Isolated CYP3A4 nucleic acid molecules and detection methods

Details Isolated CYP3A4 nucleic acid molecules and detection methods Isolated CYP3A4 nucleic acid molecules and detection methods

1998-08-31

2002-08-13

Jones, W. Gary (Department: 1631)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C536S023200, C536S023500, C536S024310

Reexamination Certificate

active

06432639

ABSTRACT:

INTRODUCTION
Cytochrome P450 enzymes are a heme-containing family that play central roles in oxidative, peroxidative and reductive metabolism of numerous endogenous and exogenous compounds, including many pharmaceutical agents. Substances known to be metabolized by P450 enzymes include steroids, bile acids, fatty acids, prostaglandins, leukotrienes, biogenic amines, retinoids, lipid hydroperoxides, phytoalexins, pharmaceuticals, environmental chemicals and pollutants. P450 substrates also include natural plant products involved in flavor, odor, flower color, and the response to wounding. P450 enzymes and other drug-metabolizing enzymes maintain steady-state levels of endogenous ligands involved in ligand-modulated transcription of genes effecting growth, apoptosis, differentiation, cellular homeostasis, and neuroendocrine functions. The metabolism of foreign chemicals by P450 enzymes can produce toxic metabolites, some of which have been implicated as agents responsible for birth defects and tumor initiation and progression.
The P450 gene superfamily is likely to have evolved from an ancestral gene present before the prokaryote/eukaryotedivergence. The number of individual P450 genes in any mammalian species is estimated at 60 to 200. The CYP2C and CYP3A subfamilies are unique in that they are present in large amounts in human liver microsomes, and there are many forms in each subfamily. Several human cDNAs encoding CYP3A proteins have been identified. The most important of these are CYP3A4, CYP3A5 and CYP3A7. CYP3A4 and CYP3A7 genes are 87% homologous by amino acid and 95% homologous by nucleotide sequence, while CYP3A4 and CYP3A5 are only 88% homologous in the coding region. CYP3A4 and CYP3A7 are 91% homologous in the 5′-flanking sequences, differing by the presence of a unique P450NF specific element (NFSE) and a P450HFLa specific element (HFLaSE), respectively (Hashimoto et al, 1993).
It has been shown that polymorphisms in the CYP2D6 gene correlates with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine (Sachse et al. (1997)
Am. J. Hum. Genet
. 60:248-295).
The CYP3A subclass catalyzes a remarkable number of oxidation reactions of clinically important drugs such as quinidine, warfarin, erythromycin, cyclosporin A, midazolam, lidocain, nifedipine, and dapsone. Current estimates are that more than 60% of clinically used drugs are metabolized by the CYP3A4 enzyme, including such major drug classes as calcium channel blockers, immunosuppressors, macrolide antibiotics and anticancer drugs, see Brian et al. (1990)
Biochemistry
29:11280-11292.
Expression profiles for each member of this family varies significantly. CYP3A4 is expressed in all adult human liver and intestine, accounting for more than 50% of total P450 in both organs. Expression is inducible in vivo and in vitro by numerous compounds such as rifampicin, barbiturates and glucocorticoids. In kidney, CYP3A4 is expressed polymorphically. CYP3A4 expression is sex-influenced, as females have 24% greater expression than males. CYP3A5 is detected in 10-30% of Caucasian adult livers, and expressed constitutively in adult kidney. CYP3A5 expression does not appear to be sex-influenced and only moderately inducible by xenobiotics both in vivo and in vitro. CYP3A7 is expressed in fetal liver but only in 25% of adult livers. Molecular mechanisms responsible for the developmentally specific expression of CYP3A's are unknown.
Since the rates of metabolism of drugs, toxins, etc. can depend on the amounts and kinds of P450s expressed in a tissue, variation in biological response may be determined by the profile of expression of P450s in each person. Analysis of genetic polymorphisms that lead to altered expression and enzyme activity are therefore of interest.
SUMMARY OF THE INVENTION
Genetic sequence polymorphisms are identified in the human CYP3A4 gene. Nucleic acids comprising the polymorphic sequences are used in screening assays, and for genotyping individuals. The genotyping information is used to predict the rate of metabolism for CYP3A4 substrates, and the effect that CYP3A4 modulators will have on such metabolism. The information allows better prediction of drug interactions, and effective dose for an individual.
DATABASE REFERENCES FOR NUCLEOTIDE SEQUENCES
Genbank accession no. S74700 provides the CYP3A5 5′ genomic region. Genbank accession no. D11131 provides a partial sequence of the human cytochrome P450IIIA4 gene. Genbank accession no. M18907 (cDNA) provides the cDNA sequence of a human CYP3A4 allele. Sequences of the CYP3A4 gene are provided in the SEQLIST as follows: cDNA sequence as SEQ ID NO:1, the encoded polypeptide as SEQ ID NO:2, the promoter region as SEQ ID NO:3, intron 3 as SEQ ID NO:4, intron 4 as SEQ ID NO:5, intron 6 as SEQ. ID NO:6, exon 7, intron 7 as SEQ ID NO:7, intron 10as SEQ ID NO:8, intron 11 as SEQ ID NO:9.
DESCRIPTION OF THE SPECIFIC EMBODIMENTS
Pharmacogenetics is the linkage between an individual's genotype and that individual's ability to metabolize or react to a therapeutic agent. Differences in metabolism or target sensitivity can lead to severe toxicity or therapeutic failure by altering the relation between bioactive dose and blood concentration of the drug. Relationships between polymorphisms in metabolic enzymes or drug targets and both response and toxicity can be used to optimize therapeutic dose administration.
Genetic polymorphisms are identified in the human CYP3A4 gene. Nucleic acids comprising the polymorphic sequences are used to screen patients for altered metabolism for CYP3A4 substrates, potential drug-drug interactions, and adverse/side effects, as well as diseases that result from environmental or occupational exposure to toxins. The nucleic acids are used to establish animal, cell culture and in vitro cell-free models for drug metabolism.
DEFINITIONS
It is to be understood that this invention is not limited to the particular methodology, protocols, cell lines, animal species or genera, constructs, and reagents described, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a construct” includes a plurality of such constructs and reference to “the CYP3A4 nucleic acid” includes reference to one or more nucleic acids and equivalents thereof known to those skilled in the art, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs unless clearly indicated otherwise.
CYP3A4 Polymorphic Sequences. It has been found that, specific sites in the CYP3A4 gene sequence are polymorphic, i.e. within a population, more than one nucleotide (G, A, T, C) is found at a specific position. Polymorphisms may provide functional differences in the genetic sequence, through changes in the encoded polypeptide, changes in mRNA stability, binding of transcriptional and translation factors to the DNA or RNA, and the like. The polymorphisms are also used as single nucleotide polymorphisms (SNPs) to detect genetic linkage to phenotypic variation in activity and expression of the particular protein.
SNPs are generally biallelic systems, that is, there are two alleles that an individual may have for any particular marker. SNPs, found approximately every kilobase, offer the potential for generating very high density genetic maps, which will be extremely useful for developing haplotyping systems for genes or regions of interest, and because of the nature of SNPs, they may in fact be the polymorphisms associated with the disease phenotypes under study. The low mutation rate of SNPs also makes them excellent markers for studying complex genetic traits.
In order to provide an una

Affiliated with

Also associated with

Rating

Isolated CYP3A4 nucleic acid molecules and detection methods does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Isolated CYP3A4 nucleic acid molecules and detection methods, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Isolated CYP3A4 nucleic acid molecules and detection methods will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2892369