Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2002-08-30
2004-09-28
Johannsen, Diana B. (Department: 1634)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S019000, C435S091200, C536S023200, C536S023500, C536S024310
Reexamination Certificate
active
06797477
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the identification of various polymorphisms in the cytochrome P450 2A6 gene and methods and reagents for genotyping and phenotyping individuals using such polymorphisms.
BACKGROUND OF THE INVENTION
The mammalian liver contains enzymes that convert various chemical compositions to products which can more easily be eliminated from the body. One enzyme system which plays a major role in determining the rate of elimination of these drugs is cytochrome P450. The cytochrome P450's are among the major constituent proteins of the liver mixed function monooxygenases. They play a central role in the metabolism of steroids, the detoxification of drugs and xenobiotics, and the activation of procarcinogens. Without cytochrome P450 and related enzymes, naturally occurring and man-made foreign chemicals would accumulate in the body. Additionally, the biological effects of some chemicals are due solely to metabolites generated by cytochrome P450 and/or related enzymes. Metabolism by cytochrome P450 enzymes is often the rate-limiting step in pharmaceutical elimination. For example, most phase I metabolism of drugs and environmental pollutants is performed by cytochrome P450 enzymes. In this process, one or more water-soluble groups (such as hydroxyl) are introduced into the fat-soluble parent molecule, thereby rendering it vulnerable to attack by the phase II conjugating enzymes. The increased water-solubility of phase I and especially phase II products permits ready excretion. Consequently, factors that lessen the activity of cytochrome P450 enzymes usually prolong the effects of pharmaceuticals, whereas factors that increase cytochrome P450 activity have the opposite effect.
The phenobarbital-inducible P450 gene, CYP2A6 or CYP4502A6, is a member of a multigene family located on by human chromosome 19. Induction by phenobarbital is mediated almost entirely at the level of transcription. P450 enzymes, as well as other so called “drug-metabolizing” enzymes, play an important role in maintaining the steady-state levels of endogenous ligands involved in ligand-modulated transcription of genes effecting homeostasis, growth, differentiation, and neuroendocrine functions.
Genetic polymorphisms of cytochrome P450 enzymes result in subpopulations of individuals that are distinct in their ability to perform particular drug biotransformation reactions. These phenotypic distinctions have important implications for selection of drugs. For example, a drug that is safe when administered to the majority of humans may cause intolerable side-effects in an individual suffering from a defect in a cytochrome P450 enzyme required for detoxification of the drug. Alternatively, a drug that is effective in most humans may be ineffective in a particular subpopulation because of the lack of a particular cytochrome P450 enzyme required for conversion of the drug to a metabolically active form. Accordingly, it is important for both drug development and clinical use to screen drugs to determine which cytochrome P450 enzymes are required for activation and/or detoxification of the drug.
It is also important to identify those individuals who are deficient in a particular P450 enzyme. This type of information has been used to advantage in the past for developing genetic assays that predict phenotype and thus predict an individual's ability to metabolize a given drug. Information such as this would be of particular value in determining the likely side effects and therapeutic failures of various drugs and routine phenotyping could be recommended for certain categories of patients.
Wood and Conney, Science, 1974, vol. 185, pages 612-614, found that basal and phenobarbital-induced rates of hepatic metabolism of coumarin to 7-hydroxycoumarin were markedly higher in DBA-2J mice than in other strains and that intermediate activities in hybrids indicated codominant inheritance. They suggested that there could be similar variability in man. Kratz, Europ. J. Clin. Pharm., 1976, vol. 10, pages 133-137, studied coumarin 7-hydroxylase activity in human liver obtained by needle biopsy. A 4-fold range of enzymatic activity was observed and Kratz suggested that the difference was due to genetic differences between sample donors. Kratz excluded individuals taking drugs that might induce enzyme activity from the study. Yamano et al., Biochemistry, 1990, vol. 29, pages 1322-1329, reported a variant allele of the CYP2A6 gene termed *2 that had a single nucleotide substitution that resulted in an amino acid substitution of a histidine for a leucine at position 160. The variant allele was found to encode an unstable and catalytically inactive enzyme. Fernandez-Salguero et al., Am. J. Hum. Genet., 1995, vol. 57, pages 651-660, reported the genomic sequence for the CYP2A6, CYP2A7, and CYP2A 13 genes, in addition to 2 pseudogenes truncated after exon 5, located on 19q13.2. They also identified three different CYP2A6 alleles: the functional CYP2A6 allele, referred to as *1; the variant-1 allele that had a single base mutation of a T to an A resulting in a substitution of a histidine for a leucine in exon 3, referred to as *2; and the variant-2 allele which was formed by gene conversion between the wildtype CYP2A6 and CYP2A7 genes in exons 3, 6, and 8, referred to as *3.
Four different deletion mutants resulting in an absence of enzyme activity have been described by prior investigators. Oscarson et al., FEBS Lett., 1999, vol. 448, pages 105-110), described the structure of a novel CYP2A locus, referred to as *4A, in which the entire CYP2A6 gene had been deleted thereby disrupting CYP2A6-dependent metabolism. They proposed that this allele was generated by an unequal crossover event between the 3-prime flanking region of the CYP2A6 and CYP2A7 genes. A “D-type” deletion mutant lacking the CYP2A6 gene region from intron 5 to exon 9, referred to as *4B, was described by Nonoya et al., Pharmacogenetics, 1998, vol.8, pages 239-249. An “E-type” mutant referred to as *4C was also identified by Nonoya et al., J. Pharmacol Exp Ther., 1999, vol.289, pages 437-442 in which exons 1, 8, and 9 of CYP2A6 gene were deleted. Oscarson et al., FEBS Lett., 1999, vol. 460, pages 321-327, identified a fourth type of deletion mutant referred to as *4D that they suggested resulted from unequal crossover event with a junction at either intron 8 or exon 9. In addition to characterizing the new deletion mutant CYP2A6*4D, Oscarson et al. also reported a new variant referred to as *5 that was a single nucleotide change of G to T at position 1436, resulting in a substitution of a valine for a glycine at codon 479. This variant allele resulted in a poor metabolizer phenotype. In addition, they found a new wild type variant referred to as *1B that resulted from a gene conversion in the 3′ flanking region of the CYP2A6 gene.
It has been established in the art that nicotine is inactivated by c-oxidation to cotinine. Tyndale, PCT Publication No. WO 98/03171, published Jan. 29, 1998, disclosed that inhibitors of the enzyme encoded by the CYP2A6 gene cause a decrease in nicotine metabolism. It has been suggested in the art that the enzyme encoded by the CYP2A6 gene may affect smoking patterns by mediating the metabolism of nicotine (Vineis et al., in Metabolic Polymorphisms and Susceptibility to Cancer, IARC Scientific Publication No. 148, 1999). Pianezza et al., Nature, 1998, vol. 393, page 750, disclosed that smokers carrying two null CYP2A6 alleles consumed fewer cigarettes. Oscarson et al., FEBS Lett., 1998, vol. 438, pages 201-205, however, indicated that Pianezza et al. used an erroneous method to measure the association of the genotype to the phenotype and therefore additional studies need to be performed to correctly determine the true phenotype of individuals that are genetically CYP2A6 defective. London et al., Lancet, 1999, vol. 353, pages 898-899, also disclosed that polymorphism in the CYP2A6 gene has little influence on the propensity to smoke cigarettes. Seller and Tyndale, PCT Publication No. WO 99/2
Guida Marco
Hall Jeff
Genaissance Pharmaceuticals, Inc.
Johannsen Diana B.
Shaner Sandra L.
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