Patent Application
20110131671
The present invention relates to a polymorphic CYP2C8 polynucleotide. Moreover, the invention relates to genes or vectors comprising the polynucleotides of the invention and to a host cell genetically engineered with the polynucleotide or gene of the invention. Further, the invention relates to methods for producing molecular variant polypeptides or fragments thereof, methods for producing cells capable of expressing a molecular variant polypeptide and to a polypeptide or fragment thereof encoded by the polynucleotide or the gene of the invention or which is obtainable by the method or from the cells produced by the method of the invention. Furthermore, the invention relates to an antibody which binds specifically the polypeptide of the invention. Moreover, the invention relates to a transgenic non-human animal. The invention also relates to a solid support comprising one or a plurality of the above mentioned polynucleotides, genes, vectors, polypeptides, antibodies or host cells. Furthermore, methods of identifying a polymorphism, identifying and obtaining a pro-drug or drug or an inhibitor are also encompassed by the present invention. In addition, the invention relates to methods for producing of a pharmaceutical composition and to methods of diagnosing a disease. Further, the invention relates to a method of detection of the polynucleotide of the invention. Furthermore, comprised by the present invention are a diagnostic and a pharmaceutical composition. Even more, the invention relates to uses of the polynucleotides, genes, vectors, polypeptides or antibodies of the invention. Finally, the invention relates to a diagnostic kit.
Cytochrome P450 enzymes are metabolic enzymes differentially expressed in several tissues. Cytochrome P450 2C mRNA was detected in abundance in hepatic tissue, to a lesser extend in extrahepatic tissues, e.g. kidney, adrenal gland, brain, uterus, mammary gland, ovary and duodenum, but neither in testes nor ovary (Klose, J Biochem Mol Toxicol 13 (1999), 289-95). Of the CYP2C subfamily, clustered on chromosome 10q24.1 (Gray, Genomics 28 (1995), 328-32), CYP2C9 and 2C19 are those which gained major interest due to their prominent role in metabolizing therapeutic drugs. Differential breakdown of their substrates led to the identification of alleles for poor (PM) or extensive metabolizers (EM). Nevertheless, the existence of minor CYP2C8 genes was known and characterized to display about 90% amino acid homology (Goldstein, Pharmacogenetics 4 (1994), 285-99). Only recently, the genomic sequence of CYP2C8, spanning a 31 kb region, was published. Interestingly, the gene is involved in intergenic splicing with CYP2C18 composed of 9 exons (Finta, Genomics 63 (2000), 433-8).
Arachidonic acid is one major endogenous substrate for CYP2C8 and specificially epoxidated to equivalent forms of 11, 12- and 14, 15-epoxides. Concerning xenobiotical substrates CYP2C8 represents the isoform with the narrowest substrate specificity. The anticancer drug taxol (paclitaxel), also well known to be a substrate for MDR-1 (Mechetner, Clin Cancer Res. 4 (1998), 389-398), is known to be the prototype. Several other drugs, e.g. verapamil (Tracy, Br J Clin Pharmacol. 47 (1999), 545-52) and rosiglitazone (Malinowski, Clin Ther. 22 (2000), 1151-68) are preferable substrates for CYP2C8 in comparison to other CYP2Cs or CYP3As. Drugs like benzphetamine, retinoic acid, tolbutamide, benzo(a)pyrene, carbamazepine and R-ibuprofen represent a minor contribution of CYP2C8 (Wrighton, J Clin Invest. 80 (1987), 1017-22; Relling, J Pharmacol Exp Ther. 252 (1990), 442-7; Hamman, Biochem Pharmacol. 54 (1997), 33-41; Kerr, Biochem Pharmacol. 47 (1994), 1969-79; Yun, Cancer Res. 52 (1992), 1868-74; Leo, Arch Biochem Biophys 259 (1987), 241-9). So far, the enzymatic induction has only be observed by phenobarbital and rifampicin (Morel, Eur J. Biochem. 191 (1990), 437-44). Thum and Borlak (Thum and Borlak, Br J Pharmacol 130 (2000), 1745-52) found a strong correlation between tissue specific gene expression and enzyme activity. Increased CYP2C8 mRNA expression within the right heart ventricle might explain for the lack of efficacy of cardioselective drugs like verapamil. In a porcine system, Fisslthaler (Fisslthaler, Nature 401(1999), 493-7; Fisslthaler, Semin Perinatol 24 (2000), 15-9; Fisslthaler, Circ Res. 88 (2001), 44-51) could show that CYP2C8 meets all criteria for the coronary endothelium-derived hyperpolarisation factor synthase acting on vascular smooth muscle cells prior to dilation.
Since the mRNA has been published, first single nucleotide polymorphisms (SNPs) in exons 3, 5 and 8 were reported in an abstract (Goldstein, Microsomes and Oxidation, Stresa (2000), Italy): an exchange in position 139 of Arg to Lys (exon 3) could be linked to a SNP in exon 8 (Lys399Arg), occurring primarily in Caucasians, and correlated to poor metabolizing phenotype (PM). Exon 5 displays a mutation (Iso269Phe) that is associated with poor metabolizing enzyme restricted to African-Americans. The regulation of the 2Cs is supposed to be modified by polymorphisms in the untranslated region. Regarding CYP2C8, two previously unidentified transcription regulatory factor sites for C/EBP and HPF-1, but no relevant SNPs were identified by Goldstein and coworkers (Goldstein, Microsomes and Oxidation, Stresa Italy (2000), Italy) in that region.
However, means and methods for reliable and improved diagnosing and treating a variety of diseases and disorders or for predicting and overcoming undesired drug effects or interactions based on dysfunctions or dysregulations of cytochrome 2C8 variants were not available yet but are nevertheless highly desirable. Thus, the technical problem underlying the present invention is to comply with the above specified needs.
The solution to this technical problem is achieved by providing the embodiments characterized in the claims.
Accordingly, the present invention relates to a polynucleotide comprising a polynucleotide selected from the group consisting of:
In the context of the present invention the term “polynucleotides” or the term “polypeptides” refers to different variants of a polynucleotide or polypeptide. Said variants comprise a reference or wild type sequence of the polynucleotides or polypeptides of the invention as well as variants which differ therefrom in structure or composition. Reference or wild type sequences for the polynucleotides are GenBank accession No: NM—000770.1 for mRNA. Reference or wild type sequence for the polynucleotide of the invention is for the 5′UTR: GenBank accession No: AF136830.1; for exon 1: GenBank accession No: AF136831.1; for exon 2: GenBank accession No: AF136832.1 and AF136833.1; for exon 3: GenBank accession No: AF136833.1; for exon 4: GenBank accession No: AF136834.2 and AF136835.1; for exon 5: GenBank accession No: AF136836.1 and AF136837.1; for exon 6: GenBank accession No: AF136838.1 and AF136839.1; for exon 7: GenBank accession No: AF136840.1 and AF136841.1; for exon 8: GenBank accession No: AF136842.1 and AF136843.1; for exon9/3′UTR: GenBank accession No: AF136844.1 and AF136845.1; partly in combination with NM—000770.1 (mRNA). Reference or wild type sequence for the polypeptide of the CYP2C8 gene is GenBank accession No: GI: 13787189. In the context of the present invention the term “5′UTR” refers to the untranslated region 5′ to the ATG start codon including the 5′ upstream region encompassing the promoter. The term “3′UTR” refers to the untranslated region 3′ to the Stop codon.
The differences in structure or composition usually occur by way of nucleotide or amino acid substitution(s), addition(s) and/or deletion(s). Preferred substitution in accordance with the present invention are a T to G substitution at a position corresponding to position 1668 (GenBank accession No: AF136830.1), a G to A substitution at a position corresponding to position 831 (GenBank accession No: AF136830.1), a G to T substitution at a position corresponding to position 309 (GenBank accession No: AF136838.1) and 232 (GenBank accession No: AF136840.1) of the CYP2C8 gene. Preferred deletions in accordance with the invention are an AT deletion at a position corresponding to position 1397 to 1398 (GenBank accession No: AF136830.1) and a deletion of at least one A at a position corresponding to position 329 (GenBank accession No: AF136833.1) of the CYP2C8 gene.
In accordance with the present invention it has also been found that a deletion of the nucleotide A at a position corresponding to position 329 (GenBank accession No: AF136833.1) of the CYP2C8 gene leads to an altered C-terminus of the protein encoding a CYP2C8 polypeptide wherein said polypeptide comprises an amino acid substitution of T to P at a position corresponding to position 159 of the CYP2C8 polypeptide (GenBank accession No: GI: 13787189). In accordance with the present invention it has also been found that a substitution of a G to a T at a position corresponding to position 309 (GenBank accession No: AF136838.1) of the CYP2C8 gene leads to a polypeptide wherein said polypeptide comprises an amino acid substitution of E to a premature termination (stop) at a position corresponding to position 274 of the CYP2C8 polypeptide (Gen Bank accession No: GI: 13787189) and a substitution of the nucleotide G to an A at a position corresponding to position 1135 (GenBank accession No: NM—000770.1) of the CYP2C8 gene leads to a polypeptide wherein said polypeptide comprises an amino acid substitution of G to S at a position corresponding to position 365 of the CYP2C8 polypeptide (GenBank accession No: GI: 13787189). This will alter the structure or confirmation of the protein and will abolish the activity of the drug metabolizing enzyme.
Preferably, said nucleotide substitution(s), addition(s) or deletion(s) comprised by the present invention result(s) in one or more changes of the corresponding amino acid(s) of the polypeptides of the invention.
The variant polynucleotides and polypeptides also comprise fragments of said polynucleotides or polypeptides of the invention. The polynucleotides and polypeptides as well as the aforementioned fragments thereof of the present invention are characterized as being associated with a CYP2C8 dysfunction or dysregulation comprising, e.g., insufficient and/or altered metabolism. Said dysfunctions or dysregulations referred to in the present invention cause a disease or disorder or a prevalence for said disease or disorder. Preferably, as will be discussed below in detail, said disease is a deficiency in the metabolism of certain drugs which are metabolized by CYP2C8, e.g. Taxol, Verapamil, or any other disease caused by a dysfunction or dysregulation due to a polynucleotide or polypeptides of the invention, also referred to as CYP2C8 gene associated diseases in the following.
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 a CYP2C8 dysfunction or dysregulation. Thus, said hybridizing polynucleotides are also associated with said dysfunctions and dysregulations. Preferably, said polynucleotides capable of hybridizing to the polynucleotides of the invention or parts thereof which are associated with CYP2C8 dysfunctions or dysregulations are at least 70%, at least 80%, at least 95% or at least 100% identical to the polynucleotides of the invention or parts thereof which are associated with CYP2C8 dysfunctions or dysregulations. 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 analyzing 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 a CYP2C8 dysfunction or dysregulation under stringent hybridization conditions, i.e. which do not cross hybridize to unrelated polynucleotides such as polynucleotides encoding a polypeptide different from the CYP2C8 polypeptides of the invention.
The term “corresponding” as used herein means that a position is not only determined by the number of the preceding nucleotides and amino acids, respectively. The position of a given nucleotide or amino acid 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 or amino acids elsewhere in the gene or the polypeptide. Thus, under a “corresponding position” in accordance with the present invention it is to be understood that nucleotides or amino acids may differ in the indicated number but may still have similar neighboring nucleotides or amino acids. Said nucleotides or amino acids which may be exchanged, deleted or comprise additional nucleotides or amino acids are also comprised by the term “corresponding position”. Said nucleotides or amino acids 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, as well as encode functional domains or motifs of the protein of the invention.
By, e.g., “position 1271 to 1273” it is meant that said polynucleotide comprises one or more deleted nucleotides which are deleted between positions 1271 and position 1273 of the corresponding wild type version of said polynucleotide. The same applies mutatis mutandis to all other position numbers referred to in the above embodiment which are drafted in the same format.
By, e.g., “position 180/181” it is meant that said polynucleotide comprises one or more additional nucleotide(s) which are inserted between positions 180 and position 181 of the corresponding wild type version of said polynucleotide. The same applies mutatis mutandis to all other position numbers referred to in the above embodiment which are drafted in the same format, i.e. two consecutive position numbers separated by a slash (/).
In accordance with the present invention, the mode and population distribution of genetic variations in the CYP2C8 gene has been analyzed by sequence analysis of relevant regions of the human said gene 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 the CYP2C8 gene, can easily be purified from individual blood samples. These individual DNA samples are then used for the analysis of the sequence composition of the alleles of the CYP2C8 gene that are present in the individual which provided the blood sample. The sequence analysis was carried out by PCR amplification of relevant regions of said genes, subsequent purification of the PCR products, followed by automated DNA sequencing with established methods (e.g. ABI dye terminator cycle sequencing).
One important parameter that had to be considered in the attempt to determine the individual genotypes and identify novel variants of the CYP2C8 gene 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 polymorphisms in the CYP2C8 gene (homozygous and heterozygous) are described in the examples below.
Over the past 20 years, genetic heterogeneity has been increasingly recognized as a significant source of variation in drug response. Many scientific communications (Meyer, Ann. Rev. Pharmacol. Toxicol. 37 (1997), 269-296 and West, J. Clin. Pharmacol. 37 (1997), 635-648) 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, Nature Biotechnology, 15 (1997), 954-957; Marshall, Nature Biotechnology, 15 (1997), 1249-1252). 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 example, and characterization of disease (Bertz, Clin. Pharmacokinet. 32 (1997), 210-256; Engel, J. Chromatogra. B. Biomed. Appl. 678 (1996), 93-103). For the founders 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 drugs, ineffective drugs and drugs with side effects.
The mutations in the variant genes of the invention sometime result in amino acid deletion(s), insertion(s) and in particular in substitution(s) either alone or in combination. It is of course also possible to genetically engineer such mutations in wild type genes or other mutant forms. Methods for introducing such modifications in the DNA sequence of said genes are well known to the person skilled in the art; see, e.g., Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.
For the investigation of the nature of the alterations in the amino acid sequence of the polypeptides of the invention may be used such as BRASMOL that are obtainable from the Internet. Furthermore, folding simulations and computer redesign of structural motifs can be performed using other appropriate computer programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput. Appl. Biosci. 11 (1995), 675-679). Computers can be used for the conformational and energetic analysis of detailed protein models (Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med. Biol. 376 (1995), 37-45). These analysis can be used for the identification of the influence of a particular mutation on metabolizing, binding and/or transport of drugs.
Usually, said amino acid deletion, addition or substitution in the amino acid sequence of the protein encoded by the polynucleotide of the invention is due to one or more nucleotide substitution, insertion or deletion, or any combinations thereof. Preferably said nucleotide substitution, insertion or deletion may result in an amino acid substitution of F to L at position corresponding to position 263 of the CYP2C8 polypeptide (GenBank accession No: GI: 13787189). The polypeptides encoded by the polynucleotides of the invention have altered biological or immunological properties due to the mutations referred to in accordance with the present invention. Examples for said altered properties are stability of the polypeptides which may be effected or an altered substrate specificity or even a complete loss of the capability of metabolizing certain drugs.
The mutations in the CYP2C8 gene detected in accordance with the present invention are listed in Table 2. The methods of the mutation analysis followed standard protocols and are described in detail in the Examples. In general such methods are to be used in accordance with the present invention for evaluating the phenotypic spectrum as well as the overlapping clinical characteristics of diseases or conditions related to dysfunctions or dysregulations and diseases related to the poor or extensive metabolism (PM or EM) certain drugs. Advantageously, the characterization of said mutants may form the basis of the development of a diagnostic assay that is able to predict a patients efficacy to metabolize a drug for instance in anticancer treatment (taxol) or cardiovascular deficiencies (verapamil). Said methods 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.
Also comprised by the polynucleotides referred to in the present invention are polynucleotides which comprise at least two of the polynucleotides specified herein above, i.e. polynucleotides having a nucleotide sequence which contains all four mutations comprised by the above polynucleotides or listed in Table 2 below (haplotype: positions 831, 1397 to 1398 of GenBank accession No: AF136830.1, position 270 of GenBank accession No: AF136833.1, and position 206 of (GenBank accession No: AF136842.1). In accordance with the present invention it is also preferred to detect only one of the above mentioned polymorphisms of the haplotype, said one polymorphism being indicative for the presence of the other polymorphisms of the haplotype. Thus, in order to detect the presence of the above mentioned haplotype it is sufficient to determine the presence of any one of the polymorphisms comprised by said haplotype. Moreover, the polynucleotides referred to above allow the study of synergistic effects of said mutations in the CYP2C8 gene and/or a polypeptide encoded by said polynucleotide on the pharmacological profile of drugs in patients who bear such mutant forms of the gene or similar mutant forms that can be mimicked by the above described proteins. It is expected that the analysis of said synergistic effects provides deeper insights into the onset of CYP2C8 dysfunctions or dysregulations or diseases related to altered drug transport as described supra. From said deeper insight the development of diagnostic and pharmaceutical compositions related to CYP2C8 dysfunctions or dysregulations or diseases related to impaired drug metabolism will greatly benefit.
As is evident to the person skilled in the art, the genetic knowledge deduced from the present invention can now be used to exactly and reliably characterize the genotype of a patient. Advantageously, diseases or a prevalence for a disease which are associated with CYP2C8 dysfunction or dysregulation, e.g. diseases associated with arachidonic acid metabolism referred to herein can be predicted and preventive or therapeutical measures can be applied accordingly. Moreover in accordance with the foregoing, in cases where a given drug takes an unusual effect, a suitable individual therapy can be designed based on the knowledge of the individual genetic makeup of a subject with respect to the polynucleotides of the invention and improved therapeutics can be developed as will be further discussed below.
In general, the CYP2C8 “status”, defined by the expression level and activity of the CYP2C8 protein, can be variable in normal tissue, due to genetic variations/polymorphisms. The identification of polymorphisms associated with altered CYP2C8 expression and/or activity is important for the prediction of drug metabolism and subsequently for the prediction of therapy outcome, including side effects of medications. Therefore, analysis of CYP2C8 variations indicative of CYP2C8 function, is a valuable tool for therapy with drugs, which are substrates of CYP2C8 and has, thanks to the present invention, now become possible.
In line with the foregoing, preferably, the polynucleotide of the present invention is associated with an incompatibility or a disease related to arachidonic acid metabolism, cancer or cardiovascular diseases.
The term “cancer” used herein is very well known and characterized in the art. Several variants of cancer exist and are comprised by said term as meant in accordance with the invention. For a detailed list of symptoms which are indicative for cancer it is referred to text book knowledge, e.g. Pschyrembel. The term “cardiovascular disease” as used herein refers to those diseases known in the art and described in detail in standard text books, such as Pschyrembel or Stadman. Examples for cardiovascular diseases are hypertension or atherosclerosis. The inefficacy or complete loss to epoxidate arachidonoic acid is referred to as disease of the arachidonic acid metabolism.
In a further embodiment the present invention relates to a polynucleotide which is DNA or RNA.
The polynucleotide of the invention may be, e.g., DNA, cDNA, genomic DNA, RNA or synthetically produced DNA or RNA 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.
The invention furthermore relates to a gene comprising the polynucleotide of the invention.
It is well known in the art that genes comprise structural elements which encode an amino acid sequence as well as regulatory elements which are involved in the regulation of the expression of said genes. Structural elements are represented by exons which may either encode an amino acid sequence or which may encode for RNA which is not encoding an amino acid sequence but is nevertheless involved in RNA function, e.g. by regulating the stability of the RNA or the nuclear export of the RNA.
Regulatory elements of a gene may comprise promoter elements or enhancer elements both of which could be involved in transcriptional control of gene expression. It is very well known in the art that a promoter is to be found upstream of the structural elements of a gene. Regulatory elements such as enhancer elements, however, can be found distributed over the entire locus of a gene. Said elements could be reside, e.g., in introns, regions of genomic DNA which separate the exons of a gene. Promoter or enhancer elements correspond to polynucleotide fragments which are capable of attracting or binding polypeptides involved in the regulation of the gene comprising said promoter or enhancer elements. For example, polypeptides involved in regulation of said gene comprise the so called transcription factors.
Said introns may comprise further regulatory elements which are required for proper gene expression. Introns are usually transcribed together with the exons of a gene resulting in a nascent RNA transcript which contains both, exon and intron sequences. The intron encoded RNA sequences are usually removed by a process known as RNA splicing. However, said process also requires regulatory sequences present on a RNA transcript said regulatory sequences may be encoded by the introns.
In addition, besides their function in transcriptional control and control of proper RNA processing and/or stability, regulatory elements of a gene could be also involved in the control of genetic stability of a gene locus. Said elements control, e.g., recombination events or serve to maintain a certain structure of the DNA or the arrangement of DNA in a chromosome.
Therefore, single nucleotide polymorphisms can occur in exons of a gene which encode an amino acid sequence as discussed supra as well as in regulatory regions which are involved in the above discussed process. The analysis of the nucleotide sequence of a gene locus in its entirety including, e.g., introns is in light of the above desirable. The polymorphisms comprised by the polynucleotides of the present invention can influence the expression level of CYP2C8 protein via mechanisms involving enhanced or reduced transcription of the CYP2C8 gene, stabilization of the gene's RNA transcripts and alteration of the processing of the primary RNA transcripts.
Therefore, in a furthermore preferred embodiment of the gene of the invention a nucleotide deletion, addition and/or substitution results in altered expression of the variant gene compared to the corresponding wild type gene.
In another embodiment the present invention relates to a vector comprising the polynucleotide of the invention or the gene of the invention.
Said vector may be, for example, a phage, plasmid, viral or retroviral vector. Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host/cells.
The polynucleotides or genes of the invention may be joined to a vector containing selectable markers for propagation in a host. Generally, a plasmid vector is introduced in a precipitate such as a calcium phosphate precipitate, or in a complex with a charged lipid or in carbon-based clusters. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.
In a more preferred embodiment of the vector of the invention the polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof.
Expression of said polynucleotide comprises transcription of the polynucleotide, preferably into a translatable mRNA. Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers. Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOX1 or GAL1 promoter in yeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells. Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. In this context, suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL). Preferably, said vector is an expression vector and/or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population. Methods which are well known to those skilled in the art can be used to construct recombinant viral 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). Alternatively, the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
The term “isolated fractions thereof” refers to fractions of eukaryotic or prokaryotic cells or tissues which are capable of transcribing or transcribing and translating RNA from the vector of the invention. Said fractions comprise proteins which are required for transcription of RNA or transcription of RNA and translation of said RNA into a polypeptide. Said isolated fractions may be, e.g., nuclear and cytoplasmic fractions of eukaryotic cells such as of reticulocytes.
The present invention furthermore relates to a host cell genetically engineered with the polynucleotide of the invention, the gene of the invention or the vector of the invention.
Said host cell may be a prokaryotic or eukaryotic cell; see supra. The polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally. In this respect, it is also to be understood that the recombinant DNA molecule of the invention can be used for “gene targeting” and/or “gene replacement”, for restoring a mutant gene or for creating a mutant gene via homologous recombination; see for example Mouellic, Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene Targeting, A Practical Approach, Oxford University Press.
The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal, mammalian or, preferably, human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term “prokaryotic” is meant to include all bacteria which can be transformed or transfected with a polynucleotide for the expression of a variant polypeptide of the invention. Prokaryotic hosts may include gram negative as well as gram positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. A polynucleotide coding for a mutant form of variant polypeptides of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Methods for preparing fused, operably linked genes and expressing them in bacteria or animal cells are well-known in the art (Sambrook, supra). The genetic constructs and methods described therein can be utilized for expression of variant polypeptides of the invention in, e.g., prokaryotic hosts. In general, expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host. The expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells. The transformed prokaryotic hosts can be grown in fermentors and cultured according to techniques known in the art to achieve optimal cell growth. The proteins of the invention can then be isolated from the grown medium, cellular lysates, or cellular membrane fractions. The isolation and purification of the microbially or otherwise expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies.
Thus, in a further embodiment the invention relates to a method for producing a molecular variant CYP2C8 polypeptide or fragment thereof comprising culturing the above described host cell; and recovering said protein or fragment from the culture.
In another embodiment the present invention relates to a method for producing cells capable of expressing a molecular variant CYP2C8 polypeptide comprising genetically engineering cells with the polynucleotide of the invention, the gene of the invention or the vector of the invention.
The cells obtainable by the method of the invention can be used, for example, to test drugs according to the methods described in D. L. Spector, R. D. Goldman, L. A. Leinwand, Cells, a Lab manual, CSH Press 1998. Furthermore, the cells can be used to study known drugs and unknown derivatives thereof for their ability to complement the deficiency caused by mutations in the CYP2C8 gene. For these embodiments the host cells preferably lack a wild type allele, preferably both alleles of the CYP2C8 gene and/or have at least one mutated from thereof. Ideally, the gene comprising an allele as comprised by the polynucleotides of the invention could be introduced into the wild type locus by homologous replacement. Alternatively, strong overexpression of a mutated allele over the normal allele and comparison with a recombinant cell line overexpressing the normal allele at a similar level may be used as a screening and analysis system. The cells obtainable by the above-described method may also be used for the screening methods referred to herein below.
Furthermore, the invention relates to a polypeptide or fragment thereof encoded by the polynucleotide of the invention, the gene of the invention or obtainable by the method described above or from cells produced by the method described above.
In this context it is also understood that the variant polypeptide of the invention can be further modified by conventional methods known in the art. By providing said variant proteins according to the present invention it is also possible to determine the portions relevant for their biological activity or inhibition of the same. The terms “polypeptide” and “protein” as used herein are exchangeable. Moreover, what is comprised by said terms is standard textbook knowledge.
The present invention furthermore relates to an antibody which binds specifically to the polypeptide of the invention.
Advantageously, the antibody specifically recognizes or binds an epitope containing one or more amino acid substitution(s) as defined above. Antibodies against the variant polypeptides of the invention can be prepared by well known methods using a purified protein according to the invention or a (synthetic) fragment derived therefrom as an antigen. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Köhler and Milstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. In a preferred embodiment of the invention, said antibody is a monoclonal antibody, a polyclonal antibody, a single chain antibody, human or humanized antibody, primatized, chimerized or fragment thereof that specifically binds said peptide or polypeptide also including bispecific antibody, synthetic antibody, antibody fragment, such as Fab, Fv or scFv fragments etc., or a chemically modified derivative of any of these. Furthermore, antibodies or fragments thereof to the aforementioned polypeptides can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. These antibodies can be used, for example, for the immunoprecipitation and immunolocalization of the variant polypeptides of the invention as well as for the monitoring of the presence of said variant polypeptides, for example, in recombinant organisms, and for the identification of compounds interacting with the proteins according to the invention. For example, surface plasmon resonance as employed in the BIAcore system can be used to increase the efficiency of phage antibodies which bind to an epitope of the protein of the invention (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).
In a preferred embodiment the antibody of the present invention specifically recognizes an epitope containing one or more amino acid substitution(s) resulting from a nucleotide exchange as defined supra.
Antibodies which specifically recognize modified amino acids such as phospho-Tyrosine residues are well known in the art. Similarly, in accordance with the present invention antibodies which specifically recognize even a single amino acid exchange in an epitope may be generated by the well known methods described supra.
In light of the foregoing, in a more preferred embodiment the antibody of the present invention is monoclonal or polyclonal.
The invention also relates to a transgenic non-human animal comprising at least one polynucleotide of the invention, the gene of the invention or the vector of the invention as described supra.
The present invention also encompasses a method for the production of a transgenic non-human animal comprising introduction of a polynucleotide or vector of the invention into a germ cell, an embryonic cell, stem cell or an egg or a cell derived therefrom. The non-human animal can be used in accordance with the method of the invention described below and may be a non-transgenic healthy animal, or may have a disease or disorder, preferably a disease caused by at least one mutation in the gene of the invention. Such transgenic animals are well suited for, e.g., pharmacological studies of drugs in connection with variant forms of the above described variant polypeptides since these polypeptides or at least their functional domains are conserved between species in higher eukaryotes, particularly in mammals. Production of transgenic embryos and screening of those can be performed, e.g., as described by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993), Oxford University Press. The DNA of the embryos can be analyzed using, e.g., Southern blots with an appropriate probe or based on PCR techniques.
A transgenic non-human animal in accordance with the invention may be a transgenic mouse, rat, hamster, dog, monkey, rabbit, pig, frog, nematode such as Caenorhabditis elegans, fruitfly such as Drosophila melanogaster or fish such as torpedo fish or zebrafish comprising a polynucleotide or vector of the invention or obtained by the method described above, preferably wherein said polynucleotide or vector is stably integrated into the genome of said non-human animal, preferably such that the presence of said polynucleotide or vector leads to the expression of the variant polypeptide of the invention. It may comprise one or several copies of the same or different polynucleotides or genes of the invention. This animal has numerous utilities, including as a research model for cardiovascular research and therefore, presents a novel and valuable animal in the development of therapies, treatment, etc. for diseases caused by cardiovascular diseases. Accordingly, in this instance, the mammal is preferably a laboratory animal such as a mouse or rat.
Thus, in a preferred embodiment the transgenic non-human animal of the invention is a mouse, a rat or a zebrafish.
Numerous reports revealed that said animals are particularly well suited as model organisms for the investigation of the drug metabolism and its deficiencies or cancer. Advantageously, transgenic animals can be easily created using said model organisms, due to the availability of various suitable techniques well known in the art.
The invention also relates to a solid support comprising one or a plurality of the polynucleotide, the gene, the vector, the polypeptide, the antibody or the host cell of the invention in immobilized form.
The term “solid support” as used herein refers to a flexible or non-flexible support that is suitable for carrying said immobilized targets. Said solid support may be homogenous or inhomogeneous. For example, said solid support may consist of different materials having the same or different properties with respect to flexibility and immobilization, for instance, or said solid support may consist of one material exhibiting a plurality of properties also comprising flexibility and immobilization properties. Said solid support may comprise glass-, polypropylene- or silicon-chips, membranes oligonucleotide-conjugated beads or bead arrays.
The term “immobilized” means that the molecular species of interest is fixed to a solid support, preferably covalently linked thereto. This covalent linkage can be achieved by different means depending on the molecular nature of the molecular species. Moreover, the molecular species may be also fixed on the solid support by electrostatic forces, hydrophobic or hydrophilic interactions or Van-der-Waals forces. The above described physico-chemical interactions typically occur in interactions between molecules. For example, biotinylated polypeptides may be fixed on a avidin-coated solid support due to interactions of the above described types. Further, polypeptides such as antibodies, may be fixed on an antibody coated solid support. Moreover, the immobilization is dependent on the chemical properties of the solid support. For example, the nucleic acid molecules can be immobilized on a membrane by standard techniques such as UV-crosslinking or heat.
In a preferred embodiment of the invention said solid support is a membrane, a glass- or polypropylene- or silicon-chip, are oligonucleotide-conjugated beads or a bead array, which is assembled on an optical filter substrate.
Moreover, the present invention relates to an in vitro method for identifying a polymorphism said method comprising the steps of:
The term “prevalence” as used herein means that individuals are be susceptible for one or more disease(s) which are associated with CYP2C8 dysfunction or dysregulation or could already have one or more of said disease(s). Thereby, one CYP2C8 associated disease can be used to determine the susceptibility for another CYP2C8 associated disease. Moreover, symptoms which are indicative for a prevalence for developing of a disease are very well known in the art and have been sufficiently described in standard textbooks such as Pschyrembel.
Advantageously, polymorphisms according to the present invention which are associated with CYP2C8 dysfunction or dysregulation or one or more disease(s) based thereon should be enriched in subgroups of individuals which have a prevalence for said diseases versus subgroups which have no prevalence for said diseases. Thus, the above described method allows the rapid and reliable detection of polymorphism which are indicative for one or more CYP2C8 associated disease(s) or a susceptibility therefor. Advantageously, due to the phenotypic preselection a large number of individuals having no prevalence might be screened for polymorphisms in general. Thereby, a reference sequences comprising polymorphisms which do not correlate to one or more CYP2C8 associated disease(s) can be obtained. Based on said reference sequences it is possible to efficiently and reliably determine the relevant polymorphisms.
In a further embodiment the present invention relates to a method for identifying and obtaining a pro-drug or a drug capable of modulating the activity of a molecular variant of a CYP2C8 polypeptide comprising the steps of:
The term “compound” in a method of the invention includes a single substance or a plurality of substances which may or may not be identical.
Said compound(s) may be chemically synthesized or produced via microbial fermentation but can also be comprised in, for example, samples, e.g., cell extracts from, e.g., plants, animals or microorganisms. Furthermore, said compounds may be known in the art but hitherto not known to be useful as an inhibitor, respectively. The plurality of compounds may be, e.g., added to the culture medium or injected into a cell or non-human animal of the invention.
If a sample containing (a) compound(s) is identified in the method of the invention, then it is either possible to isolate the compound from the original sample identified as containing the compound, in question or one can further subdivide the original sample, for example, if it consists of a plurality of different compounds, so as to reduce the number of different substances per sample and repeat the method with the subdivisions of the original sample. It can then be determined whether said sample or compound displays the desired properties, for example, by the methods described herein or in the literature (Spector et al., Cells manual; see supra). Depending on the complexity of the samples, the steps described above can be performed several times, preferably until the sample identified according to the method of the invention only comprises a limited number of or only one substance(s). Preferably said sample comprises substances of similar chemical and/or physical properties, and most preferably said substances are identical. The methods of the present invention can be easily performed and designed by the person skilled in the art, for example in accordance with other cell based assays described in the prior art or by using and modifying the methods as described herein. Furthermore, the person skilled in the art will readily recognize which further compounds may be used in order to perform the methods of the invention, for example, enzymes, if necessary, that convert a certain compound into a precursor. Such adaptation of the method of the invention is well within the skill of the person skilled in the art and can be performed without undue experimentation.
Compounds which can be used in accordance with the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligands, peptidomimetics, PNAs and the like. Said compounds may act as agonists or antagonists of the invention. Said compounds can also be functional derivatives or analogues of known drugs. Methods for the preparation of chemical derivatives and analogues are well known to those skilled in the art and are described in, for example, Beilstein, Handbook of Organic Chemistry, Springer edition New York Inc., 175 Fifth Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley, New York, USA. Furthermore, said derivatives and analogues can be tested for their effects according to methods known in the art or as described. Furthermore, peptide mimetics and/or computer aided design of appropriate drug derivatives and analogues can be used, for example, according to the methods described below. Such analogs comprise molecules that may have the basis structure of known CYP2C8 substrates, inhibitors and/or modulators.
Appropriate computer programs can be used for the identification of interactive sites of a putative inhibitor and the polypeptides of the invention by computer assistant searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computer systems for the computer aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemistry 25 (1986), 5987-5991. The results obtained from the above-described computer analysis can be used in combination with the method of the invention for, e.g., optimizing known inhibitors, analogs, antagonists or agonists. Appropriate peptidomimetics and other inhibitors can also be identified by the synthesis of peptidomimetic combinatorial libraries through successive chemical modification and testing the resulting compounds, e.g., according to the methods described herein. Methods for the generation and use of peptidomimetic combinatorial libraries are described in the prior art, for example in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. Furthermore, the three-dimensional and/or crystallographic structure of said compounds and the polypeptides of the invention can be used for the design of peptidomimetic drugs (Rose, Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558). It is very well known how to obtain said compounds, e.g. by chemical or biochemical standard techniques. Thus, also comprised by the method of the invention are means of making or producing said compounds. In summary, the present invention provides methods for identifying and obtaining compounds which can be used in specific doses for the treatment of specific forms of CYP2C8 associated diseases.
The above definitions apply mutatis mutandis to all of the methods described in the following.
In a further embodiment the present invention relates to a method for identifying and obtaining an inhibitor of the activity of a molecular variant of a CYP2C8 polypeptide comprising the steps of:
In a preferred embodiment of the method of the invention said cell is a cell, obtained by the method of the invention or can be obtained from the transgenic non-human animal as described supra.
In a still further embodiment the present invention relates to a method of identifying and obtaining a pro-drug or drug capable of modulating the activity of a molecular variant of a CYP2C8 polypeptide comprising the steps of:
Advantageously, in said method said measuring step comprises measuring the formation of a second complex of said protein and said inhibitor candidate. Preferably, said measuring step comprises measuring the amount of said first molecule that is not bound to said protein.
In a particularly preferred embodiment of the above-described method of said first molecule is a agonist or antagonist or a substrate and/or a inhibitor and/or a modulator of the polypeptide of the invention, e.g., with a radioactive or fluorescent label.
In a still another embodiment the present invention relates to a method of identifying and obtaining an inhibitor capable of modulating the activity of a molecular variant of a CYP2C8 polypeptide comprising the steps of:
In a preferred embodiment of the method of the invention said measuring step comprises measuring the formation of a second complex of said protein and said compound.
In another preferred embodiment of the method of the invention said measuring step comprises measuring the amount of said first molecule that is not bound to said protein.
In a more preferred embodiment of the method of the invention said first molecule is labeled.
The invention furthermore relates to a method for the production of a pharmaceutical composition comprising the steps of the method as described supra; and the further step of formulating the compound identified and obtained or a derivative thereof in a pharmaceutically acceptable form.
The therapeutically useful compounds identified according to the methods of the invention can be formulated and administered to a patient as discussed above. For uses and therapeutic doses determined to be appropriate by one skilled in the art and for definitions of the term “pharmaceutical composition” see infra.
Furthermore, the present invention encompasses a method for the preparation of a pharmaceutical composition comprising the steps of the above-described methods; and formulating a drug or pro-drug in the form suitable for therapeutic application and preventing or ameliorating the disorder of the subject diagnosed in the method of the invention.
Drugs or pro-drugs after their in vivo administration are metabolized in order to be eliminated either by excretion or by metabolism to one or more active or inactive metabolites (Meyer, J. Pharmacokinet. Biopharm. 24 (1996), 449-459). Thus, rather than using the actual compound or inhibitor identified and obtained in accordance with the methods of the present invention a corresponding formulation as a pro-drug can be used which is converted into its active in the patient. Precautionary measures that may be taken for the application of pro-drugs and drugs are described in the literature; see, for review, Ozama, J. Toxicol. Sci. 21 (1996), 323-329).
In a preferred embodiment of the method of the present invention said drug or prodrug is a derivative of a medicament as defined hereinafter.
The present invention also relates to a method of diagnosing a disorder related to the presence of a molecular variant of the CYP2C8 gene or susceptibility to such a disorder comprising determining the presence of a polynucleotide or the gene of the invention in a sample from a subject.
In accordance with this embodiment of the present invention, the method of testing the status of a disorder or susceptibility to such a disorder can be effected by using a polynucleotide gene or nucleic acid of the invention, e.g., in the form of a Southern or Northern blot or in situ analysis. Said nucleic acid sequence may hybridize to a coding region of either of the genes or to a non-coding region, e.g. intron. In the case that a complementary sequence is employed in the method of the invention, said nucleic acid molecule can again be used in Northern blots. Additionally, said testing can be done in conjunction with an actual blocking, e.g., of the transcription of the gene and thus is expected to have therapeutic relevance. Furthermore, a primer or oligonucleotide can also be used for hybridizing to one of the above mentioned CYP2C8 gene or corresponding mRNAs. The nucleic acids used for hybridization can, of course, be conveniently labeled by incorporating or attaching, e.g., a radioactive or other marker. Such markers are well known in the art. The labeling of said nucleic acid molecules can be effected by conventional methods.
Additionally, the presence or expression of variant CYP2C8 gene can be monitored by using a primer pair that specifically hybridizes to either of the corresponding nucleic acid sequences and by carrying out a PCR reaction according to standard procedures. Specific hybridization of the above mentioned probes or primers preferably occurs at stringent hybridization conditions. The term “stringent hybridization conditions” is well known in the art; see, for example, Sambrook et al., “Molecular Cloning, A Laboratory Manual” second ed., CSH Press, Cold Spring Harbor, 1989; “Nucleic Acid Hybridisation, A Practical Approach”, Hames and Higgins eds., IRL Press, Oxford, 1985. Furthermore, the mRNA, cRNA, cDNA or genomic DNA obtained from the subject may be sequenced to identify mutations which may be characteristic fingerprints of mutations in the polynucleotide or the gene of the invention. The present invention further comprises methods wherein such a fingerprint may be generated by RFLPs of DNA or RNA obtained from the subject, optionally the DNA or RNA may be amplified prior to analysis, the methods of which are well known in the art. RNA fingerprints may be performed by, for example, digesting an RNA sample obtained from the subject with a suitable RNA-Enzyme, for example RNase T1, RNase T2 or the like or a ribozyme and, for example, electrophoretically separating and detecting the RNA fragments as described above. Further modifications of the above-mentioned embodiment of the invention can be easily devised by the person skilled in the art, without any undue experimentation from this disclosure; see, e.g., the examples. An additional embodiment of the present invention relates to a method wherein said determination is effected by employing an antibody of the invention or fragment thereof. The antibody used in the method of the invention may be labeled with detectable tags such as a histidine flags or a biotin molecule.
The invention relates to a method of diagnosing a disorder related to the presence of a molecular variant of a CYP2C8 gene or susceptibility to such a disorder comprising determining the presence of a polypeptide or the antibody of the invention in a sample from a subject.
In a preferred embodiment of the above described method said disorder is a cancer or cardiovascular disease.
In a preferred embodiment of the present invention, the above described method is comprising PCR, ligase chain reaction, restriction digestion, direct sequencing, nucleic acid amplification techniques, hybridization techniques or immunoassays. Said techniques are very well known in the art.
Moreover, the invention relates to a method of detection of the polynucleotide or the gene of the invention in a sample comprising the steps of
The invention also relates to an in vitro method for diagnosing a disease comprising the steps of the method described supra, wherein binding of said polynucleotide or gene to said immobilized targets on said solid support is indicative for the presence or the absence of said disease or a prevalence for said disease.
The invention furthermore relates to a diagnostic composition comprising the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention.
In addition, the invention relates to a pharmaceutical composition comprising the polynucleotide, the gene, the vector, the polypeptide or the antibody of the invention. These pharmaceutical compositions comprising, e.g., the antibody may conveniently be administered by any of the routes conventionally used for drug administration, for instance, orally, topically, parenterally or by inhalation. Acceptable salts comprise acetate, methylester, HCl, sulfate, chloride and the like. The compounds may be administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate to the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable character or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables. The carrier(s) must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, either a solid or liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid and the like. Exemplary of liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax.
The dosage regimen will be determined by the attending physician and other clinical factors; preferably in accordance with any one of the above described methods. As is well known in the medical arts, dosages for any one patient depends upon many factors, including the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment.
Furthermore, the use of pharmaceutical compositions which comprise antisense-oligonucleotides which specifically hybridize to RNA encoding mutated versions of the polynucleotide or gene according to the invention or which comprise antibodies specifically recognizing a mutated polypeptide of the invention but not or not substantially the functional wild-type form is conceivable in cases in which the concentration of the mutated form in the cells should be reduced.
In another embodiment the present invention relates to the use of the polynucleotide, the gene, the vector, the polypeptide, the polynucleotides having at a position corresponding to position 270 (exon 3) and 206 (exon 8) (GenBank accession No: AF136833.1 and AF136842.1, respectively) of the CYP2C8 gene (GenBank accession No: GI: 13787189) an A instead of a G in position 270 and a G instead of an A in position 206, or the antibody of the invention for the preparation of a diagnostic composition for diagnosing a disease.
Thanks to the present invention the particular drug selection, dosage regimen and corresponding patients to be treated can be determined in accordance with the present invention. The dosing recommendations will be indicated in product labeling by allowing the prescriber to anticipate dose adjustments depending on the considered patient group, with information that avoids prescribing the wrong drug to the wrong patients or at the wrong dose.
In a further embodiment the present invention relates to the use of the polynucleotide, the gene, the vector, the polypeptide, the polynucleotides having at a position corresponding to position 117 in exon 5 (GenBank accession No: AF136837.1) of the CYP2C8 gene (GenBank accession No: GI: 13787189) a T instead of an A, or the antibody of the invention for the preparation of a pharmaceutical composition for treating a disease.
In a more preferred embodiment of the use of the present invention said disease is an incompatibility or disease related to arachidonic acid metabolism, cancer or cardiovascular disease.
Finally, the present invention relates to a diagnostic kit for detection of a single nucleotide polymorphism comprising the polynucleotide, the gene, the vector, the polypeptide, the antibody, the host cell, the transgenic non-human animal or the solid support of the invention.
The kit of the invention may contain further ingredients such as selection markers and components for selective media suitable for the generation of transgenic cells and animals. The kit of the invention can be used for carrying out a method of the invention and could be, inter alia, employed in a variety of applications, e.g., in the diagnostic field or as research tool. The parts of the kit of the invention can be packaged individually in vials or other appropriate means depending on the respective ingredient or in combination in suitable containers or multicontainer units. Manufacture of the kit follows preferably standard procedures which are known to the person skilled in the art. The kit may be used for methods for detecting expression of a mutant form of the polypeptides, genes or polynucleotides in accordance with any one of the above-described methods of the invention, employing, for example, immunoassay techniques such as radio-immunoassay or enzyme-immunoassay or preferably nucleic acid hybridization and/or amplification techniques such as those described herein before and in the Examples as well as pharmacokinetic studies when using non-human transgenic animals of the invention.
The figures illustrate the invention:
Correlation of the SNP C104G (Exon 5, 1264M) with reduced protein levels of CYP2C8. Expression levels of 14 individuals were determined by Western Blot analysis and LC-MS using verapamil as specific substrate. The boxplots show the distribution of samples according to the genotype at amino acid position 264. The genotype-phenotype correlation is significant (p=0.037, N=14).
Correlation of the SNP −370 relative to the start codon ATG with increased expression levels as detected by western blotting, using the drug taxol (paclitaxel) as specific substrate. As shown in the boxplots the genotype-phenotype correlation is significant between homozygous wild type and heterozygous mutant samples (p=0.044, N=20). (One homozygous sample was analyzed and yielded expression levels >400).
Correlation of the SNP at position −370 in the untranslated region based on deviates from mean values of CYP2C8 expression levels from two independent sample collectives (N=62). The phenotype of the patients was determined by LC/MS and Western Blot. The phenotype-genotype correlation is significant as shown in the boxplots (p=0.017 for homozygous vs wildtype and p=0.071 for heterozygous vs wildtype).
Correlation of the allele including the linked SNPs G-1207A, delAT-640/41 (both in the promoter), G270A (exon 3), and A206G (exon 8) with a poor metabolizer phenotype (PM). However, no homozygous individuals for this allele could yet be phenotyped. The data shown in the box plot do only show a trend (p=0.071; N=18) but no significance.
Transfection of LS174T cells with either a CYP2C8 wild type promoter construct or two SNP(s) containing promoter fragments (G-1207A and -640 to 641delAT or T-370G). Using the eukaryotic pGL3 expression vector mean values of six independent transfection assays were analysed following normalisation of 2C8 wild type activity to 100%.
Computer modeled CYP2C8 enzyme structure of the protein variants Thr 159 Pro (frameshift), Glu 274 Stop and Gly 365 Ser. Dark grey represents the unchanged structure of the variant protein, the light grey prepresents the missing amino acids of the CYP2C8 variant structure. The circle indicates the active site of the enzyme with the altered amino acid Gly 365 Ser in dark.
Reference or wild type GenBank sequences for the polynucleotides, polypeptide and mRNA according to the present invention
The invention will now be described by reference to the following biological Examples which are merely illustrative and are not constructed as a limitation of the scope of the present invention.
Genomic DNA was obtained by standard ion exchange chromatography techniques (Qiagen kits for isolation of genomic DNA from blood). Blood from all the individuals tested (volunteers from Parexel, Berlin and the Institute for Clinical Pharmacology, Stuttgart) was obtained under consideration of all legal, medical and bureaucratical requirements. Further samples from other ethnic populations (e.g. Caucasian, Japanese, African-American) were purchased from commercial sources.
By using polymerase chain reaction (PCR) with specific oligonucleotide primers, two for each fragment, defined DNA-fragments containing specific parts of the human CYP2C8 gene were obtained. These specific oligonucleotide primers were designed to bind to sequences upstream and downstream of the various exons as well as in the 5 prime region of the CYP2C8 gene. The resulting DNA fragments did not contain codogenic parts alone but also sequences covering the intronic parts located at the exon-intron boundaries. These sites are known to be important for correct processing and subsequent expression of the protein encoding mRNA, a process called “splicing”. Commercially synthesized oligonucleotide primer pairs that were purified by affinity chromatography were optimized for each of the 9 exon and 4 promoter fragments of the human CYP2C8 gene. The sequences for each primer are listed in table 1.
Polymerase chain reactions were performed under conditions that were optimized for each of the nine fragments and a promoter region covering about 2 kb upstream of the initiation codon for mRNA translation. PCRs were carried out for all exons in a volume of 50 μl. 10-50 ng of template DNA were added to standard PCR-buffer containing 1.5 mM MgCl2, 200 μM dNTPs and 1 U Taq-polymerase (all from Qiagen, Hilden) as well as 10-40 pMol of primers (MWG Biotech, Munich). All PCR reactions were performed on identical conditions at a Perkin Elmer thermocycler (Modell 9700) with an initial denaturation step of 94° C. for 2 min, followed by 34 cycles for PCR-fragment generation with 45 s denaturation at 94° C., 45 s of annealing at 62° C. and 1 min at 72° C. for elongation. The exact location of the primers and size of the desired fragments are also listed in table 1.
The defined DNA fragments containing specific parts of the CYP2C8 gene, exon as well as some intron sequences at the inton-exon boundaries were processed to remove nonincorporated nucleotides and buffer components that might otherwise interfere with the subsequent determination of the individual CYP2C8 genotype by direct cycle sequencing. For this purification, standard ion exchange chromatography techniques were used (Qiagen kits for PCR-fragment purification). For all fragments sufficient yields of purified fragments, suitable for direct DNA sequencing analysis were obtained. Aliquots of purified fragments were subjected to direct sequence analysis of the CYP2C8 gene in an ABI 3700 capillary sequencer.
For sequence analysis of relevant regions of the human CYP2C8 gene from many different individuals, PCR amplifications of the relevant regions of the gene were carried out (pimers see table 1) following purification of the PCR products and sequencing with established methods (ABI dye terminator cycle sequencing). Since the individual genetic makeup is represented by two copies of any gene (diploidy), great care has to be taken in the evaluation of the sequences not to unambiguously identify homozygous, but also heterozygous sequence variation. Therefore, in cases where no clear discrimination could be detected, forward and reverse sequencing was performed. Moreover, for the discovery of complete and defined alleles, e.g. in linkage equilibrium, it is necessary to cover all exons as well as the promoter region to provide a comprehensive basis for the phenotype prediction of individual SNPs.
For the evaluation of CYP2C8 variations in the human population, sequence analyses of the relevant regions, including a 2 kb promoter fragment and all exons of the gene were carried out from genomic DNA of each 48 Caucasian, Japanese and African-American individuals. The sequences were subjected to a computer analysis programme (Phredphrap™, Perkin Elmer) and inspected manually for the occurrence of DNA sequences deviating from recently published CYP2C8 sequences that were considered to represent the “wild type” sequences in this work.
Because population genetics enables a calculation of the expected frequency of homozygous vs. heterozygous alleles of a defined gene (Hardy Weinberg formula: 2p e2+2pq+2q e2=1), it was possible to confirm predicted distributions of homozygous vs. heterozygous alleles and deviations from the experimental findings. This serves as experimental control that a detected sequence variation indeed represents a novel allele.
Several new CYP2C8 sequence variations were discovered and experimentally confirmed using this approach which are shown in table 2. 18 polymorphisms are located in the 5′ untranslated region/promoter of the gene (GenBank accession No: AF136830.1). 22 new polymorphisms could be found in sequences of introns 1, 2, 3, 4 and 8 (GenBank accession Nos: AF136832.1, AF136833.1, AF136835.1 AF136843.1 and AF136844.1) and one in the 3′ untranslated region of the gene (GenBank accession No: AF136845.1).
Furthermore, three particular nucleotide changes in exon 3 (position 334 at exon/intron boundary, GenBank accession No: 163833.1) and exon 8 (position 30 and 87, GenBank accession No: AF136843.1) were detected that do not change the amino acid sequence. In exon 3 (position 329, GenBank accession No: AF136833.1), exon 4 (position 243, GenBank accession No: AF136842.2 and position 13 of GenBank accession No: AF136835.1), exon 5 (position 42, 101 and 104, GenBank accession No: AF136837.1), exon 6 (position 309, GenBank accession No: AF136838.1) and 7 (position 1135, GenBank accession No: NM—000770.1 and position 232, GenBank accession No: AF136840.1) nine SNPs could be identified that change the protein sequence as shown in table 4, where the deviative amino acid is typed in a bold style. These novel, and already published CYP2C8 SNPs serve as markers for the characterization of the CYP2C8 status in patients.
The positions of the novel CYP2C8 SNPs, including the exact novel sequence context are listed in table 2. The deviative base in the sequence is typed underlined and in a bold style.
The anticancer drug taxol (paclitaxel) can be considered to be the prototype for CYP2C8 and it's isoform 6-hydroxypaclitaxel as diagnostic substrate. Furthermore, verapamil represents another specific substrate for CYP2C8 that is methylated to different metabolites, e.g D-702 and D-703 or desalkylated to D-617 (i.e. a substrate of CYP3A4). The generation of a specific metabolite used to selectively proof the functional activity of CYP2C8 (LC-MS) is shown in
To validate a possible correlation of the single nucleotide polymorphisms G-1207A, delAT-640 to -641, and/or T-370G (all 5′UTR/Promoter) and/or C104G (exon 5) with a certain phenotype, biopsies from patients of two different studies were analyzed. The functional characterization of the CYP2C8 gene had been determined from enterocyte preparations of the duodenum and the liver.
Using these analytical tools, phenotypically characterized samples that have been treated with taxol (N=22) or verapamil (N=15, 44) were subjected to genotyping. Either of the collectives showed that two SNPs G-1207A, delAT-640 to -641 of the 5′ untranslated region are in linkage disequilibrium and, in combination with SNPs G270A (exon 3) and A206G (exon 8), represent a new allele (haplotype) that is mainly defined by the presence of the two novel promoter SNPs G-1207A and delAT-640 to -641.
The eukaryotic promoter region of a gene is composed of several regulatory elements, e.g enhancer, silencer and other responsive elements. Here, single nucleotide polymorphisms exhibit significant influence. Regarding cytochrome P450-enzymes induction mechanisms, e.g. transcription factors like C/EBP, HPFs or barbiebox-sites identified in CYP2C9 (Klose, J Biochem Mol Toxicol 13 (1999), 289-95) are important since temporary expression is required. SNPs change or interfere with such elements and can alter promoter action and/or transcription activity. The novel SNP identified at position −1207 most probably abolishes transcription factor binding that has selectively been shown for the binding of tissue specific sterol regulatory element binding protein 1 (SREBP-1, Vallett, J Biol. Chem. 271 (1996), 12247-53). Reduced expression has been shown if position −1207 does not correspond to the wild type. The correlation of this new allele with CYP2C8 enzyme activity is displayed in
Another polymorphism located further downstream at position 1668 in the 5-prime untranslated region (position −370 relative to the ATG-start codon) could be identified to significantly increase the expression level as shown in
Independently, a further validation was carried out by genotyping 22 samples corresponding to individual liver extracts, in which the metabolism of taxol was assessed. The data confirm results as presented in examples 8 and 9.
In another embodiment the present invention relates to a polymorphism in exon 5 at position C104G (GenBank accession No: AF136837.1). This change correlates with a reduced protein concentration analyzed from genotyped samples (
CYP2C8-polymorphisms can be detected not only by sequencing but also by various other means. As one alternative to the sequencing methodology, genotyping can be performed with PCR fragments to be processed by one restriction endonuclease specificially cutting at a region, composed of a unique sequence of 4-6 nucleotides. Due to the limited length of a PCR-fragment sometimes advantage can be taken of this specificity if it discriminates mutant and wild type, i.e. resulting in digested or un digested fragments (Table 5). Regarding the present invention this was the case for fragments of the promoter region 4 (position −370, double cutter AcsI for wild type and single cutter in the mutant), that indicates the respective allele, exon 3 (position 275, single cutter Sapl for CYP2C8-mutant), and exon 5 (position 104, single cutter ClaI for wild type). Depending of the fragments' specific SNP-region the restriction pattern unambiguously reflects either the wild type, the heterozygous or homozygous mutant. As defined by the primers listed in table 1, the exons screened for result in the following RFLP-fragments:
The screen for SNPs in the CYP2C8 gene in the genomes of different ethnic groups yielded a number of polymorphisms listed in table 2. 48 samples were analyzed from each of the ethnic populations Caucasian, Japanese and African-American, respectively. Within this collection, the large number of SNPs in the untranslated region could be considered to potentially influence the protein level.
Furthermore, several polymorphisms show extensive inter ethnical discrepancies (Table 3) between various ethnical groups. The 57 new polymorphisms identified in the CYP2C8 gene will complement the existing knowledge and contribute to a more comprehensive understanding of the gene, avoiding problems in drug response and, concerning other ethnical groups, thus facilitating “bridging studies” that could be of interest when projecting data from these embodiment.
Three promoter SNPs were tested for their contribution to different expression levels by transfection assays using the LS174T cell line. Promoter fragments containing the wild type or the SNPs at positions corresponding to positions G-1207A and delAT-640 to -641 (GenBank accession No: AF136830.1) or at position corresponding to position T-370G (GenBank accession No: AF136830.1) were introduced into a commercial mammalian expression vector. The plasmid harbours standard sequences for the propagation in eukaryotic cells including the reporter gene luciferase that is controlled by the integrated promoter sequence. Following sequence verification of each DNA-insert, cells were cotransfected with β-galactosidase, harvested after 48 h and analysed for luciferase activity. Promoter activities (%) are shown in
Protein extracts have been prepared from human liver samples. The protein levels of CYP2C8 were analysed by western blot using samples genotyped for SNPs G-1207A, delAT-640 to -641, T-370G (all GenBank accession No: AF136830.1) and C104G (GenBank accession No: AF136837.1). Table 5 shows the effects of different genotypes on the expression levels of CYP2C8 (pmol/mg) normalized for the wild type (=100%). Results are in total agreement with the functional data described by the promoter-reporter assays in example 8. The promoter SNP in position T-370G confers to increased levels of the CYP2C8 protein (150%), whereas polymorphisms G-1207A and delAT-640 to -641 in contrast show a reduced protein expression (72%). In combination with SNPs G-1207A and delAT-640 to -641, or C104G alone the polymorphism T-370G differentially influences protein levels as indicated by arrow. Here, the presence of two SNPs with significant frequency leads to combined effects. Therefore, considerations for reliable phenotype prediction as a result from genotyping must depend on multiple SNP-analyses. The data indicate that the SNP in position −370, which by itsself is responsible for up regulation of the CYP2C8-protein level, shows no significant CYP2C8 increase if it is combined with SNPs G-1207A and delAT-640 to -641 (5′UTR), which by themselves reduce the expression. The combined expression level of 119% is barely higher than in the homozygous wildtype situation. Vice versa, in combination with the C104G allele (exon 5) the increased expression due to the SNP at position −370 is compensated by the SNP C104G to normal expression levels (97%) compared to the wild type. This reflects the strong impact of SNP C104G alone on the protein level (see
In another embodiment the present invention relates to a polymorphisms in exon 3, exon 6 and 7 at positions 329 (GenBank accession No: AF136833.1), 309 (GenBank accession No: AF136838.1) rand 1135 (GenBank accession No: NM—000770.1) respectively. The delA change in position 329 causes a frameshift abolishing the C-terminal part of the protein. The G309T change in position 309 results in a premature termination at amino acid position 274 of the protein. Both variant transcripts encode for polypeptides that will loose their function and are therefore most likely poor metabolizer alleles (
§has been detected in a phenotyped sample (Caucasian sample).
| Number | Date | Country | Kind |
|---|---|---|---|
| 01112899.8 | Jun 2001 | EP | regional |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10479225 | Apr 2004 | US |
| Child | 12880001 | US |
