FIELD OF THE INVENTION
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The present invention relates generally to means and methods of diagnosing and treating the phenotypic spectrum as well as the overlapping clinical characteristics with several forms of inherited abnormal expression and/or function of the cytochrome P-450 (CYP)2D6 gene. In particular, the present invention relates to polynucleotides of molecular variant promoters of the CYP2D6 gene which, for example, are associated with abnormal drug response or individual predisposition to several common diseases and disorders caused by drug under- or overmetabolization, and to vectors comprising such polynucleotides. Furthermore, the present invention relates to host cells comprising such polynucleotides or vectors. Moreover, the present invention relates to methods for identifying and obtaining drug candidates for therapy of disorders related to the malfunction of the CYP2D6 gene as well as to methods of diagnosing the status of such disorders. The present invention furthermore provides kits comprising oligonucleotides hybridizing to the CYP2D6 promoter and/or being capable of being extended into this region useful for diagnosing subjects that are for example ultrarapid or intermediate metabolizer of drugs.
Several documents are cited throughout the text of this specification. Each of the documents cited herein (including any manufacturer's specifications, instructions, etc.) are hereby incorporated herein by reference; however, there is no admission that any document cited is indeed prior art as to the present invention.
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OF THE INVENTION
Cytochrome P450 CYP2D6 belongs to the CYP2 family of P450s and is the only functionally active isozyme of the CYP2D subfamily in humans. It is involved in the metabolism of up to 25% of all therapeutically used drugs (Hardman et al, 1995). The gene encoding its synthesis is located in the CYP2D locus at q13.1 on the long arm of chromosome 22 (Eichelbaum et al., 1987). It is part of a gene cluster containing also two pseudogenes CYP2D7P and CYP2D8P (Kimura et al., 1989). Like other members of the human CYP2 gene family, the CYP2D genes consist of 9 exons and 8 introns. The enzyme exhibits a common genetic polymorphism (Meyer and Zanger, 1997). In fact it was the first cytochrome P450 enzyme for which a genetic polymorphism was demonstrated which was named the debrisoquine/sparteine polymorphism based on the two substrates involved in its discovery (Mahgoub et al., 1977; Eichelbaum et al., 1979). Depending on the metabolic handling of these two probe drugs, between 5 and 10% of subjects of European populations have a severely impaired capacity to form the major metabolites 4-hydroxydebrisoquine and 2-dehydrosparteine. These subjects were designated as poor metabolizers (PM), the remainder of the population being so-called extensive metabolizers (EM). The trait ‘poor metabolism’ is inherited in an autosomal recessive fashion, i.e. PMs are carriers of two non-functional alleles. The molecular basis of this polymorphism has been extensively investigated and more than 30 functional and non-functional alleles have been described which allow to predict the PM phenotype in Caucasians with an estimated 99% reliability. (Daly et al., 1996 and CYP Allele Nomenclature Web-Site: http://www.imm.ki.se/CYPalleles/cyp2d6.htm).
Over 50-fold variability in CYP2D6 activity exists among extensive metabolizers (genetically carriers of at least one functional allele) which has led to the designation of the most rapid “extensive” phenotype as “ultrarapid” (UM) and the slowest as “intermediate” metabolizer (IM). There is evidence in the literature that these sub-phenotypes are clinically relevant. Individuals with the UM phenotype are at risk to experience therapeutic failure due to abnormally fast clearance of the drug whereas IMs may be comparable to PMs in their risk to develop adverse side effects and toxicity.
A molecular explanation for the UM phenotype was provided by the discovery of the CYP2D6 gene duplication, which however only applies to a fraction of UMs (Johansson et al., 1993; Dahl et al., 1995). Two CYP2D6 alleles previously described result in lower enzyme activity and cause the IM phenotype in individuals not carrying a normal functional allele. However, both of these alleles (*9: Broly and Meyer, 1993; *10: Yokota et al., 1993) occur with a frequency of only 2% in the Caucasian population and only about 20% of the IMs have informative genotypes involving these two alleles (i.e *9/*0, *10/*0 and *10/*10; Sachse et al., 1997; Griese et al., 1998). 80% of the IMs have therefore “uninformative” genotypes, i.e. genotypes that are also associated with the normal extensive or the ultrarapid metabolizer phenotypes. It has therefore remained unclear whether the IM sub-phenotype has a genetic basis or whether it is an epigenetic phenomenon.
It is clear that naturally occurring mutations, if they exist can have effects on drug metabolization and efficacy of therapies with drugs. It is unknown, however, how many of such variations exist, and with what frequency and at what positions in the human CYP2D6 genes.
Accordingly, means and methods for diagnosing and treating a variety of forms of individual drug intolerability and inefficacy of drug therapy which result from CYP2D6 gene polymorphisms that interfere e.g., with chemotherapeutic treatment of diseases was hitherto not sufficiently available but are nevertheless highly desirable.
Thus, the technical problem of the present invention is to comply with the needs described above.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
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OF THE INVENTION
The present invention is based on the finding of novel, so far unknown variations in the nucleotide sequences of the CYP2D6 gene promoter and the population distribution of these alleles. Based upon the knowledge of these novel sequences diagnostic tests and reagents for such tests were designed for the specific detection and genotyping of CYP2D6 promoter alleles in humans, including homozygous as well as heterozygous, frequent as well as rare alleles of the CYP2D6 promoter. The determination of the CYP2D6 promoter allele status of humans with such tests is useful for the optimization of therapies with the numerous substrates of CYP2D6.
In a first embodiment, the invention provides polynucleotides of molecular variant CYP2D6 gene promoter and embodiments related thereto such as vectors and host cells transferred therewith.
In yet another embodiment, the invention provides methods for therapy of disorders related to acquired drug hypo- or hypersensitivity as well as methods of diagnosing the status of such disorders.
In a further embodiment, the invention provides kits comprising the above-described polynucleotides or vectors containing the same.
The novel variant forms of CYP2D6 gene according to the invention provide the potential for the development of a pharmacodynamic profile of drugs for a given patient.
DESCRIPTION OF THE INVENTION
The finding and characterization of variations in the CYP2D6 genes, and diagnostic tests for the discrimination of different CYP2D6 alleles in human individuals provide a very potent tool for improving the therapy of diseases with drugs that are targets of the CYP2D6 gene product, and whose metabolization is therefore dependent on CYP2D6 activity. The diagnosis of the individual allelic CYP2D6 status permits a more focused therapy, e.g., by opening the possibility to apply individual dose regimens of drugs. It may also be useful as prognostic tool for therapy outcome. Furthermore, diagnostic tests to genotype CYP2D6 will improve therapy with established drugs and help to correlate genotypes with drug activity or side effects.
Thus, the present invention provides a way to exploit molecular biology and pharmacological research for drug therapy while bypassing their potential detrimental effects which are due to expression of variant expression of the CYP2D6 gene.
Accordingly, the invention relates to a polynucleotide selected from the group consisting of:
(a) a molecular variant polynucleotide having the nucleic acid sequence of SEQ ID NO: 1, wherein at nucleotide position corresponding to nucleotide position −1584 of the CYP2D6 promotor as shown in FIG. 1, is a G;
(b) a molecular variant polynucleotide capable of hybridizing to the CYP2D6 promotor as shown in FIG. 1, wherein said polynucleotide is having at a position corresponding to position -1584 of the CYP2D6 promoter as shown in FIG. 1 at least one nucleotide deletion, addition and/or substitution; and
(c) a molecular variant polynucleotide capable of hybridizing to the CYP2D6 promoter as shown in FIG. 1, wherein said polynucleotide is having at a position corresponding to position −1584 of the CYP2D6 promotor as shown in FIG. 1, a G.
In the context of the present invention the term “molecular variant” CYP2D6 promoter, gene or protein as used herein means that said CYP2D6 promoter, gene or protein differs from the wild type CYP2D6 promoter, gene or protein by way of nucleotide substitution(s), addition(s) and/or deletion(s) (Genomic sequences of the CYP2D6 gene including the promoter are described, for example in Genbank, http://www.ncbi.nlm.nih.gov/Genbank/GenbankOverview.html [accession number M33388]).
The term “corresponding” as used herein means that a position is not only determined by the number of the preceding nucleotides. The position of a given nucleotide in accordance with the present invention which may be deleted, substituted or comprise one or more additional nucleotide(s) may vary due to deletions or additional nucleotides elsewhere in the promotor or gene. Thus, under a “corresponding position” in accordance with the present invention it is to be understood that nucleotides may differ in the indicated number but may still have similar neighboring nucleotides. Said nucleotides which may be exchanged, deleted or comprise additional nucleotides are also comprised by the term “corresponding position”. Said nucleotides may for instance together with their neighbors form sequences which may be involved in the regulation of gene expression, stability of the corresponding RNA or RNA editing.
The term “hybridizing” as used herein refers to polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with altered expression of the variant CYP2D6 gene compared to the corresponding wild type gene. Thus, said hybridizing polynucleotides are also associated with said altered expression of the variant CYP2D6 gene compared to the corresponding wild type gene. Therefore, said polynucleotides may be useful as probes in Northern or Southern Blot analysis of RNA or DNA preparations, respectively, or can be used as oligonucleotide primers in PCR analysis dependent on their respective size. Also comprised by the invention are hybridizing polynucleotides which are useful for analysing DNA-Protein interactions via, e.g., electrophoretic mobility shift analysis (EMSA). Preferably, said hybridizing polynucleotides comprise at least 10, more preferably at least 15 nucleotides in length while a hybridizing polynucleotide of the present invention to be used as a probe preferably comprises at least 100, more preferably at least 200, or most preferably at least 500 nucleotides in length.
It is well known in the art how to perform hybridization experiments with nucleic acid molecules, i.e. the person skilled in the art knows what hybridization conditions s/he has to use in accordance with the present invention. Such hybridization conditions are referred to in standard text books such as Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. Preferred in accordance with the present inventions are polynucleotides which are capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with altered expression of the variant CYP2D6 gene compared to the corresponding wild type gene under stringent hybridization conditions, i.e. which do not cross hybridize to unrelated polynucleotides such as polynucleotides that may not alter expression of the variant CYP2D6 gene compared to the corresponding wild type gene.
In accordance with the present invention, the mode and population distribution of novel so far unidentified genetic variations in the CYP2D6 gene have been analyzed by sequence analysis of relevant regions of the human CYP2D6 genes from many different individuals. It is a well known fact that genomic DNA of individuals, which harbor the individual genetic makeup of all genes, including CYP2D6 can easily be purified from individual blood samples. These individual DNA samples are then used for the analysis of the sequence composition of the CYP2D6 gene alleles that are present in the individual which provided the blood sample. All previously reported studies on the CYP2D6 polymorphism were restricted and focused to the coding sequence which comprises 9 exons. This work represents the first systematic mutation analysis of the promoter region of the gene. The aim was to identify mutations, if any that are linked to altered enzyme activity in vivo, based on the assumption that promoter mutations may affect gene transcription which may result in higher or lower mRNA levels and thus lead to higher or lower amounts of enzyme expressed in liver. Surprisingly, mutations in the CYP2D6 promoter could be found that are associated with enhanced and reduced CYP2D6 enzyme activity, respectively. The sequence analysis was carried out by PCR amplification of relevant regions of the CYP2D6 gene, subsequent purification of the PCR products, followed by automated DNA sequencing with established methods; see the examples. In particular a subgroup of 10 to 15% of Caucasians are termed phenotypical “intermediate metabolizers” of drug substrates of CYP2D6, because they have severely impaired yet residual in vivo function of this cytochrome P450. Genotyping based on the currently known CYP2D6 alleles does not predict this phenotype. In accordance with the present invention, a systematic search through 1.6 kb of the CYP2D6 5′-flanking sequence revealed 6 mutations of which three were exclusively associated with the functional CYP2D6*2 allele and one of these co-segregated with increased CYP2D6*2 activity in a family study. In a representative population sample the median urinary metabolic ratio (MRs) for sparteine oxidation was over 4-fold reduced in individuals with the novel variant allele (*2[−1584G]: MRS=0.53, N=27) compared to individuals lacking the mutation (*2[−1584C]: MRS=2.33, N=12; P<0.0001). This first functional promoter variant of the CYP2D6 gene has an estimated frequency of 20% to 25% in the general population and allows to establish a genotype for the identification of over 50% of Caucasians with the intermediate metabolizer phenotype.
One important parameter that had to be considered in the attempt to determine the individual CYP2D6 genotype and identify novel CYP2D6 variants by direct DNA-sequencing of PCR-products from human blood genomic DNA is the fact that each human harbors (usually, with very few abnormal exceptions) two gene copies of each autosomal gene (diploidy). Because of that, great care had to be taken in the evaluation of the sequences to be able to identify unambiguously not only homozygous sequence variations but also heterozygous variations. The details of the different steps in the identification and characterization of novel CYP2D6 gene polymorphisms (homozygous and heterozygous) are described in the examples below.
The mutations in the CYP2D6 gene detected in accordance with the present invention are illustrated in FIG. 1 and Table 2, respectively. The methods of the mutation analysis followed standard protocols and are described in detail in the examples. In general such methods to be used in accordance with the present invention for evaluating the phenotypic spectrum as well as the overlapping clinical characteristics with other forms of drug metabolization and altered tolerance to drugs in patients with mutations in the CYP2D6 gene encompass for example haplotype analysis, single-strand conformation polymorphism analysis (SSCA), PCR and direct sequencing. On the basis of thorough clinical characterization of many patients the phenotypes can then be correlated to these mutations as well as to mutations that had been described earlier. As is evident to the person skilled in the art this new molecular genetic knowledge can now be used to exactly characterize the genotype of the index patient where a given drug takes an unusual effect and of his family.
Over the past 20 years, genetic heterogeneity has been increasingly recognized as a significant source of variation in drug response. Many scientific communications (Meyer and Zanger, 1997; West et al., 1997) have clearly shown that some drugs work better or may even be highly toxic in some patients than in others and that these variations in patient\'s responses to drugs can be related to molecular basis. This “pharmacogenomic” concept spots correlations between responses to drugs and genetic profiles of patient\'s (Marshall, 1997a and 1997b). In this context of population variability with regard to drug therapy, pharmacogenomics has been proposed as a tool useful in the identification and selection of patients which can respond to a particular drug without side effects. This identification/selection can be based upon molecular diagnosis of genetic polymorphisms by genotyping DNA from leukocytes in the blood of patient. For the providers of health care, such as health maintenance organizations in the US and government public health services in many European countries, this pharmacogenomics approach can represent a way of both improving health care and reducing overheads because there is a large cost to unnecessary therapies, ineffective drugs and drugs with side effects.
In a preferred embodiment, the present invention relates to polynucleotides of molecular variant CYP2D6 gene promoters, wherein the nucleotide deletion, addition and/or substitution result in altered expression of the variant CYP2D6 gene compared to the corresponding wild type gene.
The polynucleotide of the invention may be, e.g., DNA, genomic DNA or synthetically produced DNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination. Preferably said polynucleotide is part of a vector, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide of the invention. Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions. Methods which are well known to those skilled in the art can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, N.Y. (1994). The present invention furthermore relates to host cells transformed with a polynucleotide or vector of the invention. Said host cell may be a prokaryotic or eukaryotic cell.
With the variant CYP2D6 polynucleotides and vectors of the invention, it is now possible to study in vivo and in vitro the efficiency of drugs in relation to particular mutations in the CYP2D6 gene of a patient and the affected phenotype. Thus, a particular object of the present invention concerns drug/pro-drug selection and formulation of pharmaceutical compositions for the treatment of diseases which are amenable to chemotherapy taking into account the polymorphism of the variant form of the CYP2D6 gene promoter that cosegregates with the affected phenotype of the patient to be treated. This allows the safe and economic application of drugs which for example were hitherto considered not appropriate for therapy of, e.g., cancer due to either their side effects in some patients and/or their unreliable pharmalogical profile with respect to the same or different phenotype(s) of the disease. The means and methods described herein can be used, for example, to improve dosing recommendations and allows the prescriber to anticipate necessary dose adjustments depending on the considered patient group.