Hannu Raunio

Inhibition and induction of human cytochrome P450 enzymes: current status

Variability of drug metabolism, especially that of the most important phase I enzymes or cytochrome P450 (CYP) enzymes, is an important complicating factor in many areas of pharmacology and toxicology, in drug development, preclinical toxicity studies, clinical trials, drug therapy, environmental exposures and risk assessment. These frequently enormous consequences in mind, predictive and pre-emptying measures have been a top priority in both pharmacology and toxicology. This means the development of predictive in vitro approaches. The sound prediction is always based on the firm background of basic research on the phenomena of inhibition and induction and their underlying mechanisms; consequently the description of these aspects is the purpose of this review. We cover both inhibition and induction of CYP enzymes, always keeping in mind the basic mechanisms on which to build predictive and preventive in vitro approaches. Just because validation is an essential part of any in vitro–in vivo extrapolation scenario, we cover also necessary in vivo research and findings in order to provide a proper view to justify in vitro approaches and observations.

CYP3A4 allelic variants with amino acid substitutions in exons 7 and 12 : Evidence for an allelic variant with altered catalytic activity

To determine the existence of mutant and variant CYP3A4 alleles in three racial groups and to assess functions of the variant alleles by complementary deoxyribonucleic acid (cDNA) expression.

Prodrugs—from Serendipity to Rational Design

The prodrug concept has been used to improve undesirable properties of drugs since the late 19th century, although it was only at the end of the 1950s that the actual term prodrug was introduced for the first time. Prodrugs are inactive, bioreversible derivatives of active drug molecules that must undergo an enzymatic and/or chemical transformation in vivo to release the active parent drug, which can then elicit its desired pharmacological effect in the body. In most cases, prodrugs are simple chemical derivatives that are only one or two chemical or enzymatic steps away from the active parent drug. However, some prodrugs lack an obvious carrier or promoiety but instead result from a molecular modification of the prodrug itself, which generates a new active compound. Numerous prodrugs designed to overcome formulation, delivery, and toxicity barriers to drug utilization have reached the market. In fact, approximately 20% of all small molecular drugs approved during the period 2000 to 2008 were prodrugs. Although the development of a prodrug can be very challenging, the prodrug approach represents a feasible way to improve the erratic properties of investigational drugs or drugs already on the market. This review introduces in depth the rationale behind the use of the prodrug approach from past to present, and also considers the possible problems that can arise from inadequate activation of prodrugs.

Diagnosis of polymorphisms in carcinogen-activating and inactivating enzymes and cancer susceptibility--a review.

Up to 90% of all cancers are possibly caused by environmental factors, such as tobacco smoke, diet and occupational exposures. The majority of chemical carcinogens require metabolic activation before they interact with cellular macromolecules and can cause cancer initiation. The xenobiotic-metabolising machinery contains two main types of enzymes: the phase-I cytochromes P-450 (CYP) mediating oxidative metabolism, and phase-II conjugating enzymes. Several phase-I and phase-II genes have recently been cloned and identified in humans. Many of them show polymorphism and have been suggested to contribute to individual cancer susceptibility as genetic modifiers of cancer risk. Altered phenotypes and genotypes in the CYP subfamilies CYP1A1, CYP2D6 and CYP2E1 have been associated with tobacco smoke-induced lung cancer and other cancers. Defective glutathione S-transferase (GST) and N-acetyltransferase (NAT) enzymes have been associated with an increased risk of developing lung and bladder cancer. There are also several studies in each category in which no associations have been found. The risk of developing lung cancer is dramatically (up to 40-fold) elevated in subpopulations having simultaneously high-risk genotypes in CYP1A1 and GSTM1. There are several difficulties in this area of research. First, many of the observed restriction-fragment length polymorphisms (RFLPs) are due to mutations in introns or other silent areas of DNA, raising the possibility that any associations found between RFLPs and cancer occur only by chance. Second, biologically plausible mechanisms linking genotypes and cancer are lacking in most of the observed cases.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Expression and Regulation of Xenobiotic- Metabolizing Cytochrome P450 (CYP) Enzymes in Human Lung

Pathogenesis of lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is tightly linked to exposure to environmental chemicals, most notably tobacco smoke. Many of the compounds associated with these diseases require an enzymatic activation to exert their deleterious effects on pulmonary cells. These activation reactions are mostly catalyzed by cytochrome P450 (CYP) enzymes. Interindividual differences in the in situ activation and inactivation of chemical toxicants may contribute to the risk of developing lung diseases associated with these compounds. This review summarizes in detail the expression of individual CYP forms in human pulmonary tissue and gives a view on the significance of the pulmonary expression of CYP enzymes. The localization of individual CYP enzymes in various cell types of human lung and the emerging field of regulation of human pulmonary CYP enzymes are discussed. At least CYP1A1 (in smokers), CYP1B1, CYP2B6, CYP2E1, CYP2J2, and CYP3A5 proteins are expressed in human lung, and also other CYP forms are likely to be expressed. Xenobiotic-metabolizing CYP enzymes are mostly expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages in human lung, although individual CYP forms have different patterns of localization in pulmonary tissues. Problems in animal to human lung toxicity extrapolation and several specific aspects requiring more detailed assessment are identified.

Expression of xenobiotic-metabolizing cytochrome P450 forms in human adult and fetal liver

Expression of human cytochrome P450 (CYP) genes in human adult and fetal liver were studied using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. In adult liver mRNA of CYPs 1A1, 1A2, 2A6/2A7, 2B6/2B7, 2C8-19, 2D6, 2E1, 3A3/3A4 and 3A7 were detected while CYPs 2F1 and 4B1 were absent. In fetal liver mRNA of CYPs 2C8, 2D6, 3A3/3A4 and 3A7 were found but all other forms studied were undetectable. The results provide a comprehensive qualitative picture of the expression of CYP genes in families CYP1 through CYP4 in human adult and fetal liver.

Expression of xenobiotic-metabolizing cytochrome P450 Forms in human full-term placenta

The expression of individual xenobiotic-metabolizing cytochrome P450 (CYP) genes in human placenta was studied at the mRNA level by reverse transcriptase-polymerase chain reaction (RT-PCR). mRNAs of CYP1A1, CYP2E1, CYP2F1, CYP3A3/4, CYP3A5, and CYP4B1 were detected by RT-PCR, and CYP1A2, CYP2A6/7, CYP2B6/7, CYp2C8-19, CYP2D6, and CYp3A7 were not detected. Several enzyme activity assays and immunoblasts were used to further characterize expression of forms producing detectable mRNA transcripts. The catalytic activities of 7-ethoxycoumarin O-deethylase (ECOD), 7-ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) were substantially increased in response to maternal cigarette smoking, and paralleled the amount of CYP1A1 mRNA and protein. Aromatase activities were slightly lower in placentas exposed to cigarette smoke compared with nonexposed placentas. These data show that several xenobiotic-metabolizing CYP genes are expressed in human placenta at a low level. The significant of such low-level expression is unknown, but it may have local physiological or toxic consequences.

Xenobiotic-Metabolizing Cytochrome P450 Enzymes in the Human Feto-Placental Unit: Role in Intrauterine Toxicity

Practically all lipid-soluble xenobiotics enter the conceptus through placental transfer. Many xenobiotics, including a number of clinically used drugs, are known to cause unwanted effects in the embryo or fetus, including in utero death, initiation of birth defects, and production of functional abnormalities. It is well established that numerous xenobiotics are not necessarily toxic as such, but are enzymatically transformed in the body to reactive and toxic intermediates. The cytochrome P450 (CYP) enzymes are known to catalyze oxidative metabolism of a vast number of compounds, including many proteratogens, procarcinogens, and promutagens. About 20 xenobiotic-metabolizing CYP forms are known to exist in humans. Most of these forms are most abundant in the liver, but examples of exclusively extrahepatic CYP forms also exist. Unlike rodents, the liver of the human fetus and even embryo possesses relatively well-developed metabolism of xenobiotics. There is experimental evidence for the presence of CYP1A1, CYP1B1, CYP2C8, CYP2D6, CYP2E1, CYP3A4, CYP3A5, and CYP3A7 in the fetal liver after the embryonic phase (after 8 to 9 weeks of gestation). Significant xenobiotic metabolism occurs also during organogenesis (before 8 weeks of gestation). Also, some fetal extrahepatic tissues, most notably the adrenal, contain substantial levels of CYP enzymes. The full-term human placenta is devoid of many CYP activities present in liver. Placental CYP1A1 is highly inducible by maternal cigarette smoking. Other forms present in full-term placenta include CYP4B1 and CYP19 (steroid aromatase), which also contribute to the oxidation of some xenobiotics. At earlier stages of pregnancy, the placenta may express a wider array of CYP genes, including CYP2C, CYP2D6, and CYP3A7. Due to the small size of the fetus and low abundance of CYPs in placenta, the contribution of feto-placental metabolism to overall gestational pharmacokinetics of drugs is probably minor. In contrast, several toxic outcomes have been ascribed to altered metabolic patterns in the feto-placental unit, including a putative association between reduced placental oxidative capacity and birth defects. Examples of human teratogens that are substrates for CYP enzymes include thalidomide, phenytoin, ethanol, and several hormonal agents. Recent studies have improved our understanding of the expression and regulation of individual CYP genes in the fetus and placenta, and the stage is set for applying this knowledge with more precision to the role of xenobiotic metabolism in abnormal intrauterine development in humans.

Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism

The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C‐oxidase. A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed. Two defective alleles have hitherto been identified, one of which is very common in Asian populations. Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption. Here, we have re‐evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3′ flanking region of 30–40% of CYP2A6*1 alleles results in genotype misclassification. In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects. In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles. One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D). The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events. We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.