Urs A. Meyer

Genotyping of poor metabolisers of debrisoquine by allele-specific PCR amplification

A method for genotyping poor metabolisers of debrisoquine is based on specific polymerase chain reaction (PCR) amplification of parts of mutant genes for hepatic cytochrome P450IID6. Analysis by restriction fragment length polymorphism allowed identification of only 25% of poor metabolisers, but when it was combined with allele-specific PCR over 95% of poor metabolisers could be identified. The PCR method also allowed the identification of heterozygous carriers of mutant alleles.

Induction of Drug Metabolism: The Role of Nuclear Receptors

Induction of drug metabolism was described more than 40 years ago. Progress in understanding the molecular mechanism of induction of drug-metabolizing enzymes was made recently when the important roles of the pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), two members of the nuclear receptor superfamily of transcription factors, were discovered to act as sensors for lipophilic xenobiotics, including drugs. CAR and PXR bind as heterodimeric complexes with the retinoid X receptor to response elements in the regulatory regions of the induced genes. PXR is directly activated by xenobiotic ligands, whereas CAR is involved in a more complex and less well understood mechanism of signal transduction triggered by drugs. Most recently, analysis of these xenobiotic-sensing nuclear receptors and their nonmammalian precursors such as the chicken xenobiotic receptor suggests an important role of PXR and CAR also in endogenous pathways, such as cholesterol and bile acid biosynthesis and metabolism. In this review, recent findings regarding xenosensors and their target genes are summarized and are put into an evolutionary perspective in regard to how a living organism has derived a system that is able to deal with potentially toxic compounds it has not encountered before.

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.

Nomenclature for N-acetyltransferases.

A consolidated classification system is described for prokaryotic and eukaryotic N-acetyltransferases in accordance with the international rules for gene nomenclature. The root symbol (NAT) specifically identifies the genes that code for the N-acetyltransferases, and NAT* loci encoding proteins with similar function are distinguished by Arabic numerals. Allele characters, denoted by Arabic numbers or by a combination of Arabic numbers and uppercase Latin letters, are separated from gene loci by an asterisk, and the entire gene-allele symbols are italicized. Alleles at the different NAT* loci have been numbered chronologically irrespective of the species of origin. For designation of genotypes at a single NAT* locus, a slash serves to separate the alleles; in phenotype designations, which are not italicized, alleles are separated by a comma.

Nutritional Regulation of Hepatic Heme Biosynthesis and Porphyria through PGC-1α

Inducible hepatic porphyrias are inherited genetic disorders of enzymes of heme biosynthesis. The main clinical manifestations are acute attacks of neuropsychiatric symptoms frequently precipitated by drugs, hormones, or fasting, associated with increased urinary excretion of delta-aminolevulinic acid (ALA). Acute attacks are treated by heme infusion and glucose administration, but the mechanisms underlying the precipitating effects of fasting and the beneficial effects of glucose are unknown. We show that the rate-limiting enzyme in hepatic heme biosynthesis, 5-aminolevulinate synthase (ALAS-1), is regulated by the peroxisome proliferator-activated receptor gamma coactivator 1alpha (PGC-1alpha). Elevation of PGC-1alpha in mice via adenoviral vectors increases the levels of heme precursors in vivo as observed in acute attacks. The induction of ALAS-1 by fasting is lost in liver-specific PGC-1alpha knockout animals, as is the ability of porphyrogenic drugs to dysregulate heme biosynthesis. These data show that PGC-1alpha links nutritional status to heme biosynthesis and acute hepatic porphyria.

Genomic organization of the human CYP3A locus: identification of a new, inducible CYP3A gene.

Proteins encoded by the human CYP3A genes metabolize every second drug currently in use. The activity of CYP3A gene products in the general population is highly variable and may affect the efficacy and safety of drugs metabolized by these enzymes. The mechanisms underlying this variability are poorly understood, but they include gene induction, protein inhibition and unknown genetic polymorphisms. To better understand the regulation of CYP3A expression and to provide a basis for a screen of genetic polymorphisms, we determined and analysed the sequence of the human CYP3A locus. The 231 kb locus sequence contains the three CYP3A genes described previously (CYP3A4, CYP3A5 and CYP3A7), three pseudogenes as well as a novel CYP3A gene termed CYP3A43. The gene encodes a putative protein with between 71.5% and 75.8% identity to the other CYP3A proteins. The highest expression level of CYP3A43 mRNA is observed in the prostate, an organ with extensive steroid metabolism. CYP3A43 is also expressed in several other tissues including liver, where it can be induced by rifampicin. CYP3A43 transcripts undergo extensive splicing. The identification of a new member of the CYP3A family and the characterization of the full CYP3A locus will aid efforts to identify the genetic variants underlying its variable expression. This, in turn, will lead to a better optimization of therapies involving the numerous substrates of CYP3A proteins.

Overview of enzymes of drug metabolism.

Most pharmacologically active molecules are lipophilic and remain un-ionized or only partially ionized at physiological pH. Biotransformation means that a lipid-soluble xenobiotic or endobiotic compound is enzymatically transformed into polar, water-soluble, and excretable metabolites. The major organ for drug biotransformation is the liver. The metabolic products often are less active than the parent drug or inactive. However, some biotransformation products (metabolites) may have enhanced activity or toxic effects. Thus biotransformation may include both “detoxication” and “toxication” processes. One of the major enzyme systems that determines the organisms capability of dealing with drugs and chemicals is represented by the cytochrome P450 monooxygenases. Studies in the last 15 years have provided evidence that cytochrome P450 occurs in many different forms or “isozymes” which differ in spectral, chemical, and immunological properties and have different substrate affinities. These isozymes also differ in their regulation and tissue distribution. Recombinant DNA studies indicate that between 40 and 60 structural genes code for different cytochrome P450 isozymes in a single organism. Other enzyme systems include dehydrogenases, oxidases, esterases, reductases, and a number of conjugating enzyme systems including glucuronosyltransferases, sulfotransferases, glutathione S-transferases, etc. Environmental and genetic factors cause interindividual and intraindividual differences in drug metabolism and may alter the balance between toxification and detoxification reactions. Genetic polymorphisms lead to subpopulations of patients with decreased, absent, or even increased activities of certain reactions (e.g., CYP2D6, CYP2C19, N-acetyltransferase polymorphism). Environmental factors such as other drugs, steroids, dietary factors, alcohol, and cigarette smoke can induce or inhibit drug-metabolizing enzymes and cause intraindividual variation.

Pregnane X Receptor Is a Target of Farnesoid X Receptor

The pregnane X receptor (PXR) is an essential component of the bodys detoxification system. PXR is activated by a broad spectrum of xenobiotics and endobiotics, including bile acids and their precursors. Bile acids in high concentrations are toxic; therefore, their synthesis is tightly regulated by the farnesoid X receptor, and their catabolism involves several enzymes regulated by PXR. Here we demonstrate that the expression of PXR is regulated by farnesoid X receptor. Feeding mice with cholic acid or the synthetic farnesoid X receptor (FXR) agonist GW4064 resulted in a robust PXR induction. This effect was abolished in FXR knock-out mice. Long time bile acid treatment resulted in an increase of PXR target genes in wild type mice. A region containing four FXR binding sites (IR1) was identified in the mouse Pxr gene. This region was able to trigger an 8-fold induction after GW4064 treatment in transactivation studies. Deletion or mutation of single IR1 sites caused a weakened response. The importance of each individual IR1 element was assessed by cloning a triple or a single copy and was tested in transactivation studies. Two elements were able to trigger a strong response, one a moderate response, and one no response to GW4064 treatment. Mobility shift assays demonstrated that the two stronger responding elements were able to bind FXR protein. This result was confirmed by chromatin immunoprecipitation. These results strongly suggest that PXR is regulated by FXR. Bile acids activate FXR, which blocks synthesis of bile acids and also leads to the transcriptional activation of PXR, promoting breakdown of bile acids. The combination of the two mechanisms leads to an efficient protection of the liver against bile acid induced toxicity.

Identification of three cytochrome P450 isozymes involved in N-demethylation of citalopram enantiomers in human liver microsomes.

Using in vitro techniques, the present study demonstrates that CYP2D6, and 3A4 are involved in N-demethylation of citalopram (CIT) enantiomers. Human liver microsome incubations performed with specific inhibitors of these three CYP isozymes have shown up to 60% inhibition of demethylcitalopram production. cDNA expressed human cytochrome P-450 3A4, 2C19 and 2D6 isozymes, but not CYP1A2, were identified to be involved in N-demethylation of CIT enantiomers. Kinetics using cDNA expressed CYP2C19 and CYP3A4 show K(m) values in the same range: 198 microM, 211 microM for CYP2C19 and 169 microM, 163 microM for CYP3A4 for S- and R-CIT demethylation, respectively. In contrast, kinetics using cDNA expressed CYP 2D6 show a K(m) of 18 microM and 22 microM for S- and R-CIT demethylation, respectively. Nevertheless, kinetics using cDNA expressed CYP2C19 and 3A4 have a range of Vmax values ten times higher than that of CYP2D6. For this reason, intrinsic clearance values (Vmax/K(m)) for S- and R-CIT were within a small range for these three isozymes: 0.25 to 0.39 microliter h-1 x pmol-1 of CYP. CYP2D6 has an opposite stereoselectivity in the biotransformation of CIT enantiomers than CYP2C19 and 3A4; the S/R ratios of the intrinsic clearance were 0.71, 1.57 and 1.37, respectively. Taking into account that CYP isozymes are expressed at various levels, CYP2D6, which is expressed at lower levels than CYP2C19 and CYP3A4, plays a minor role in the biotransformation of CIT enantiomers. These results confirm that the use of cDNA expressed CYP isozymes is a potent tool for the measurement of kinetic constants and help to predict clearance modifications of CIT enantiomers, especially in poor metabolizers of mephenytoin (with a CYP2C19 deficiency) or patients comedicated with potent CYP2C19 or 3A4 inhibitor(s). For instance, fluvoxamine (100 microM) inhibits CIT N-demethylation by 64% in microsomes.

Omics and Drug Response

A new generation of technologies commonly named omics permits assessment of the entirety of the components of biological systems and produces an explosion of data and a major shift in our concepts of disease. These technologies will likely shape the future of health care. One aspect of these advances is that the data generated document the uniqueness of each human being in regard to disease risk and treatment response. These developments have reemphasized the concept of personalized medicine. Here we review the impact of omics technologies on one key aspect of personalized medicine: the individual drug response. We describe how knowledge of different omics may affect treatment decisions, namely drug choice and drug dose, and how it can be used to improve clinical outcomes.