance, but also future therapies using these enzymes as drug targets can be envisioned. This review gives an up-to-date description of our current knowledge in these areas.

Human drug metabolising cytochrome P450 enzymes: properties and polymorphisms

The cytochrome P450s are responsible for about 75% of phase I dependent drug metabolism and for the metabolism of a huge amount of dietary constituents and endogenous chemicals. The human has 59 active genes, and 6 of those encode important drug metabolising enzymes. About 40% of cytochrome P450 dependent drug metabolism is catalysed by polymorphic enzymes and such drug P450 interactions are frequently seen in adverse drug reaction reports. In this contribution an update of human cytochrome P450 enzymology and pharmacogenetics is given with particular emphasis on CYP1B1, CYP2B6, CYP2E1 and CYP3As.

Activation of protein kinase C by lipoxin A and other eicosanoids. Intracellular action of oxygenation products of arachidonic acid

Arachidonic acid, linolenic acid and 14 different oxygenated fatty acid derivatives were tested as activators of human protein kinase C in vitro using histone as substrate. Lipoxin A (5,6,15L-trihydroxy-7,9,11,13-eicosatetraenoic activated the kinase in the presence of calcium at 30 fold lower concentration (1 microM) than did arachidonic acid or 1,3-dioleoylglycerol. The methyl ester of lipoxin A and the free acids of leukotriene B4 as well as two lipoxin B isomers were without effect. In contrast, linolenic acid, leukotriene C4, certain mono- and dihydroxylated eicosanoids and one lipoxin B isomer had stimulatory effects, albeit at higher concentrations. The substrate specificity of protein kinase C activated by lipoxin A proved to be different from that of the phosphatidylserine or phorbol ester activated kinase. Results of the present study suggest that arachidonic acid derived oxygenation products, in particular lipoxin A, may serve as intracellular activators of protein kinase C.

CHARACTERISATION AND PCR-BASED DETECTION OF A CYP2A6 GENE DELETION FOUND AT A HIGH FREQUENCY IN A CHINESE POPULATION

Cytochrome P450 2A6 is an important human hepatic P450 which activates pre‐carcinogens, oxidises some drugs and constitutes the major nicotine C‐oxidase. In fact, results have been presented in the literature which suggested a relationship between the distribution of defective CYP2A6 alleles and smoking behaviour as well as cigarette consumption. In the present report, we describe the structure of a novel CYP2A locus where the whole CYP2A6 gene has been deleted, resulting in an abolished cytochrome P450 2A6‐dependent metabolism. The origin of this locus is apparently due to an unequal crossover event between the 3′‐flanking region of the CYP2A6 and CYP2A7 genes. A rapid PCR‐based method for the detection of the CYP2A6del allele was developed and the allele frequency was 15.1% among 96 Chinese subjects, but only 1.0% in Finns (n=100) and 0.5% in Spaniards (n=100). In the Chinese population, we did not detect any CYP2A6*2 alleles using an improved genotyping procedure, in contrast to the 11–20% previously reported. It is concluded that genotyping for the CYP2A6del allele is of great importance in studies correlating, for example, smoking behaviour, pre‐carcinogen activation or drug metabolism to the CYP2A6 genotype, in particular when oriental populations are investigated.

Carbon tetrachloride-induced lipid peroxidation dependent on an ethanol-inducible form of rabbit liver microsomal cytochrome P-450

Treatment of rats with ethanol or rabbits with either imidazole or pyrazole, agents known to induce the ethanol‐inducible form of liver microsomal cytochrome P‐450 (P‐450 LMeb), caused, compared to controls, 3–25‐fold enhanced rates of CCl4‐dependent lipid peroxidation or chloroform production in isolated liver microsomes. No significant differences were seen when the rate of CCl4‐dependent lipid peroxidation was expressed relative to the amount of P‐450 LMeb in the various types of microsomal preparations. In reconstituted membranous systems, this type of P‐450 was a 100‐fold more effective catalyst of CCl4 metabolism than either of the cytochromes P‐450 LM2 or P‐450 LM4. It is proposed that the induction of this isozyme provides the explanation on a molecular level for the synergism seen of ethanol on CCl4‐dependent hepatotoxicity.