Biomarkers for estimating efficacy of aliskiren as hypertensive agent

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, particularly to therapy and pharmacology, particularly to pharmacogenetic analysis, and concerns detecting genetic polymorphisms being efficacy markers of aliskiren as an antihypertensive agent. Aliskiren is used for preparing a drug for treating hypertension, for reducing mean derived systolic pressure and for reducing diastolic pressure. And aliskiren therapy is applied in a group of patients specified by genetic polymorphisms in biomarker genes where the aliskiren efficacy is indicated by genetic polymorphisms - SNP_4769 as specified in SEQ ID NO:1 in an angiotesin-converting enzyme (ACE) gene, SNP1445 as specified in SEQ ID NO:2 in an angiotensin II receptor type 2 (AGTR2) gene, and SNP_4795 as specified in SEQ ID NO:3 in an AGTR2 gene .

EFFECT: invention provides a method for determining sensitivity of a hypertensive individual to the aliskiren therapy, and application of a gene product of a gene specified in a group including the angiotesin-converting enzyme (ACE) gene and angiotensin II receptor type 2 (AGTR2) gene as a drug target.

6 cl, 1 dwg, 7 tbl, 2 ex

 

The technical field to which the invention relates.

The present invention mainly relates to analytical testing tissue samples in vitro, more specifically to the identification of genetic polymorphisms, which are markers of the effectiveness of aliskiren as an antihypertensive agent.

Background of invention

The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure and volume homeostasis. Renin is secreted by the kidneys in response to decreased circulating blood volume and low blood pressure and cleaves the substrate angiotensinogen education inactive Decapeptide of angiotensin I (Ang I). Ang I is converted to the active oktapeptid Ang II angiotensin-converting enzyme (ACE). Ang II interacts with cellular receptors inducyruya vasoconstriction and the release of catecholamines from the medulla of the adrenal glands and presinapticheskih nerve endings. It also induces aldosterone secretion and reabsorption of sodium. In addition, Ang II inhibits the release of renin, thereby providing feedback to the system. Therefore, Ang II acts on different levels (for example, at the level of the vascular network, the sympathetic nervous system, the cortex and the medulla of the adrenal glands) to increase the resistance of the blood vessels and increase Romanova pressure.

The renin-angiotensin system (RAS) can be blocked at different levels. As renin inhibitors block the RAS at a higher level than the ACF inhibitors and antagonists of Ang II, they have a different effect on the components of the RACES. After administration of the renin inhibitor blocked the education and Ang I, and Ang II. Despite the fact that after the suppression of the ACE only blocked the formation of Ang II, the levels of Ang I rise. Thus, Ang I still able to be converted to Ang II and other angiotensinii peptides on other metabolic pathways, for example, through hematol system.

Aliskiren (SPP100) is ones antihypertensive agent with a low molecular weight (609,8). Cm. Wood J.M., and others, Biochem. Biophys. Res. Commun. 308, 2003, s-705. Its mechanism of action is distinct from the mechanism of action of other commercial antihypertensive agents. Aliskiren blocks the renin-angiotensin system (RAS) at its first rate-limiting step. In vitro aliskiren is a potent inhibitor of human renin (IC50=0.6 nmole). In vivo aliskiren, administered orally (P.O.) or intravenously (I.V) in several studies on grungowych monkeys, depleted in sodium, has caused a complete suppression of plasma renin activity (AED), a steady decrease in mean arterial pressure (SBP) and a significant increase in plasma concentrations of acting is about, and total renin. In humans, the concentrations of aliskiren plasma rapidly increased after administration, reaching peak levels within 3-5 hours of Magnitude, and Cmaxand AUC increased with increasing dose, but not linearly. The half-life of aliskiren takes approximately 25 h, and its bioavailability is about 2.7%.

Traditional medical approaches to diagnosis and treatment of disease based only on clinical data, or in addition to them also for diagnostic testing. This conventional approach often leads to the choice of treatment method that is optimal for the effectiveness of prescribed drug therapy or to minimize the likelihood of side effects for a specific subject. The way a specific therapeutic diagnostics (also known as theranostics) refers to expanding the field of medical technology, which provides the tests that are applicable for the diagnosis of the disease, the choice of adjusted treatment regimen and monitoring the response of a subject to treatment. That is theranostic applicable for prediction and response assessment of a specific subject on the drug, i.e. is individualized medicine. Tests theranostic also applicable for the selection of subjects to ensure that with high probability will have a therapeutic effect in the treatment or DL is finding out early and objective evidence of efficacy of treatment in specific subjects so what treatment can be changed with minimal delay.

Progress in pharmacogenetics, establishing correlations between responses to particular drugs and genetic profiles of individual patients, based on the development of novel theranostic approaches. In this regard, in the art there is a need to assess the patient variations in gene sequence and gene expression. The General form of the genetic profile is based on the identification of variations in the DNA sequence, called single nucleotide polymorphisms (SNPS), which are one of the types of genetic mutations that lead to variations in patients ' individual responses to medicine. From this it follows that in this area there is a need for identification and description of genetic mutations such as SNPS that can be used to identify the genotypes of the subjects associated with responses to drugs.

Brief description of the invention

The present invention meets existing in this field of technology needs. Established the important correlation between polymorphisms in the gene for angiotensin-converting enzyme (ACE)polymorphisms in the gene for the receptor 2 angiotensin II type (AGTR2) and reduction of clinical parameters srednechsrochnoj diastolic and systolic values of blood pressure with subsequent treatment aliskiren, used as an antihypertensive agent. These effects are not observed when treatment with irbesartan and placebo, but they are specific in relation to treatment aliskiren.

Thus, the present invention provides for the use of aliskiren to obtain drugs for the treatment of hypertension in the group in a certain way selected patients. Patients intended to treat, select the group on the basis of genetic polymorphisms in biomarker genes present in patients. Biomarker genes are gene angiotensin-converting enzyme (ACE) gene and receptor 2 angiotensin II type (AGTR2). These genetic polymorphisms are indicators of the effectiveness of aliskiren in the treatment of hypertension.

The present invention also provides a diagnostic method for determining the response of individuals with hypertension on treatment aliskiren, based on the identity of the nucleotide pair at one or more polymorphic genetic loci of the present invention.

The present invention also provides theranostics a method of treating hypertension in a particular individual. Antihypertensive agent is administered to the individual, if the nucleotide pair at the polymorphic genetic locus according to the present invention shows that the individual is responsible antigipertenzivny agent. In one of the embodiments of the present invention antihypertensive agent is aliskiren. Another variant therapeutic treatment of an individual apply, if the nucleotide pair at the polymorphic genetic locus present invention shows that the individual does not respond to antihypertensive agent.

The present invention generally provides a method of reducing diastolic blood pressure during day time (DCDD) for outpatients. In a specific embodiment, the present invention provides theranostics a method of reducing the average specific diastolic blood pressure (ADCD).

In addition, the present invention provides a method of reducing systolic blood pressure during day time for outpatients (SCDD). In a specific embodiment, the present invention provides theranostics a method of reducing the average specific systolic blood pressure (SOCKD).

The present invention also provides a method of selecting an individual for inclusion in a clinical trial to determine the effectiveness of antihypertensive agent for the treatment of hypertension. An individual may be included in the study, if the genotype of evidence of the et on the effectiveness of antihypertensive agent in the treatment of hypertension particular individual. An individual may be excluded from the study if the genotype of the individual does not confirm the effectiveness of antihypertensive agent for the treatment of hypertension in the individual.

The present invention provides kits for the practical implementation of the methods of the present invention. The present invention also provides a method of using gene product of the angiotensin converting enzyme (ACE) gene product of angiotensin II receptor, type 2 (AGTR2) as targets for the search for new medicines.

Brief description of drawing

The drawing represents a histogram showing the proportion of individuals selected by genotype ON responding to treatment (responders) in all three treatment groups by Tegaserod together in the group with the highest dose of aliskiren (600 mg), under treatment with irbesartan and in the placebo group. The signatures under the histograms of the upper row refers to the allele ARTICLE, and the bottom number refers to the TT allele.

Detailed description of the invention

Retrospective pharmacogenetic analysis was performed to assess possible links between genetic variation and the clinical treatment. In clinical treatment aliskiren, administered at doses of 75, 150 or 300 mg once daily, was proven an effective antihypertensive and the enta in the treatment of patients with hypertension, expressed in degree from mild to moderate, which resulted in a statistically significant reduction in systolic blood pressure during the day (SCDD). All doses of active treatment were statistically more effective than placebo, which was reflected in the decrease in the average particular diastolic blood pressure (ADCD) at the end point of clinical treatment in the group of patients selected for this treatment, 8 weeks, as well as in the treatment group according to the Protocol by the end of the clinical study. Similar results reduction SDCD were achieved in the application of aliskiren at a dose of 150 mg and irbesartan at a dose of 150 mg. Cm. the following example 1.

By setting the average specific systolic blood pressure (SOCKD) at the endpoint of the clinical study, all doses of active treatment were statistically more effective than placebo. The efficiency of aliskiren at doses of 300 and 600 mg statistically significantly higher than the result of placebo and irbesartan at the endpoint of the clinical study. A similar decrease SOCKD was achieved when applying aliskiren at a dose of 150 mg and irbesartan at a dose of 150 mg Dose of aliskiren 300 and 600 mg cause the greatest decrease in blood pressure. Cm. the following example 1.

When conducting pharmacogenetic analysis investigated the 48 SNPs in 12 genes of the system renin-aldosterone (RAS) or genes for which you previously installed their involvement in the regulation of blood pressure. Important relationships were observed between a polymorphism in the gene for angiotensin-converting enzyme (ACE) (ONP, SEQ ID NO:1), two polymorphisms in the gene for the angiotensin II receptor, type 2 (AGTR2) (ONP, SEQ ID NO:2 and ONP, SEQ ID NO:3) and clinical parameters decrease of the average diastolic and systolic blood pressure. These effects were not observed when treatment with irbesartan or placebo in the present study, presumably they are specific for aliskiren.

The nucleotide sequence ANP (SEQ ID NO:1) is the following:

AGGACTTCCC AGCCTCCTCT TCCTGCTGCT CTGCTACGGG CACCCTCTGC TGGTCCCCAG CCAGGAGGCA/Y CCCAACAGGT GACAGTCACC CATGGGACAA GCAGCCAGGC AACAACCAGC AGCCAGACAA SSSSSSS

ONP is coding SNPS, which changes the amino acid sequence of the Proline to serine at codon 32 enzyme ACF.

The nucleotide sequence ANP (SEQ ID NO:1) is the following:

TGGAAACTTC ATTTTTTTTG TTTGAGATTT ATTTGAATGA GCTGTTATGA TTGGAGACAG TGAGAATTTC AGATTAATGT TTTGCAGACA AAAAAAAACC TCTCTGGAAA GCTGGCAAGG GTTCATAAGT CAGCCCTAGA ATTATGTAGG TTGAAGGCTC CCAGTGGACA GACCAAACAT ATAAGAAGGA AACCAGAGAT CTGGTGCTAT TACGTCCCAG CGTCTGAGAG AACGAGTAAG CACAGAATTC AAAGCATTCT GCAGCCTGAA TTTTGAAGGT AAGTATGAAC AATTTATATA TAATTTACTT GGAAAGTAGA ACATACATTA AATGAAAATA TTTTTTATGG ATGAACTTCT GTTTTTCCTG TGTTTTAACA CTGTATTTTG CAAAACTCCT/R AATTATTTAG CTGCTGTTTC TCTTACAGGA GTGTGTTTAG GCACTAAGCA AGCTGATTTA TGATAACTGC TTTAAACTTC AACAACCAGT AAGTCTTCAA GTGGAATTTA TTATTGATTC TTTTATGTTA ATTTGTTAGG TCAAAAGAAA AATCTTTAGA GCAAAATAAA AGTTTTGCTC TTTATTAGGA GGTTCTTTAG ATATTACACT TTTAATTGGG TAGCTTATTT GCATGTATTT TGAAACTATC TAAAGTAAAT AGTGTTTCCT TTGTATGCTT ATCTTAGCT AATGTGTTTT TTTTTTTGGT TTTAAAATAA TGCTTCTAGT GAAAAAAATC ACAAAAACCT CAACACTGTA ACGTTTGAGA GCAACGGCTA TTCAGTTCGG TTAAACCGAA

ONP is untranslated region of the endogenous gene AGTR2 (see tablb).

The nucleotide sequence ANP (SEQ ID NO:1) is the following:

sasasa of adasasd ttgagaactg ggaaagcatc gcactacaac tgctactgcc attaaccaca ttgtcctgga tgcccaagag cttaagagcc cacttaccta cctggtacac tgctactaca actgacatct gagaaagcca cccaaaggaa caagaatttc cctgtctgga accaacagaa ttgtcactat/R ttctgtacca gatcccaagg atacacatgc ttagcttact attactacca ctgaaacttg caaaagaacc catcaagcat tccattcccc agcacaaatt catcagtttc tatcaataac ctcacaatgc cacacagagg aatagacaga tactactaag gctgtttata gccaatgaaa tcatacacag tcttcacca

ANP is present in the genomic region of the gene AGTR2 (see tablb). Certain objects, methods, implementation options and properties of the present invention is described below in greater or lesser degree of detail for real understanding of the present invention. In General, such a description provides a new use of polynucleotide variants, SNPS, applicable for the diagnosis and treatment of subjects in need. Thus, the various objects of the present invention relate to polynucleotides, polynucleotide encoding variations of the present invention genes ACF and AGTR2. Various objects of the present invention also relates to diagnostic/theranostics methods and kits employ polynucleotide variations of the present invention to identify individuals predisposed to the disease, or to classify individuals according to their ability to respond to drug among the STV, on side effects or optimal drug dose. In addition, the present invention provides methods of estimation connection and a computer system for accumulation and analysis of data related polynucleotide variations of the present invention. Below are various specific embodiments of the present invention, which illustrate the above objects.

Definitions. Below are definitions of some terms used in the present invention. Definitions of other terms can be found in the dictionary of the U.S. Department of energy, office of science, the human genome project <http://www.ornl.gov/sci/techresources/Human_Genome/glossary/>. Medical terms are presented Chobanian and others in Hypertension 42, 2003, SS-1252. With the definition of hypertension Association of cardiologists in the US can be found on the website <http://www.americanheart.org/presenter.jhtml?identifier=4623>. All references to these sources are included in the present invention.

In the context of the present invention, the term "allele" means a specific localization on the chromosome (locus) forms of a gene or DNA sequence.

In the context of the present invention the term "antibody" includes, but is not limited to, polyclonal antibodies, monoclonal antibodies, humanized or chimeric antibodies and biologically functional fragments of the antibodies, the residual binding fragment of the antibody with the protein.

In the context of the present invention "clinical response" means any one or all of the following concepts: quantitative measurement of response, no response and harmful response (i.e. side effects).

In the context of the present invention the term "clinical investigation" means any investigation undertaken to collect clinical data on responses to a particular treatment, and includes, but is not limited to, stages I, II and III clinical studies. Use standard methods to identify groups of patients and the inclusion of entities.

In the context of the present invention, the term "effective amount" means an amount of compound sufficient to achieve a desired therapeutic and/or prophylactic effect, such as the number, which prevents the appearance of symptoms associated with hypertension, or lowers their manifestation. In the preferred embodiment implementation of the present invention in this connection is aliskiren.

The number of connections, enter the subject, depends on the type and severity of the disease and the condition of the individual, such as General health, age, sex, body weight and resistance to drugs. It can also depend on the degree, severity and type of disease. The person skilled in the art can determine the pouring of the required dose, depending on these and other factors. Typically, an effective amount of the compounds of the present invention, sufficient for achieving a therapeutic or prophylactic effect, varies about 0.000001 mg/kg body weight per day to about 10,000 mg/kg of body weight per day. Preferably doses ranging approximately from 0.0001 mg/kg of body weight per day to about 100 mg/kg of body weight per day. Compounds of the present invention can be administered in combination with each other or with one or more additional pharmaceutical compounds. In a preferred embodiment of the present invention an effective amount of aliskiren 75, 150 or 300 mg at introduction once a day.

In the context of the present invention the term "expression" means one or more of the following concepts, but they are not limited to: transcription of the gene and the formation of mRNA, splicing and other processing of mRNA, precursor for the formation of Mature mRNA, mRNA stability, translation of Mature mRNA and protein (including the use of codon and tRNA availability) and glycosylation and/or other modifications broadcast, if they are necessary for proper expression and function.

In the context of the present invention, the term "gene" means the segment of DNA that contains all the information for the regulated b is sinteza product RNA, including promoters, exons, introns and other noncoding region controlling the expression.

In the context of the present invention the term "genotype" means nefrasovoy 5'→3' sequence of nucleotide pairs detected in one or more polymorphic sites of locus pairs of homologous chromosomes of an individual. In the context of the present invention the term "genotype" refers to the complete genotype or sub-genotype.

In the context of the present invention the term "locus" means a location on a chromosome or DNA molecule corresponding to a gene or physical or phenotypic property.

In the context of the present invention the term "ACF-modulating agent" or "AGTR2-modulating agent" means any compound that changes (i.e. increases or decreases) the expression level or biological activity of the polypeptide ACF or polypeptide AGTR2, respectively, in the absence of modulating agent. The modulating agent may be a small molecule compound, a polypeptide, a hydrocarbon, lipid, nucleotide, or a combination of them. The modulating agent may be an organic compound or inorganic compound.

In the context of the present invention the term "mutant" means any heritable change in the wild type, resulting from a mutation, for example adnanul autigny polymorphism. In the present description the term "mutant" is used interchangeably with the terms "marker"and "biomarker" and "target".

In the context of the present invention the term "medical condition" means, but is not limited to, any condition or disease that is manifested in the form of one or more physical and/or physiological symptoms, which is shown treatment, and includes the previously or newly described diseases and other disorders. In a preferred embodiment of the present invention, the medical condition is hypertension.

In the context of the present invention the term "pair nucleotide" means a nucleotide found in the polymorphic site of two copies of the chromosome of the individual.

In the context of the present invention, the term "polymorphic site" refers to the position in the locus at which at least two different sequences detected in the group with the highest frequency of occurrence is not more than 99%.

In the context of the present invention, the term "polymorphism" means any variant of a sequence occurring in a population with a frequency of >1%. Variant sequences may be present with a frequency substantially greater than 1%, for example 5%, 10% or more. In addition, this concept can be applied to the variant consequently the STI, find the individual at a polymorphic site. The polymorphisms include nucleotide substitutions, insertions, deletions and microsatellites, which may not necessarily lead to apparent differences in gene expression or protein function.

In the context of the present invention the term "polynucleotide" means any RNA or DNA, which can be unmodified or modified RNA or DNA. To polynucleotides include, without any limitations of single and Dunaeva DNA, DNA that is a mixture of single and double strand regions, single - and Dunaeva RNA, RNA, a mixture of single and double strand regions, hybrid molecules, DNA and RNA that may be single-stranded or, more typically, Dantewada, or a mixture of single and double strand regions. In addition, the concept of "polynucleotide" refers to technetium regions comprising RNA or DNA or both RNA and DNA. The concept of "polynucleotide" also includes molecules of DNA or RNA that contains one or more modifications, which are based on DNA or RNA, modified with the purpose of stability or for other reasons.

In the context of the present invention the term "polypeptide" means any polypeptide comprising two or more amino acids connected by peptide bonds or modified peptide bonds, i.e peptide the s isostere. The term polypeptide refers to compounds with a short circuit, commonly called peptides, glycopeptides or oligomers, and to compounds with long chain, commonly called proteins. Polypeptides may contain amino acids that differ from the 20 amino acids encoded by the genes. The polypeptides include amino acid sequences modified either by natural processes, for example, post-translational processing, or by chemical modifications are well known in the art. Such modifications are detailed in the guidelines for more detail in the monographs, but also by the huge number of scientific publications.

In the context of the present invention, the term "nucleic acid SNPS" means a nucleic acid sequence, which is variable nucleotide, and in other respects the sequence identical to the sequence of individuals and groups of individuals, thus it exists in the form of alleles. Such nucleic acid SNPS contain preferably from about 15 to about 500 nucleotides in length. Nucleic acid SNPS can be part of a chromosome, or they can be an exact copy of part of a chromosome, for example, by amplification of this part of the chromosome in the PCR or konyrov the deposits. Nucleic acid SNPS in the present description are simply called "SNPS". SNPS reflects the variability of the nucleotide at one position of the genome, in which two different bases found in human populations with appreciable frequency (i.e. >1%). SNPS may be present in a gene or intergenic regions of the genome. Probes SNPS according to the present invention are oligonucleotides, complementary to the nucleic acid SNPS.

In the context of the present invention the term "subject" means preferably a mammal such as human, but can also mean an animal, such as a pet (e.g., dogs, cats and other), farm animals (e.g. cows, sheep, pigs, horses and others) and laboratory animal (e.g., monkeys, for example, macaques-Griboedov, rats, mice, Guinea pigs and others).

In the context of the present invention the introduction of the agent or drug to a subject or patient means the introduction, produced or yourself or another person. You should also be aware that different methods of treating or preventing diseases in the nature of "fundamental", which means common, but not only, treatment or prevention, which achieved some biological or medically relevant results.

Identification and description of options the purpose of gene sequences. In connection with the prevalence and wide distribution of SNPS can be an effective and important tools for the localization of genes involved in painful human condition. See, for example, Wang and others, Science, 280, 1998, SS-1082. It is obvious that the risk of many common diseases and metabolism of drugs used to treat these diseases largely depends on the underlying genomic variation, although the effects of some of the options may not be significant.

SNPS called "allelic"because it contains a polymorphism, with some members of the species can have a sequence without mutations (i.e. natural allele)and the other sequence with a mutation (i.e. a variant or mutant allele).

The Association between the presence of polymorphism and a specific phenotype does not necessarily indicate that the UNP is the cause of this phenotype. The Association can exist only because of the genomic proximity SNPS and those genetic factors that are actually responsible for this phenotype, for example, SNPS, and these genetic factors can be closely spaced. Thus, SNPS may be in nonequilibrium coupling ("NA") with the "true" functional variant. NA (also called allelic Association) was observed in t is x cases when alleles at two different locations of the genome associated to a greater extent than anticipated. Therefore, SNPS can serve as a marker, which is close to the mutation causing a particular phenotype.

In the described polymorphic sites present invention for convenience is considered the semantic chain gene. However, the specialist is obvious that the molecules of nucleic acid containing the gene can be complementary donativum molecules and, hence, the reference to a specific site of the semantic circuit also applies to the corresponding site complementary antisense chain. Thus, reference may be made to the same polymorphic site in any of the two circuits and the oligonucleotide can be designed to hybreed specifically any of the circuits on the target region containing the polymorphic site. Therefore, the present invention also includes a single-stranded polynucleotide that complementary semantic chain of genomic variants described in the present invention.

Identification and description of SNPS. Can be used many different methods to identify and describe the SNPS, including the analysis of single-strand conformational polymorphisms (ANCP), heteroduplex analysis by denaturing high-performance liquid HRO what ecografia (DASH), direct DNA sequencing and computational methods. Shi and others, Clin. Chem. 47, 2001, SS-172. There is a huge available sequence information presented in the database.

The most common modern methods of typing SNPS include hybridization, for primers and methods of cleavage. Each of these methods should be combined with the detection system. Methods of detection include fluorescent polarization (Chan and others, Genome Res. 9, 1999, s-499), luminometric detection of the release of phosphate (persecutione) (Ahmadiian and other, Anal. Biochem. 280, 2000, SS-110), studies of fission-based energy transfer fluorescence resonance, DASH and mass spectrometry (Shi, Clin. Chem. 47, 2001, s-172, US 6300076 B1). Other methods of detection and description of the SNPS described in patents US 6297018 and 6300063.

Polymorphisms can also be detected using commercially available products, such as technology INVADER™ (company Third Wave Technologies Inc. Madison, Wisconsin, USA). In this study, specific oligonucleotide located higher up the chain "invader" and partially overlapping located below the circuit probe together form a specific structure upon binding with a complementary DNA matrix. This structure is recognized and cut at a specific site specific enzymes Rasse the population, resulting in the released 5'-fragment of the oligonucleotide probe. This fragment then acts as oligonucleotide "invader" in relation to synthetic secondary targets and secondary fluorescently labeled signal probes present in the reaction mixture. Cm. also Ryan D. and others, Molecular Diagnosis 4, 1999, s-144, Lyamichev V. and others, Nature Biotechnology 17, 1999, SS-296, patents US 5846717 and 6001567.

Identity polymorphisms can also be identified by a method of detecting erroneous mating grounds, including, but not limited to, a method of protection from RNase using ribosomal (Winter and others, Proc. Natl. Acad. Sci. USA 82. 1985, s, Meyers and others, Science 230, 1985, s) and proteins that recognize erroneous pairing nucleotides, such as protein mutS E. coli (Modrich P., Ann Rev Genet 25, 1991, SS-253). In another method variant alleles can be identified by analysis of single-strand conformation polymorphism (CPC) (Orita and others, Genomics 5, 1989, SS-879, Humphries and others in kN.: "Molecular Diagnosis of Genetic Diseases" Ed. by Elles R., 1996, SS-340) or denaturing gradient gel electrophoresis (DGGE) (Wartell and others, Nucl. Acids. Res. 18, 1990, s-2706, Sheffield and others, Proc. Natl. Acad. Sci. USA 86, 1989, SS-236). Polymerase-mediated primer extended method can also be used to identify polymorphisms. Several such methods have been described in the patent and scientific literature, these include the method of "genetic is a mini bit of analysis" (WO 92/15712) and ligase/polymerase-mediated method, genetic bit analysis (US 5679524). Similar methods are described in WO 91/02087, WO 90/09455, WO 95/17676 and US 5302509 and 5945283. Extended primers containing the polymorphism can be detected by the method of mass spectrometry (MCC) as described in US 5605798. The other primer is extended method is allele-specific polymerase chain reaction (PCR) (Ruaflo and others, Nucl. Acids. Res. 17, 1989, s), Ruafio and others, Nucl. Acids. Res. 19, 1991, s-6882), WO 93/22456, Turki, etc., J. Clin. Invest. 95, 1995, s-1641). In addition, different polymorphic sites can be studied simultaneously amplificatoare different regions of nucleic acid, using a set of allele-specific primers, as described in published PCT patent application WO 89/10414.

Haplodiploidy and genotyping oligonucleotides. The present invention provides methods and compositions for haplodiploidy and/or genotyping of genes of the individual. In the context of the present invention the terms "genotype" and "haplotype" refers to a genotype or haplotype containing the nucleotide pair or nucleotide, respectively, which are contained in one or more new polymorphic sites described in the present invention, and may optionally also include a nucleotide pair or nucleotide contained in one or more additional polymorphic sites in the gene. Additional polymorphic sites may be known polymorphic sites Il the sites, found later.

Compositions of the present invention contain nucleotide probes and primers designed for specific hybridization with one or more target regions containing polymorphic site or attached to it. Oligonucleotide compositions of the present invention are applicable in the methods of haplodiploidy and/or genotyping of genes of the individual. Methods and compositions for the installation of the genotype or haplotype of the individual new polymorphic sites described in the present invention is applicable to study the effect of polymorphisms in the etiology of a disease associated with the expression and function of a protein to study the effectiveness of targeting drugs, predicting the sensitivity of an individual to the disease associated with the expression and function of the protein, and prediction of the reactivity of the individual to medicines aimed at the gene product.

Genotypically oligonucleotides of the present invention can be immobilized or synthesized on a solid surface, such as a microchip, spheres or glass plates. See, for example, WO 98/20020 and WO 98/20019.

Genotypically oligonucleotides can hybridisierung with a target area located at a distance of one or more nucleotides that are lower in the chain one or more new the x polymorphic sites, identified in the present invention. Such nucleotides are applicable in a polymerase-mediated primer extended the methods of detection of one of the new SNPs discovered in the present invention and, therefore, such genotyped oligonucleotides described in this invention as a primer extended oligonucleotides".

The method of direct genotyping. described in the present invention. The way genotyping of the present invention may include the selection of a mixture of molecules of the nucleic acid of an individual including two copies of the test gene or its fragment, and the identity of nucleic pair at one or more polymorphic sites in two copies. The person skilled in the art it is obvious that two copies of the gene of an individual may be the same allele or different. In the most preferred embodiment of the present invention a method of genotyping includes determining the identity of the nucleotide pair at each polymorphic site. Usually a mixture of nucleic acid molecules isolated from the biological sample of the individual, for example from a blood sample or tissue. Suitable tissue samples are whole blood, semen, saliva, tears, urine, fecal matter, sweat, cheek scraping, skin and hair.

Below in example 1 sets the of ondov for studies of single nucleotide polymorphisms (SNPS) for the method for genotyping get to platform Assays-by-Design® firm ABI. Livak K.J., J. Marmaro, Todd J.A., Nature Genetics 9, 1995, SS-342. For genotyping according to the manufacturer's instructions using 10 ng genomic DNA.

The method of direct haplodiploidy described in this invention. The way genotyping of the present invention may include the selection of the sample of the individual molecules of nucleic acid containing one or two copies of the test gene or its fragment, and the identity of the nucleotide at one or more polymorphic sites in this copy. Direct genotyping include, for example, technology CLASPER System™ (US 5866404) or allele-specific long-range PCR (Michalotos-Beloin and others, Nucl. Acids. Res. 24, 1996, SS-4843). The nucleic acid may be isolated by the method that is used to separate two copies of the gene or fragment. The person skilled in the art it is obvious that any individual clone can only provide information on the haplotype of one of the two copies of the gene present in the individual. In one embodiment, the present invention haplotype a couple of an individual is determined by identification of phase sequence of nucleotides on one or more polymorphic sites in each copy of the gene of the individual. In a preferred embodiment, the method haplodiploidy includes the identification of phase sequence nucleot the species in each polymorphic site of each copy of the gene.

Identification of nucleotide (or nucleotide pair) at a polymorphic site in the use and method of genotyping, and method haplodiploidy can be carried out by amplification of the target regions containing polymorphic sites directly from one or two copies of a gene or its fragments, and by sequencing of the amplified regions in traditional ways. The genotype or haplotype of a gene of an individual may also be determined by hybridization of the sample nucleic acid containing one or both copies of the gene with sequences and suppositionally nucleic acid, for example, as described in WO 95/11995.

The method of indirect genotyping with the use of polymorphic sites in nonequilibrium coupling with the target polymorphism. The identity of the alleles present in any new polymorphic sites present invention can also be determined indirectly using other genotyping of polymorphic sites located in non-equilibrium coupling with the studied sites. According to the above two sites are in non-equilibrium coupling, if the presence of a particular variant in a single site indicates the presence of another variant of the second site. Stevens JC, Mol. Diag. 4, 1999, SS-317. Polymorphic sites in nonequilibrium coupling with a polymorphic sites this is about the invention can be localized in regions of the same gene, or in other genomic regions.

Amplification of the target gene. The target region can be amplified using any method of oligonucleotide-directed amplification, including, but not limited to it, polymerase chain reaction (PCR). (US 4965188), ligase chain reaction (LCR) (Wahapo and others, Proc.Natl. Acad. Sci. USA 88, 1991, SS-193, published PCT patent application WO 90/01069) and oligonucleotide ligation (OL) (Landegren, etc., Science 241, 1988, SS-1080). The oligonucleotides used in such ways as primers or probes specifically hybridize with a region of nucleic acid that contains or is attached to the polymorphic site. Typically, the oligonucleotides contain 10-35 nucleotides, preferably 15 to 30 nucleotides. Most preferably the length is 20-25 nucleotides nucleotides. The exact length of the oligonucleotide depends on many factors that are evaluated by a specialist.

Other known methods of nucleic acid amplification can be used for amplification of the target region, including amplification-based transcription (US 5130238, EP 329822, US 5169766, published PCT patent application WO 89/06700) and isothermally ways (Walker and others, Proc. Natl. Acad. Sci. USA 89. 1992, SS-396).

Hybridization of allele-specific oligonucleotide with the target gene. Polymorphism of the target area can b shall be evaluated before or after amplification, using one or more methods based on hybridization, known in the art. Typically, when implementing such methods using allele-specific oligonucleotides. Allele-specific oligonucleotides can be used as a differently labeled probe pairs, in which one component in the pair shows the complete affinity with one variant of the target sequence, and the other component shows affinity with another option. In some embodiments, implementation of the present invention can be detected more than one polymorphic site using a set of allele-specific oligonucleotides or oligonucleotide pairs. Preferably the components of this kit have a melting point not more than 5°C, more preferably not more than 2°C in hybridization to identify polymorphic sites.

Hybridization of allele-specific oligonucleotide with the target polynucleotide can be carried out with both objects in solution, or such hybridization can be carried out, if the oligonucleotide or the target polynucleotide covalently or ecovalence attached to a solid substrate. The attachment may be mediated, for example, interactions, antibody-antigen, poly-L-lysine, streptavidin, or avidin-Biotin, salt bridges, hydrophobic entries batch is s, chemical bonds, UV cross-linking, annealing, etc. Allele-specific oligonucleotide can be synthesized directly, or on a solid substrate, or attached to a solid substrate after synthesis. To hard substrates suitable for use in the detection methods of the present invention include silicone, glass, plastic, etc. that can be designed, for example, in the form of wells (96 well plates), slides, plates, membranes, fibers, grains, cups and beads. The solid substrate may be treated, coated, or may be obtained from its derivative in order to facilitate the immobilization of the allele-specific oligonucleotide or a target nucleic acid.

Determination of genotypes and haplotypes in the group and their correlation with a certain characteristic. The present invention provides a method of determining the frequency of genotype or haplotype in the group. The method comprises determining the genotype or haplotype of a gene that is present in every member of the group, in which the genotype or haplotype comprises the nucleotide pair or nucleotide detected at one or more polymorphic sites in the gene, and counting the frequency of genotype or haplotype in the group. The group may be the control group, family group, group, sex, population group, or a group defined by the first characteristic (e.g., a group of individuals with a certain studied characteristic, such as medical condition or response to therapeutic treatment).

Another object of the present invention associated with the data on the frequency of occurrence of genotypes and/or haplotypes in the control population used in the method for the identification of Association between the sign and the haplotype or genotype. The characteristic may be any detectable phenotype, including, but not limited to, the sensitivity of the disease or response to treatment. The method includes obtaining data on the research genotypes and haplotypes in the control group and the comparison of these data with the frequencies of genotypes and haplotypes in a group with a certain characteristic. Frequency data for one or two groups (control and intervention) can be obtained by genotyping or haplotypefusion each individual in the group, carried out by the methods described above. The haplotypes to be tested can be determined directly or, in another embodiment, the above described method for predicting the genotype of the haplotype.

Data on frequency of occurrence in the control and/or study groups is obtained by evaluating the previously obtained data on the frequency of occurrence, which may be in written or electronic form. For example, data on h is the frequency of occurrence can be in the form of a computer database. After obtaining data on the frequency of occurrence compared the frequency of occurrence of the studied haplotypes or genotypes in the control group and in the group with a particular attribute.

In the analysis of polymorphisms of the calculations can be performed for input adjustments for significant Association, which may be identified by chance. Applicable in the present invention, the statistical methods described in the book: "Statistical Methods in Biology", 3rd ed., Bailey NTJ, 1997, published by Cambridge Univ. Press, in kN.: Waterman M.S. "Introduction to Computational Biology", 2000, published by CRC Press in kN.: "Bioinformatics" Ed. by A.D. Baxevanis, B.F.F. Ouellette, 2001, published by John Wiley & Sons, Inc.

In another embodiment of the present invention the frequency of the haplotype in different groups define in order to determine whether they are consistent with equation hardy-Weinberg equilibrium. Cm. book: Harti D.L. and other "Principles of Population Genomics", 3rd ed., 1997, published by Sinauer Associates, Sunderland, Massachusetts.

In yet another embodiment of the present invention, a statistical analysis is performed standard tests ANOVA with correction of Bonferroni or using the bootstrap, which repeatedly simulates the correlation of genotype with phenotype and calculates the significance level. The ANOVA test is used to test the assumption whether the response to one or more characteristics or variables, measurable, or to relire with them. Cm. book: Fisher L.D. & vanBelle G. Biostatistics: A Methodology for the Health Sciences", Chapter 10, 1993, published by Wiley-lnterscience, new York.

In another embodiment of the present invention to predict haplotype pairs analysis involves the following stages. First, each of the possible haplotype pairs mapped to haplotype pairs in the control group. Usually only one of haplotype pairs in the control group mates with possible haplotype pair, and this pair is present in the individual. Sometimes only one haplotype present in the control haplotype pairs, compatible with possible haplotype pair of the individual, and in such cases the individual is assessed as containing haplotype a pair of this well-known haplotype and a new haplotype is derived by the subtraction of the known haplotype from possible haplotype pair.

In another embodiment of the present invention to identify the genotype or haplotype found in nonequilibrium coupling with interested haplotype or haplotype can be used as a surrogate marker. Genotype found in nonequilibrium coupling with another genotype, is detected, if a particular genotype or haplotype of this gene occurs more frequently in the group, which also shows a potential surrogate marker genotype, but not in the control of the second population. If the frequency of statistically significant marker genotype is an indicator of such a genotype or haplotype and can be used as a surrogate marker.

In another method of detecting correlation between the presence of the haplotype and clinical responses use forecasting models based on optimization algorithms to minimize errors, one of them is the genetic algorithm. Cm. book: "Reviews in Computational Chemistry", Ed. by Lipkowitz K.B., Boyd DB, Chapter 10, 1997, SS-73, published by VCH Publishers, new York. You can apply the methods of false recall (kN.: "The Art of Scientific Computing", Chapter 10, 1992, published by Cambridge University Press, Cambridge), neural networks (kN.: Rich, E., Knight, K. "Artificial Intelligence", 2nd ed., Chapter 10, 1991, McGraw-Hill, new York), standard methods for the reduction of the gradient (see above Press and others, Chapter 10) or can also be applied to other global or local optimization approaches (see above discussion in the work of the Judson).

In the examples below research Association of genotype-phenotype and related analyses were performed in the firm SAS (Cary, NC, USA)using software Analyst®. In tests to detect Association using a clearly defined genotypes as independent variable, without assumptions about dominance, and various variables efficiency as dependent variables. Tests continuous saveselection, used in the ANCOVA analysis, and logistic regression is used for certain dependent variables. Covariant derivatives of analysis of the Association of genotype-phenotype are: treatment, research space and the dimension of the underlying level.

Covariance analysis (ANCOVA) is repeated on the same model for each group, in which individuals are treated. In addition, the percentage of responders analyzed using a logistic regression model with factors such as the type of treatment and region, and baseline as a covariant derivative. Associations with p<0.05 all data are evaluated as significant.

Correlation of genotype or haplotype of a subject with a response to treatment. In preferred embodiments, the implementation of the present invention symptoms are sensitivity to disease, disease severity, stage of disease or response to drug use. Such methods are applicable when developing diagnostic tests and therapeutic regimens for all pharmacogenetic applications, if there is the ability to link genotype and treatment outcomes, including performance measurement, pharmacogenetic measurement and measurement of side effects.

In another preferred embodiment, the present image is the shadow, you need to sign is a clinical response, show the patient, in some embodiments, therapeutic treatment, for example, the targeting of drugs or therapeutic treatment of a medical condition.

To detect a correlation between clinical response to treatment and genotype or haplotype, genotype or haplotype receive on the basis of clinical responses of individuals in the treatment group, in which the present invention is called "clinical group. These clinical data can be obtained by analyzing the results of clinical research that has already been conducted, and/or the development and implementation of one or more new clinical studies.

Individuals enrolled in the clinical group, usually defined by the presence of the studied medical condition. This separation prospective patients can be based on the results of standard medical examinations or on one or more laboratory tests. In another embodiment, separation of patients can be made on the basis of haplodiploidy for those situations in which there is a strong correlation between haplotypes pair and susceptibility to illness or disease severity.

Investigational therapeutic treatment for each individual in the study group and the response of each ind is videum to treatment is measured on the basis of one or more pre-selected criteria. Assume that in many cases the sample may show a range of responses and that the researcher can choose the number responding groups (e.g., low, medium, high), giving different answers. In addition, genotypic and/or haplotyping gene of each individual in the study population, which may be done before or after treatment.

The obtained results are then analyzed to determine whether variations in clinical response between groups with polymorphisms statistically significant. Applicable methods of statistical analysis described L.D. Fisher, G. vanBelle in kN.: "Biostatistics: A Methodology for the Health Sciences", published by Wiley-lnterscience, 1993, new York). This analysis may also include regression calculation which polymorphic sites of the gene most have the most significant is the difference in phenotype.

After obtaining clinical data and data on polymorphisms reveal the correlation between the responses of individuals to treatment and genotype or haplotype. Correlation can be obtained in several ways. One way individuals are grouped according to genotype or haplotype (or haplotype pair) (this also applies to polymorphic group) and then the average and standard deviation of the clinical responses exhibited by representatives of each polymorphic group, otschityvayut.

From the above analysis it follows that specialist in this area, prediction of clinical response as a function of the available genotype or haplotype, can easily construct a mathematical model. Identification of the Association between clinical response and a genotype or haplotype (or haplotype pair) gene may be the basis for designing a diagnostic method to determine those individuals who can respond or not respond to treatment, or, in another embodiment, can respond at a lower level and thus may require more intensive treatment, i.e. increased dose of the drug. The diagnostic method may be in one or several forms: for example, direct DNA testing (i.e. genotyping or haplodiploidy one or more polymorphic sites in the gene), serological test or medical examination. The only requirement is that there should be good correlation between the results of the diagnostic test and the genotype or haplotype of the underlying. In a preferred embodiment of the present invention in such a diagnostic method using the above-described manner predicted by genotyping.

Assessment of facilities subject to a specific genotypic group. The specialist in this on the region of the obvious, what could be some degree of inaccuracy in this definition facilities subject. Therefore, the standard deviation of the levels of the control group can be used to obtain probabilistic determination and methods of the present invention can be applied to a wide range of possibilities, based on the definitions of the genotypic groups. Thus, as an example, but not limitation, in one embodiment of the present invention it is noted that if the measured product level of gene expression decreases to a value of 2.5 standard deviations from the average value of any of the control groups, then the specific individual can be classified as a given genotype. In another embodiment of the present invention, if the measured level of gene expression product falls within 2.0 standard deviations of the mean values of any of the control groups, then the specific individual can be classified as a given genotype. In yet another embodiment of the present invention, if the measured level of gene expression product falls within 1.5 standard deviations of the mean values of any of the control groups, then the specific individual can be attributed to gruppenname genotype. In another embodiment of the present invention, if the measured level of the gene expression product is 1.0 or less average deviations of the mean values of the levels of any of the control groups, then the specific individual can be classified as a given genotype. Thus, this method allows to determine, with varying degrees of probability, in which group a specific subject should be placed and what kind of evaluation belonging to genotype group, then this can help you determine the risk category to which the individual may be assigned.

Correlation between clinical response and a genotype or haplotype. In order to establish a correlation between clinical response to treatment and genotype or haplotype, it is necessary to obtain data on the clinical responses exhibited by representatives of under-treated group, which is called below in the present invention "clinical group. Clinical data can be obtained by analyzing the results of clinical studies that have already been carried out, and/or clinical data can be obtained in the design and conduct of one or more new clinical studies.

Standard reference levels of the product of gene expression, obtained as described in various control what's groups, can then be compared with the measured product level of gene expression in a specific patient. The product of gene expression may be specific mRNA associated with this specific genotypic group, or the product of gene expression is a polypeptide specific to this genotypic group. Then the patient may be classified and referred to the group of a specific genotype, based on how close the measured levels to reference levels of this group.

A computer system for storing or displaying data on polymorphism. The present invention also provides a computer system for storing and displaying data on the polymorphism identified for this gene. A computer system includes a processor unit, a monitor and a database containing data on polymorphism. Data on polymorphism include polymorphisms, genotypes and haplotypes identified for this gene in the control group. In a preferred embodiment of the present invention is a computer system capable of on-screen display haplotypes, organized according to their inherent evolutionary relationships. The computer may enforce any or all of the mathematical operations on the methods of the present invention. In addition, the computer can meet the ü program which visually reflects on the screen (or multiple screens) and over which the researcher can work for review and analyze large volumes of information on gene and its genomic variations, including chromosomal localization, gene structure, gene family, data on gene expression, polymorphism, data on genetic sequences, as well as clinical data for the group (for example, data on ethno-geographical origin, clinical responses, genotypes and haplotypes for one or more groups). Data on the polymorphism described in the present invention may be stored as a relational database (such as Oracle database or a set of ASCII flat files). Such data on the polymorphism can be stored on your computer's hard drive or on CD-ROM, or one or more storage devices that may be installed on the computer. For example, data may be stored in one or more devices for storing information, which can be installed on the computer. For example, data may be stored in one or more databases connected to the computer through the network.

Diagnosis of nucleic acids. Another object of the present invention provides probes SNPS applicable for the classification of subjects by type gene is on systematic variations. Probes SNPS according to the present invention are oligonucleotides that distinguish SNPS in traditional studies to distinguish alleles. In some preferred embodiments of implementing the present invention, the oligonucleotides according to this object of the present invention is complementary to one allele SNPS nucleic acid, but not to any other allele SNPS nucleic acid. The oligonucleotides in this variant implementation of the present invention can distinguish between SNPS in different ways. For example, stringent conditions of hybridization of an oligonucleotide probe specific length can hybridisierung with only one SNPS, but not with others. The oligonucleotide may be caused radioactive label, or a label in the form of fluorescent molecules. In another embodiment, the oligonucleotide of a certain length can be used as primers for PCR, the 3'-terminal nucleotide complementary to one allele containing SNPS, but not to any other allele. In this embodiment of the present invention, the presence or absence of amplification by PCR determines the haplotype SNPS.

Genomic and cDNA fragments of the present invention include at least one new polymorphic site identified in the present invention, a length of at least 10 nucleotides, and can castigat the full length gene. Preferably the fragment of the present invention is from 100 to 3000 nucleotides in length, more preferably 200-2000 nucleotides in length, and most preferably 500-1000 nucleotides in length.

The kits of the present invention. The present invention provides kits for identifying nucleic acids and polypeptides that can be applied to haplodiploidy and/or genotyping gene of the individual. These kits are applicable for the classification of individuals. Specifically, the present invention encompasses kits for detecting the presence of the polypeptide or nucleic acid corresponding to a marker of the present invention in a biological sample, for example, in any body fluids including, but not limited to, serum, plasma, lymph, bile, urine, fecal matter, cerebrospinal fluid, ascitic fluid, or blood, as well as in samples for biopsy. For example, the sample may include a labeled compound or agent capable of identify the polypeptide or mRNA encoding the polypeptide corresponding to a marker of the present invention in a biological sample and means for determining the amount of polypeptide or mRNA in the sample, for example an antibody that binds the polypeptide or an oligonucleotide probe which binds to DNA or mRNA that encodes the polypeptide. The kits can also include the instructions for the interpretation of the results, obtained with this set.

In another embodiment, the present invention provides a kit comprising at least two genotypically of the oligonucleotide, Packed in individual containers. The kit may also contain other components, such as buffer for hybridization (in which the oligonucleotides are used as probes), Packed in a separate container. In another embodiment, the oligonucleotides used for amplification of the target region, and for this set can contain in separate containers polymerase and buffer for the reaction optimized for primer-extended polymerase reaction, for example, in the case of PCR. In a preferred embodiment of the present invention, a kit may optionally include a means of selection of the sample DNA.

Sets based on the antibodies can include, for example, (1) a first antibody, for example, attached to a thick substrate, which binds to a polypeptide corresponding to a marker of the present invention, and, optionally, (2) a second antibody which binds to either the polypeptide or the first antibody and conjugates with a detectable label.

Sets on the basis of oligonucleotides may include, for example, (1) a oligonucleotide, e.g identified on the label of the oligonucleotide that's hybrid with posledovatel the ability of the nucleic acid, encodes a polypeptide corresponding to a marker of the present invention, or (2) a pair of primers used for amplification of a nucleic acid molecule corresponding to a marker of the present invention.

The kit can also include, for example, a buffering agent, a preservative, or a protein stabilizing agent. The kit may additionally include components necessary for detecting the detectable label such as an enzyme or substrate. The kit can also include a control sample or a series of control samples that can be studied and compared with the test sample. Each component of the set can be placed in a separate container and all variants of the containers can be packaged in a common package, along with instructions for analysis of the results obtained with the help of this set.

The nucleic acid sequences of the present invention. One of the objects of the present invention is one or more selected polynucleotides. The present invention also includes allelic variants, there are other natural forms selected polynucleotides that encode mutant polypeptides that are identical, homologous or related to those that encode polynucleotide. In another embodiment, unnatural variants can be obtained by the method of mutagenesis or met the DAMI direct synthesis, well known in this technical field.

Therefore, the nucleic acid sequences capable of hybridisierung with less rigidity with any sequences of a nucleic acid that encodes a mutant polypeptide of the present invention belong to the scope of the present invention. Standard stiffness terms are described in detail in standard textbooks on molecular biological cloning. See, for example, kN.: Sambrook, Fritsch, Maniatis "Molecular Cloning A Laboratory Manual" 2nd ed., 1989, published by Cold Spring Harbor Laboratory Press, kN.: "DNA Cloning", volumes I and II, Ed. by D.N. Glover, 1985, kN.: "Oligonucleotide Synthesis", edited by M.J. Gait, 1984, kN.: "Nucleic Acid Hybridization", edited by B.D. Hames, S.J. Higgins, 1984.

Characteristics of the level of gene expression. Methods of detection and measurement of mRNA levels (i.e. levels of gene transcription) and levels of polypeptide products of gene expression (i.e. levels of translation of a gene are well known in this area and include the use of nucleic acid microarrays and methods for the detection of polypeptides, including mass spectrometers and/or methods of detection and quantification of antibodies. Cm. also the book: Strachan T., Read, A., "Human Molecular Genetics, 2nd ed., 1999, published by John Wiley and Sons, Inc. Publication, New York.

Determination of the transcription of the target gene. Determining the level of expression of a gene product in a biological sample, such as tissue or W is drasti an individual's body, can be made in different ways. The term "biological sample" means a tissue, cells, biological fluids and their components obtained from the subject, as well as tissues, cells and fluids that are in the subject. In many ways revealing expression using the selected RNA. For in vitro methods any methods RNA extraction, which are not selective in the allocation of mRNA, can be used for purification of RNA from cells. See, for example, kN. "Curr. Prot. Mol. Biol.", Ed. by Ausubel and others, 1987-1999, published by John Wiley & Sons, new York.

In one of the embodiments of the present invention determine the level of expression product is mRNA of the target gene. Methods of measuring the level of specific mRNA are well known in this field and include Northern blotting, PCR reverse transcription or hybridization with oligonucleotide sequence or micropaleontology. In other preferred embodiments, the implementation of the present invention the level of expression can be performed by determining the level of expression of the gene product, protein or polypeptide in samples of tissue fluid or tissue, including, but not limited to, blood or serum. A large number of tissue samples can readily be processed using techniques well known specialist is m in this area, for example a single-stage method of RNA extraction, as described in patent US 4843155.

Selected mRNA can be used for hybridization or amplification, which include the following methods, but not limited to: southern blotting or norsen-blotting, PCR and probe sequences. One preferred method of diagnosis for the detection of mRNA levels involves contact selected mRNA with a nucleic acid molecule (probe)that can hybridisierung with mRNA that encodes the investigated gene. The probe nucleic acid can be, for example, full length cDNA or part thereof, such as an oligonucleotide probe, the length of which is at least, 7, 15, 30, 50, 100, 250 or 500 nucleotides, and which is sufficient for specific hybridization in harsh environments with mRNA or genomic DNA encoding a marker of the present invention. In the present invention described other methods suitable for the diagnosis according to the present invention. Hybridization of mRNA with the probe indicates that the marker is expressed.

In one format, the probes are immobilized on a solid surface and the mRNA molecules in contact with the probes, for example, is a set of gene Affymetrix chips (firm Affymetrix, CA, USA). The person skilled in the art can easily adapt the methods of detection of mRNA for use in detecting the level of mRNA encoded mark the Rami of the present invention.

Other methods of determining the level of mRNA corresponding to the marker of the present invention in the sample are the amplification of nucleic acid, for example, using real-time PCR (RT-PCR) (options experimental implementation is described in the patent US 4683202), ligase chain reaction (Wahapo and others, Proc. Natl. Acad. Sci. USA 88, 1991, SS-193), self-sustained replication sequence (Guatelli and others, Proc. Natl. Acad. Sci. USA 87, 1990, SS-1878), the system transcriptionally amplification, (Kwoh and others, Proc. Natl. Acad. Sci. USA 86, 1989, SS-1177), Q-beta replicase (Lizardi and others, Biol. Technology 6, 1988, s), replication type "rolling ring" (US 5854033) or any other methods of nucleic acid amplification and subsequent detection of the amplified molecules using methods known to experts in this field. Such detection scheme is especially applicable for the detection of nucleic acid molecules if such molecules are present in a low number of copies. In the context of the present invention "primers amplification" called a couple of molecules of nucleic acid, which can anneal to the 5' or 3' region of the gene (plus and minus circuit, respectively, or Vice versa) and contain a short region between them. Typically, the amplification primers comprise about 10-30 nucleotides in length and flank the region length of about 50-200 nucleotides.

Quantitative polymer is owned chain reaction real-time (RT-PCR) is one of the ways to assess levels of gene expression, for example, genes of the present invention, for example, containing SNPS and analyzed polymorphisms. In the RV-PCR using RNA-reverse transcriptase for the catalysis of the synthesis of the DNA strand in the RNA chain including chain mRNA. The formed DNA can be specifically detected and quantified, and this process can be used to determine the levels of specific mRNA. One of such ways is the way TAQMAN®, PE Applied Biosystems, Foster City, CA, USA)using 5'-nucleara activity of DNA polymerase AMPLITAQ GOLD™ for splitting a particular form of the probe during PCR. This refers to the probe TAQMAN™. Cm. Luthra and others, Am. J. Pathol. 153, 1998, SS-68, Kuimelis and others, Nucl. Acids Symp.Ser. 37, 1997, SS-256, and Mullah and others, Nucl. Acids Res. 26, 1998, s-1031. During the reaction the cleavage of the probe separates the reporter dye and the dye-muffler, resulting in increased fluorescence of the reporter. The accumulation of PCR products is detected directly by the appearance of fluorescence of the reporter dye. Held and others, Genome Res. 6, 1996, s-994. Increasing the number of starting copies of a target nucleic acid leads to more rapid significant increase in the observed fluorescence. Cm. Gibson, Heid, Williams and others, Genome Res. 6, 1996, SS-1001.

Other measurement technology status of transcription in the cell lead to the creation of pools of restriction fragments of limited clonos and for analysis by electrophoresis, for example, methods combining double restrictase splitting with phasing primers (see, for example, EP 0534858 A1), or methods of selection restriction sites, tight to a specific end of the mRNA (see, for example, Prashar, Weissman, Proc. Natl. Acad. Sci. USA 93, 1996, s-663).

Other methods statistically selected pools of cDNA, for example, by sequencing a sufficient number of bases, for example 20-50 bases in each of a large number of cDNA molecules to identify each cDNA molecule, or by sequencing short tags, for example, a length of 9-10 bases, which are formed by known positions with respect to a particular mRNA is the end of the metabolic pathway. See, for example, Velculescu, Science 270, 1995, SS-487. Levels of cDNA in the samples calculate and determine for each type of cDNA average, arithmetic mean and standard deviation using standard statistical techniques known in the art. Norman T.J. Bailey in kN.: "Biology", 3rd ed., 1995, published by Cambridge University Press.

Identification of polypeptides. Immunological methods of detection. Expression of the proteins encoded by genes of the present invention can be detected using a probe coated with a detectable label, or a label applied subsequently. The term "labeled" in regard to the probe or antibody refers to the direct application of the label by combining with the probe or antibody is m, i.e. due to the physical connection, but also to the indirect application of the label to the probe or antibody by reaction with another reagent that is directly labeled. Examples of indirect application of labels include detection of a primary antibody using a fluorescently labeled secondary antibody and probe DNA labeled with the end of Biotin, so that it can be detected with fluorescently labeled streptavidin. Typically, the probe is an antibody that recognizes the expressed protein. Various formats can be used to determine whether samples of the target protein, which binds with the antibody. Methods immunostimulant, applicable for detection of the target polypeptides of the present invention include, but are not limited to, dot-blotting, Western blotting, protein chips, the method of competitive and non-competitive protein binding, enzyme-linked immunosorbent assay (ELISA - enzyme-linked immunoabsorbent assay),

methods immunohistochemistry, the separation of fluorescently-activated cells (fluorescence activated cell sorting - FACS) and other commonly used and widely described in the patent and scientific literature methods, including many commercial methods. An experienced specialist can easily adapt known methods for detection of protein/antibodies for use in order to determine, whether Express cell Mar the EP of the present invention, and approximate concentrations of specific expressed polypeptide product in the blood or other tissues of the body. Proteins individuals can be isolated by methods well known to specialists in this field of technology. Applicable methods for isolating a protein, for example, can be the techniques described in the book: Harlow, Lane, "Antibodies: A Laboratory Manual', 1988, published by Cold Spring Harbor Laboratory Press, Cold Spring Harbor, new York.

To generate antibodies to a protein encoded by one of the described genes, possible immunizing host animals by injection with the polypeptide or a part of it. These animal hosts include, but are not limited to, rabbits, mice and rats. Various adjuvants can be used to increase the immunological response, depending on the type of owner, including, but not limited to, beta-blockers (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, plutonomies polyols, polyanion, peptides, oil emulsions, hemocyanin mollusk saucer and dinitrophenol, and potentially applicable adjuvants such vaccinal strain Bacillus Calmette-guérin (BCG) and Corynebacterium parvum.

Monoclonal antibodies (manta rays) are homogeneous populations of antibodies to a particular antigen and can be obtained by a method of providing received the e molecules of antibodies using cultured cell lines. Such methods include, but are not limited to, the hybridoma method of Kohler and Milstein, Nature, 256, 1975, SS-497, patent US 4376110, the method is a hybrid In-cell human Kosbor and others, Immunol. Today 4, 1983, p.72, Cole and others, Proc. Natl. Acad. Sci. USA 80, 1983, SS-2030 and the EBV-hybridoma method of Cole and others in kN.: "Monoclonal Antibodies and Cancer Therapy", 1985, firm Alan R. Liss, Inc., cc.77-96.

In addition, we can apply the methods developed to obtain a "chimeric antibodies" (see Morrison and others, Proc. Natl. Acad. Sci. USA 81, 1984, cc.6851-6855, Neuberger and others, Nature 312, 1984, cc.604-608 and Takeda and others, Nature 314, 1985, cc.452-454), based on splicing the genes of a mouse antibody molecule with a particular antigenic specificity genes molecule antibodies person with a specific biological activity. A chimeric antibody is a molecule in which different parts - derived molecules of different species of animals, for example, with variable or supervariables fragment derived from mouse manta rays, and a stable fragment of human immunoglobulin.

In another embodiment, the methods described for obtaining single-chain antibodies (US 4946778, Bird, Science 242, 1988, cc.423-426, Huston and others, Proc. Natl. Acad. Sci. USA 85, 1988, cc.5879-5883, Ward and others, Nature 334, 1989, cc.544-546), can be adapted to receive differently expressed gene single-chain antibodies.

Methods applicable for "humanized antibodies"can be adapted to generate antibodies to proteins, f is amentum or derived. Such methods are described in US patents 5932448, 5693762, 5693761, 5585089, 5530101, 5569825, 5625126, 5633425, 5789650, 5661016 and 5770429.

Antibodies or antibody fragments can be used in the methods, such as Western-blot or methods using immunofluorescence assay that can detect the expression of proteins. In such methods it is often desirable to mobilitat either antibody or proteins on a solid basis. Acceptable solid bases or carriers are any substrate capable of binding the antigen or antibody. The well-known bases or carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified cellulose, polyacrylamides, gabbros, and magnetite.

To simplify the detection method is applicable ELISA-sandwich", and many of its variants, each of which can be used in the present invention. In the context of the present invention a method of "sandwich" refers to all variants of the method, based on applying the two sides. And immunofluorescent, and immunodeficiencies EIA methods are widely used in the art. However, other reporter molecules, such as radioisotopes, chemiluminescent or bioluminescent molecules, can also be used. Specialist obvious how to vary the way, adapting it to the concrete is m conditions.

Monitoring protein whole genome, i.e. the "proteomics", can be performed using the design of the chips, in which binding sites comprise immobilized, preferably monoclonal antibodies, specific sets of proteins encoded by the cell genome. Preferably the antibodies are available for a significant fraction of the encoded proteins, or at least those proteins that are important for testing or reinforcement of the investigated biological network models. Above it is noted that the methods for obtaining monoclonal antibodies are well known. See, for example, Harlow, Lane in kN.: "Antibodies: A Laboratory Manual", published by Cold Spring Harbor Laboratory Press, 1988, Cold Spring Harbor, new York, USA). In a preferred embodiment of the present invention monoclonal antibodies generated against synthetic fragments of a peptide designed based on the genomic sequence of the cell. Cell proteins come into contact with so many antibodies and their binding is measured using methods known in the art.

Identification of polypeptides. Two-dimensional gel electrophoresis. Two-dimensional gel electrophoresis is well known in this area and usually involves isoelectric focusing, along with the first dimension, the resulting SDS-PAGE electrophoresis, along with the second dimension. See, for example, Hames, etc. in the book. "Gel Electrophoresis of Proteins: A Practical Approach", published by IRL Press, 1990, new York, USA), Shevchenko and others, Proc. Natl. Acad. Sci. USA 93, 1996, SS-14445, Sagliocco, etc., Yeast 12, 1996, SS-1533, Lander, Science 274, 1996, SS-539.

Identification of polypeptides. Mass spectroscopy. The identity and the level of expression of a target polypeptide can be determined by the method of mass spectroscopy. Methodology based on mass spectroscopy is applicable for the analysis of the selected target polypeptide, as well as for analysis of the target polypeptide in the biological sample. Methods of mass spectrometry for the analysis of the target polypeptide include: methods of ionization, for example, but these methods list of methods is not limited to, the method ionization, laser desorption, with the assistance of matrix-assisted laser desorption/ionization - MALDI), continuous or pulse ionization electrospray (electrospray ionization - ESI) and similar methods, such as ionspray or thermospray, and massive cluster impact (massive cluster impact - MCI). Such ion sources can be selected detection methods, which include linear or nonlinear reflectivity measurement time-of-flight of ions (time of flight - TOF)method, single or multiple quadrupole, method, single or multiple magnetic sector, a method of ion cyclotron resonance Fourier transform of the signal (Fourier transform ion cyclotron resonance - FTICR), ion trap, and combinations thereof, for example ion trap/TOF. For ions which then can be applied to various combinations of matrix/wavelengths (for example, MALDI) or combination of solvents (e.g., ESI).

For the application of mass spectroscopy, the target polypeptide can be dissolved in a solution or system reagents. The choice of a solution or system reagents, such as organic or inorganic solvent may depend on properties of the target polypeptide and a method of mass spectroscopy and is based on methods well known in the art. See, for example, the description of the method MALDI in the work Vorm and other, Anal. Chem. 61, 1994, s (1994), and ESI method in the work Valaskovic, etc., Anal. Chem. 67, 1995, s. Mass spectroscopy of peptides are also described in international patent application PCT 93/24834 and in the patent US 5792664. The solvent is chosen so as to minimize the risk of destruction of the target polypeptide at the expense of energy that occur during evaporation. To reduce the risk of fracture, for example, by immersion of the sample in the matrix. This can be applied to the matrix, which is an organic compound, such as sugar, for example, pentose or hexose, or a polysaccharide such as cellulose. Such compounds thermolytic are destroyed to CO2and H2About in a way that does not occur residues of molecules that can enter into chemical reactions. The matrix can also be an inorganic compound, such as ammonium nitrate, which destroyed almost without a trace. the label of these and other solvents known to specialists in this field. See, for example, US patent 5062935. Method of mass spectroscopy has been described in J. Phys. Chem. 88, 1984, SS-4459 and PCT application WO 90/14148, and modern methods of application are summarized in review articles. Cm. Anal. Chem. 62, 1990, SS-889 and Spectroscopy 4, 1992, SS-18.

The mass of the target polypeptide was determined using mass spectroscopy, may be correlated with the mass of the corresponding known polypeptide. For example, if the target peptide is a mutant protein, the corresponding known polypeptide may be appropriate nematanthus protein, such as protein wild type. With ESI, you can very accurately determine the molecular weight of the sample in which they are contained in femtomolar quantities, due to the presence of many ion peaks, which can be used to determine the mass. Submolecular levels of the protein reveal, for example, using the methods of mass spectroscopy ESI (Valaskovic, etc., Science 273, 1996, SS-1202) and MALDI (Li and others, J. Am. Chem. Soc. 118, 1996, SS-1663).

Method ionization, laser desorption, with the assistance of the matrix (MALDI). The level of the target protein in a biological sample, such as body fluids or tissue sample, can be measured by mass spectroscopy, including but not limited to, methods known in this field: method ionization, laser desorption, with the assistance of the matrix (MALDI), method of measuring time-of-flight (MALDI-TOF-MS), Elena surface time-of-flight laser mass spectroscopy (Surface-enhanced laser desorption/ionization time-of-flight - SELDI-TOF), which are described in detail below. Methods for MALDI well known to specialists in this field. Cm. Juhasz, etc., Analysis, Anal. Chem. 68, 1996, SS-946, and patents US 5777325, 5742049, 5654545, 5641959, 5654545 and 5760393 for descriptions and MALDI protocols delayed extraction. Also there are many ways to improve dissolution. MALDI-TOF-MS have been described Hillenkamp and others in kN.: "Biological Mass Spectrometry", edited Burlingame, McCloskey, 1990, published by Elsevier Science PubL, Amsterdam, SS-60.

Can be applied various methods of detection of the marker using mass spectroscopy. Cm. book: "Bordeaux Mass Spectrometry Conference Report" edited by Hillenkamp, 1988, SS-362, kN.: "Bordeaux Mass Spectrometry Conference Report" edited by Karas, Hillenkamp, 1988, cc.416-417, Anal. Chem. 60, 1988, cc.2299-2301, Biomed. Environ. Mass Spectrum 18, 1989, cc.841-843. Shows the use of laser beams in TOF-MS, for example, in patents US 4694167, 4686366, 4295046 and 5045694, links to which are included in the present invention in their entirety. Other methods of mass spectroscopy allow for successful evaporation of biopolymers with high molecular weight without fragmentation and allow you to analyze a large number of biological macromolecules using mass spectrometry.

Enhanced surface time-of-flight laser mass spectroscopy (SELDI). Other methods using new compositions of probe elements mass spectroscopy with surfaces that allow the probe to the active element SW is experienced in continuation trapped and accumulate specific analytes, described under the name affinity mass spectrometry (Affinity Mass Spectrometry - AMS). Cm. patents method SELDI: US 5719060, 5894063, 6020208, 6027942, 6124137 and published patent applications US 2003/0003465. Several types of new mass-spectroscopic probe elements described with reinforced surface affinity trap (Surfaces for Enhanced Affinity Capture - SEAC). Cm. Rapid Commun. Mass Spectrom. 7, 1993, cc.576-580. Probe elements SEAC successfully used for the restoration and binding of different classes of biopolymers, especially proteins, by using knowledge about the surface of the protein structures and biospecific molecular recognition. Device immobilized affinity traps on the surface of the mass spectrometric elemental probe, i.e. SEAC, determine the location and affinity (specificity) of the analyte to the surface of the probe, which increases the efficiency of analytical mass spectrometry.

Within the method of SELDI there are three different methodological choices: (1) Enhanced surface clean desorption (Surfaces for Enhanced Neat Desorption - SEND), in which the surface probe element, i.e. presenting the sample means are designed in such a way that contain adsorption energy of the molecule (Energy Absorbing Molecules - EAM) instead of "matrix" to facilitate desorption/ionization of analytes added directly (pure) to the surface. (2) a Variant of the method SEAC, in which the surface of the probes of the x elements, i.e. presenting the sample means are designed in such a way that they contain a certain chemical and/or certain biological trap to facilitate either specific or nonspecific joining or adsorption (docking or binding) of analytes to the surface of the probe using different mechanisms (mainly ecovalence). (3) Enhanced surface for photolabeling attach and release technique (Surfaces for Enhanced Photolabile Attachment and Release - SEPAR), in which the surface probe element, i.e. presenting the sample means are designed in such a way, that contain one or more types of chemically with certain crosslinking molecules that are covalently binding means. Chemical characteristics that determine the type and number of connection points photolabile molecules between SEPAR presenting means (i.e. the surface probe element) and analyzed the connection (e.g., protein) may include any or more of the number of different residues or chemical structures in the analyzed compound (for example, residues His, Lys, Arg, Tight, Phe and Cys in the case of proteins and peptides).

Other objects of the present invention with the biological activity. In various embodiments, implementation of the present invention the objects with biological action or see the si objects can be measured for to get the drug and metabolic responses. Activity of the proteins which are important for the description of cellular function, can be measured, and embodiments of the present invention may be based on such measurements. Activity measurement can be performed using any functional, biochemical or physical methods applicable to the particular described activity. If the activity is a chemical transformation, cellular protein can be contacted with the natural substrates, and the speed of transformation is measured. If the activity involves multidimensional Association units, such as the Association of activated DNA-binding complex with DNA, the number associated protein or secondary sequence Association, such as the number of transcribed mRNA, can be measured. In addition, if only the functional activity, such as control of the cell cycle, the expression of the function may be logged. If the function is known and measurable changes in the activity of proteins form response data measured by the methods of the present invention. In other and Pogranichnaya variants of implementation of the present invention data response can be formed on the basis of the evaluation of different objects biologists is a mini-state cells. Data on the response can be extracted, for example, from changes of large quantities of specific proteins and changes in the activity of certain proteins.

The example below is for a more complete description of the preferred embodiments of the present invention. This example is not intended to limit the scope of the present invention described in the claims.

Example 1. Comparison of aliskiren with placebo and irbesartan in patients with hypertension, expressed in degree from mild to moderate

Introduction and summary. Retrospective pharmacogenetic analysis is performed to assess possible links between genetic variation and the clinical treatment. Specifically 49 investigate polymorphisms in 12 genes of the renin-angiotensine-aldosterone system (RAS) or in genes that previously have shown that they are involved in the regulation of blood pressure. Significant associations observed between a polymorphism in the gene for angiotensin-converting enzyme (ACE), two polymorphisms in the gene for the angiotensin II receptor, type 2 (AGTR2), and clinical settings to lower average installed diastolic and systolic blood pressure. These effects are not found in the introduction of irbesartan and placebo, in this analysis they are likely specificmedia treatment aliskiren.

A clinical study. A multicenter, randomized, double-blind, parallel group clinical study was conducted to investigate the efficacy and safety of aliskiren compared with placebo in patients with hypertension, expressed in state from low to moderate. The study was conducted for eight weeks, i.e. the endpoint of clinical research occurs within eight weeks of this specific study. The main objective of this research is to define the actions of aliskiren in doses of 150 mg, 300 mg and 600 mg on lowering blood pressure and compare this action with the action of irbesartan at a dose of 150 mg and placebo effect in patients with hypertension, expressed in state from low to moderate. Demographic indicators in General compare all groups. The majority of patients belong to the Caucasian race, and the majority of patients does not exceed 65 years with an average age of 50 years. The distribution of men and women in the study about the same.

The first measured by the performance indicator is the change SOCKD (decrease SOCKD relative to the base level). The second measurable performance indicators are changing SOCKD (decrease SOCKD relative to the base level), the proportion choose what to say is', the decreased activity of plasma renin (BDA) and the increase of active plasma renin (ACTR) relative to the baseline.

Regarding the primary efficacy paired comparison shows that all doses of active treatment were statistically higher compared to placebo on reducing the average specific diastolic blood pressure (ADCD) endpoint 8 weeks in the treatment group and the control point of the control group. Similar results reduction SOCKD reach the dose of aliskiren 150 mg and irbesartan 150 mg Dose of aliskiren 300 and 600 mg cause the greatest decrease, but its value does not exceed the decline observed at the dose of 600 mg

Regarding secondary efficacy regarding medium-specific systolic blood pressure (SOCKD), all doses of active treatment statistically higher compared to placebo at the end point of the study. After 8 weeks aliskiren at a dose of 300 and 600 mg statistically higher compared to placebo with irbesartan in endpoint. Similar results reduction SOCKD reach the dose of aliskiren 150 mg and irbesartan 150 mg Dose of aliskiren 300 and 600 mg cause the greatest decrease, but this decrease was not increased at the dose of 600 mg

The proportion of successful response rate (expressed as reduction SOCKD < 90 mm Hg or ≥ 10 mm Hg relative to the base is level or decrease SOCKD < 140 mm Hg or ≥ 20 mm Hg relative to the base level) shows that all effective regimens at the end point of the study for treatment groups show results that are statistically higher than placebo at endpoint treatment. The results SOCKD at the endpoint of treatment is 59-67% of responders in the treatment group by aliskiren against 56% in the treatment with irbesartan and 38% for placebo. The results SOCKD at the endpoint of treatment is 57-68% of responders in the treatment group by aliskiren against 59% for irbesartan and 36% for placebo. In the treatment of observed dose-dependent increase in renin activity in groups of aliskiren (20,8, and 29.9 and 93.6 IU/ml for doses of aliskiren 150, 300 and 600 mg, respectively). Aliskiren at a dose of 150 mg leads to a greater average increase in renin compared with irbesartan dose of 150 mg (11,2 IU/ml). This is consistent with the mechanism of action of inhibitors of renin.

For patients participating in clinical research, pre-ask consent to participate in the pharmacogenetic analysis. Blood samples of all patients who gave consent, collected in different locations of the study and then sent to the firm Covance (Indianapolis, Indiana, USA). Genomic DNA of each patient are extracted from the blood by the method of the firm Covance, using the kit for DNA extraction PUREGENE™ DNA Isolation Kit (D-50K) (firm Gentra, Minneapolis, Minnesota, USA). In to enom account was genotypically 511 patients which is approximately equal shares for each group.

Pharmacogenetic analysis of interest and research plan. Retrospective pharmacogenetic analysis is performed on all the patients who agreed to provide samples for pharmacogenetic studies. The main objective is to identify associations between genetic variation and variations in treatment effectiveness by aliskiren, which will be evaluated primarily on ADKD and again on SOCKD and the proportion of responders.

Methodological approach using gene-candidates applied for the selection of 48 SNPs in 12 genes of the renin-angiotensine system (RAS) or in genes previously associated with the regulation of blood pressure (table 1). All available samples genotyping to identify each SNPS. The Association then identify the method described below.

Table 1
Candidate genes chosen for pharmacogenetic analysis
The designation of the geneThe name of the gene
RENBPthe renin-binding protein
RENrenin
ACE/td> angiotensin I converting enzyme (peptidylglycine A) 1
USEangiotensin I converting enzyme (peptidylglycine 2
AGTangiotensinogen
AGTR1the angiotensin II receptor, type 1
AGTR2the angiotensin II receptor, type 2
ABCC2/MRP2ATP-binding cassette, subfamily C (CFTR/MRP), member 2
AGTRAPthe angiotensin II receptor-associated protein
CYP11B2cytochrome P450, family 11, subfamily B, polypeptide 2, aldosteronism
TGFB1transforming growth factor, beta 1
NOS3/eNOSsynthase nitric oxide 3 (endothelial cells)

Genotyping. Studies of single nucleotide polymorphisms (SNPS) is performed with the use of information from the public dbSNP database and a proprietary database Celera/ABI. Sets of probes for geneti the investments get to a platform Assays-by-Design® firm ABI. Livak KJ, Marmaro J, & Todd JA, Nature Genetics 9: 341-2 (1995). Genotyping performed using 10 ng of genomic DNA according to the manufacturer's recommendations.

The list of polymorphisms genotyped in this analysis, including the internal code of the CPG locus, gene, metabolic pathway, links to the database and effect, results in table 2.

rs2285666
Table 2A
Genotyped polymorphisms
pg locus idGeneRs numberDescription
5284ASShCV11305436Intron
4786ASSrs2273697A>G I417V
4783ASSrs2756104Intron
4784ASSrs2756109Intron
4785ASSrs3740066 T>C I1324I
4782ASSrs717620untranslated
4797ASSrs8187710A>G Y1515C
5286ACEhCV1247681Intron
4766ACErs4293Intron
4769ACErs4317With>T P32S
4767ACErs4329Intron
4768ACErs4362C>T F1129F
400ACErs4364A>S712R
1345ACEthe insertion/deletion
4746USEIntron
1669USErs879922Intron
4773AGTrs1926723Intron
1667AGTrs4762T>NT
2AGTrs699C>T TM
4772AGTrs943580Genomic
4796AGTR1rs2638362Intron
4780AGTR1rs3772616Intron
411AGTR1rs5182C>T L191L

Table 2B
Genotyped polymorphisms
pg locus idGeneRs numberDescription
5287AGTR2hCV1841569
1445AGTR2rsl403543untranslated
186AGTR2rs5193untranslated
4795AGTR2-LDrs4497127Genomic
5288AGTRAPhCV516817Intron
4787AGTRAPrs4073574Genomic
4789CYP11B2rs4539G>A R173K
575CYP11B2rs4544With>T T339I
4788CYP11B2 rs4545A>G S435G
3541CYP11B2rs6431Intron
4792NOS3rs1007311Intron
482NOS3rs1799983G>T E298D
4791NOS3rs1800779Genomic
4464NOS3rs1800780Intron
4793NOS3rs891512Intron
5292RENrs11571092Intron
1513RENrs1464816Intron
4771RENrs3730103Intron
4794 RENrs6704321C>G P403A
4770RENBPrs2269371G>A G274D
2407RENBPrs2269372Intron
1676RENBPrs762656Genomic
5285TGFB1hCV11707868Intron
4790TGFB1rs2241717Intron
4798TGFB1rs8105161Intron

The statistical analysis. Investigation of the connection of the genotype-phenotype and other similar tests conducted in SAS (Saga, North Carolina, USA), using Analyst®.

In tests to detect Association using a clearly defined genotypes as independent variable, without assumptions about dominance, and various variables efficiency as dependent variables. Tests of continuous dependent variables, ispolzuemykh in the ANCOVA analysis, and logistic regression is used for certain dependent variables. Covariant derivatives of analysis of the Association of genotype-phenotype are: treatment, research space and the dimension of the underlying level.

Covariance analysis (ANCOVA) is repeated on the same model for each group, in which individuals are treated. In addition, the percentage of responders analyzed using a logistic regression model with factors such as the type of treatment and region, and baseline as a covariant derivative. Associations with p<0.05 all data are evaluated as significant.

Angiotensin-converting enzyme (ACE). Angiotensin-converting enzyme (ACE) performs the conversion of Ang I in the current oktapeptid Ang II. ON genotypic patients together with six other SNPs in the key enzyme of the RAS. The significant Association observed between ONP and variable the primary efficacy reduction SOCKD relative to baseline (p=0.0058) and the secondary variable - proportion of responders (p=0.001). For other secondary variable - reduction SOCKD relative to the base level, not observe significant associations, only a trend (p=0,0745).

When repeating the analysis ANCOVA for each treatment group, the Association observed only for patients in group the treatment aliskiren, but not in groups of treatment with irbesartan or placebo. Do not reveal significant associations between any of the SNPS and secondary parameters: plasma renin activity (ARA) and active renin (ACTR).

Specific effects of aliskiren shown in table 3.

72
Table 3
The impact of variations ACF and AGTR2 on ADKD, SOCKD and the percentage of responders in the treatment group by aliskiren
ONPONPONP
TTARTICLESSAAAGGGGGGAAA
N27814010669118106115
Reduction SOCKD (MND)-11,1-4,7NA-11,2-13,2-9,3-11,1margin of 13.3-9,0
P0,0058*0,0074*0,0022*
Reduction SOCKD-13,7is 6.2NA-13,9-15,7-11,8-13,6-16,3-11,5
P0,0384*0,13600,0479*
TTARTICLESS AAAGGGGGGAAA
The percentage of responders69%14%NA74%71%58%74%73%56%
P0,0024*0,13990,0382*

ONP is coding SNPS, which changes the composition of the amino acid sequence at codon 32, substituting Proline for serine in the enzyme ACF. Polymorphism of the ACE I/D (the presence or absence of repetitive sequences 287 BP Alu polymorphism in intron 16) associated with the regulation of the level and activity of ACF and its ramifications in the element of RACES. Rigat Century. etc., J. Clin. Invest. 86, 1990, SS-1346.

The angiotensin II receptor, type 2 (AGTR2). AGTR2 encodes the angiotensin II receptor, type 2. The angiotensin II receptor, type 2, is the target of several antihypertensive drugs. Si is Nala Ang II through binding to receptor type I induce compression of blood vessels and increase blood pressure. It is established that the II receptor, type 2, inhibits the activity of ACF and TERS via receptor type 1, causing vasodilatation. Hunley I.E etc., Kidney Int. 57, 2000, s-577 (2000), Ichiki T. and others, Nature 377, 1995, s-750.

The significant Association observed between SNPS AGTR2 gene (ONP and ONP) and variable the primary efficacy reduction SOCKD relative to baseline (p=0,0078 and to 0.0004, respectively). ONP also shows a significant Association with the secondary variable reduction SOCKD relative to baseline (p=0,0245), but not with other secondary parameter is the proportion of responders. ONP meanwhile, did not show significant Association with the secondary parameters. Was not established significant relationships between any of the SNPS and secondary parameters, namely the plasma renin activity (ARA) and active renin (ACTR).

During the analysis of ANCOVA for each group was set associative links only in the treatment groups by aliskiren, but not in groups of treatment with irbesartan or placebo. When repeating the analysis ANCOVA for each group of treatment Association observed only for patients in the treatment groups by aliskiren, but not in groups of treatment with irbesartan or placebo. The effect of genotype in patients who were treated by aliskiren shown above in table 3.

These two SNPS are not coded. ONP is netrans the dummy region AGTR2 gene, and ONP is located in a genomic region with non-equilibrium coupling with AGTR2 gene.

Discussion. Briefly, one variant in the gene ACF and two options in the AGTR2 gene show a significant Association with the primary parameter ADKD. Option ACF and one option AGTR2 also shows a significant Association with the secondary parameters SOCKD and the proportion of responders. The second option AGTR2 not show significant associations, but only shows this trend in relation to the secondary parameters. This effect of genotype observed only in groups in which patients were treated with aliskiren, but not in the treatment groups by obersontheim or placebo. Therefore, the above effect is a pharmacogenetic effect specific to aliskiren.

The effect option, the ACF is particularly interesting because the difference between the genotypes TT and ST is considerable (decrease SOCKD from 11.1 to 4.7 mm Hg, a decrease SOCKD from 13.7 to 6.2 mm Hg, the percentage of responders increased from 14% to 69%).

Homozygous CC genotype is missing patients, therefore, the occurrence frequency of this allele is much less than the frequency for SNPS according to databases, equal to 0.1.

In the analysis of response to treatment aliskiren depending on the genotype, the proportion of the response to the TT genotype increases to approximately 70%. Therefore, this option can help the placement of al is Sirena on the mass market antihypertensive drugs, facilitated the distribution of genotype in the General population (about 80% allelic frequency in the database SNPS, but can even be higher than what follows from this analysis), with the best percentage of responders (figure 1). It can also support the selection of a possible "better" treatment further research.

It is very likely that two SNPS in the gene AGTR2 are in nonequilibrium grip.

These reporter SNPS did not show Association with the original values ADKD and SOSD, again confirming that the genetic effect is aliskiren-related pharmacogenetic effect.

Example 2. Clinical pharmacogenetic analysis of clinical studies A

Introduction and summary. Retrospective pharmacogenetic analysis is performed to assess the potential link between genetic variation and the clinical treatment. Cm. example 1. Subsequently, the results of another clinical study (A) estimate for replication analysis. Specifically investigated the 48 SNPs in 12 genes of the renin-angiotensine system (RAS) or in genes that were previously found to be involved in the regulation of blood pressure.

In example 1 observed a significant Association between a polymorphism in the gene for angiotensin-converting enzyme (ACE), two polymorphisms of the mi in the gene for the receptor of angiotensin II, type 2 (AGTR2), and clinical parameters for reducing the average specific diastolic and systolic blood pressure. Such effects are not found in the treatment with irbesartan and placebo. In addition, we discovered that the meaningless variation ACF (Pro32Ser)associated with reduced response, the higher the occurrence frequency of the allele With a (Pro) among members of the Negroid race, compared to Caucasian (19/154 against 2/790).

In this example, all four patients with a serine residue (allele C) belong to the Negroid race. Due to the extremely small number of patients with allele Since it is impossible to conduct a study with the data SNPS on the same model as in example 1. In addition, the Association of two SNPS in AGTR2, established in example 1 was not replicated.

A clinical study in this example (A) randomizer and conduct double-blind, multicenter, multifactorial, placebo control and the application of parallel groups method for evaluating the efficacy and safety of combinations of aliskiren and valsartan compared with monotherapy individual components, or a combination of valsartan and hydrochlorothiazide (HCTZ)in patients with hypertension.

The main objectives of this example are the estimate of the effect in reducing blood pressure by using a combination of aliskiren valsartan (75/80 mg, 150/160 mg and 300/320 mg) compared with monotherapy individual components, administered for 6 weeks to patients with clinical average specific diastolic blood pressure ([SDKD] ≥ 95 mm Hg and < 110 mm Hg), and assessing the effect on reduction in blood pressure when using only one of aliskiren 75 mg, 150 mg and 300 mg compared with placebo, administered for 6 weeks to patients with clinical average specific diastolic blood pressure ([SDKD] ≥ 95 mm Hg and < 110 mm Hg). Group treatment is mainly correlated with demographic and baseline, with an average age of 56, 56% of men and 6.8% of the Negroid race.

The primary variable of efficiency change relative to baseline SOCKD treatment aliskiren compared with placebo, statistically significant in the group of patients receiving aliskiren at a dose of 300 mg Average reduction in blood pressure for the two variants monotherapy is the same as in the previous studies. However, the magnitude of the placebo effect is higher than expected, and higher than in previous studies of aliskiren. Lowering blood pressure with a combination of aliskiren and valsartan was less than with the use of hydrochlorothiazide (HCTZ) 12.5 mg/valsartan and additivity was less with a maximum dose of aliskiren 300 m is/valsartan 320 mg, than when less stringent combinations.

Pharmacogenetic analysis of objects. In a retrospective pharmacogenetic analysis includes all patients who agreed to provide samples for pharmacogenetic studies. The main objective is to identify associations between genetic variation and variations in treatment effectiveness by aliskiren, which is estimated mainly on ADKD, and SOCKD and the proportion of responders.

The approach using gene-"candidate" is used for selection of the 48 SNPs in 12 genes RACES or in genes that have previously been contacted with the regulation of blood pressure (table 4). All available samples genotyping for each SNPS. Then perform Association studies as described in example 1.

Table 4
Selection of candidate genes for pharmacogenetic analysis
The designation of the geneThe name of the gene
RENBPthe renin-binding protein
RENrenin
ACEangiotensin I converting farms is HT (peptidylglycine A) 1
USEangiotensin I converting enzyme (peptidylglycine 2
AGTangiotensinogen
AGTR1the angiotensin II receptor, type 1
AGTR2the angiotensin II receptor, type 2
ABCC2/MRP2ATP-binding cassette, subfamily C (CFTR/MRP), representative of 2
AGTRAPthe angiotensin II receptor - associated block
CYP11B2cytochrome P450, family 11, subfamily B, polypeptide 2, aldosteronism
TGFB1transforming growth factor, beta 1
NOS3/eNOSsynthase nitric oxide 3 (endothelial cells)

Samples. For patients participating in a clinical study A (see example 1) and a clinical study A, separately ask consent to participate in the conduct pharmacogenetic analysis. The blood samples of the patients who gave consent to participate, gather in some places research and then send firmw Covance (Indianapolis, Indiana, USA). Genomic DNA of each patient are extracted from the blood using the method of the firm Covance, using the kit for DNA extraction PUREGENE™ (D-50K) (firm Gentra, Minneapolis, Minnesota), and send in NIBR for genotyping. Ultimately, 511 patients participating in a clinical study in example 1 (A2201), genotypic approximately equal proportions for each group. Six hundred eighty four patients from A genotyping in the ratio 3:1 the number of patients in the placebo group and groups monotherapy aliskiren compared to other treatment groups.

The primary variables of effectiveness are changing values SOCKD (decrease SOCKD at endpoint compared to baseline levels). Secondary variables effectiveness are changing values SOCD (decrease SOCKD at endpoint compared to baseline levels), the percentage of responders, a decrease in plasma renin activity (ARA) and increased active plasma renin (ARP) relative to the original values.

Genotyping. Studies of SNPS plan, using information from the public database dbSNP and the private Celera database/ABI. Developed sets of probes for the study of haplodiploidy get to platform Assays-by-Design® firm ABI. Livak K.J., and others, Nat. Genet. 9, 1995, SS-342. For genotyping using 10 ng of genomic DNA, which is matching the manufacturer's recommendations.

Genotyped polymorphisms. The list of SNPs identified in this study, including the code of locus, a gene database reference and effect, results in table 5.

intron
Table 5
Genotyped polymorphisms
pg__idgeners_description
5284ASShCV11305436intron
4786ASSrs2273697A>G I417V
4783ASSrs2756104intron
4784ASSrs2756109intron
4785ASSrs3740066T>C I1324I
4782ASSrs717620netr Norway
4797ASSrs8187710A>G Y1515C
5286ACEhCV1247681intron
4766ACErs4293intron
4769ACErs4317C>T P32S
4767ACErs4329intron
4768ACErs4362C>T F1129F
400ACErs4364A>S712R
1345ACEthe insertion/deletion
4746USErs2285666intron
1669USErs879922
4773AGTrs1926723intron
1667AGTrs4762T>C M207T
2AGTrs699C>T TM
4772AGTrs943580genomic
4796AGTR1rs2638362intron
4780AGTR1rs3772616intron
411AGTR1rs5182C>T L191L
5287AGTR2hCV1841569
1445AGTR2rs1403543untranslated
186AGTR2rs5193 untranslated
4795AGTR2-LDrs4497127genomic
5288AGTRAPhCV516817intron
4787AGTRAPrs4073574genomic
4789CYP11B2rs4539G>A R173K
575CYP11B2rs4544With>T T339I
4788CYP11B2rs4545A>G S435G
3541CYP11B2rs6431intron
4792NOS3rs1007311intron
482NOS3rs1799983G>T E298D
4791NOS3genomic
4464NOS3rs1800780intron
4793NOS3rs891512intron
5292RENrs11571092intron
1513RENrs1464816intron
4771RENrs3730103intron
4794RENrs6704321C>G P403A
4770RENBPrs2269371G>A G274D
2407RENBPrs2269372intron
1676RENBPrs762656genomic
5285TGB1 hCV11707868intron
4790TGFB1rs2241717intron
4798TGFB1rs8105161intron

The statistical analysis. Studies of the Association of genotype-phenotype and related tests conducted in the firm SAS (Cary, NC, USA)using software Analyst®.

The tests detect associations have clearly defined genotypes as independent variable, without assumptions about dominance, and various variables efficiency as dependent variables. Tests of continuous dependent variables used in the analysis ANCOVA and logistic regression is used for clearly defined dependent variables. Note that none of the results are not fit for testing many hypotheses. The threshold p<0.05 is used to determine the valid associations. The coincidence of random variables in the analysis of the Association of the genotype-phenotype are: (1) the dose for the treatment, (2) the study area, denoted STU1A in A (see example 1) or designated REGION in A, (3) measurement of initial levels ADKD and SOCKD and (4) race. An who symbolise all samples from all three groups, in which patients were treated with aliskiren. When observing the coincidence of random variables the same analysis is carried out in groups of patients who were treated with irbesartan and placebo.

Table 6
Demographic and baseline characteristics SOCKD genotyped patients (mean ± standard error)
A (see example 1)A (this example)
The treatmentN=The body Mass indexAgeInitial level ADCDThe treatmentN=AgeInitial level ADCD
Aliskiren 150 mg9931,01±0,6754,15S 1,3098,76±0,34Aliskiren 75 mg10254,73±1,3798,69±0,28
Aliskiren 300 mg96 30,79±0,5855,93±1,03a 99.16±0,36Aliskiren 150 mg11555,83±1,1899,47±0,35
Aliskiren 600 mg10130,53±0,6555,97±1,0898,90±0,38Aliskiren 300 mg10257,09±1,2299,26±0,34
Irbesartan 150 mg9931,81±0,7256,66±1,2199,41±0,40Valsartan 80 mg3958,08±1,7799,30±0,61
Placebo10330,65±0,6058,21±1,1598,89±0,33Valsartan 160 mg3353,45±2,1598,87±0,69
Valsartan 320 mg3455,50±1,65a 99.16±0,57

A (see example I)A (this example)
The treatmentN=The body Mass indexAgeInitial level ADCDThe treatmentN=AgeInitial level ADCD
Aliskiren 75 mg and valsartan 80 mg3555,43±1,9599,73±0,64
Aliskiren 150 mg and valsartan 160 mg3456.26 vertical±1,9499,32±0,61
Aliskiren 300 mg and valsartan 320 mg4157,10±1,8698,82±0,46
Valsartan 160 mg and HCTZ 12.5 mg4155,41±1,9398,83±0,55
Placebo10855,80±1,2498,86±0,29

ACF. SNPS 4769 genotyping patients along with six other SNPs in this crucial enzyme for RACES. A significant relationship observed between SNPS 4769 and the first variable efficiency, reduced SOCKD relative to baseline (p=0.023), and with the second variable, the level of responders (p=0,0048). For another second variable, decrease the amount of SOCDEV relative to the original level, not noted a significant Association noted only a tendency (p=0,14).

When repeating the analysis ANCOVA with the same model the La of each of the treatment groups Association revealed for groups treatment aliskiren, but not for a group of treatment with irbesartan or placebo group. Specific effects of aliskiren shown in table 7. Insignificant relationship is found between SNPS and secondary parameters of plasma renin activity (ARA) and active renin (ARP).

Table 7
The impact of the ACF and AGTR2 variations on ADKD, SOCKD and the percentage of responders in all treatment groups by aliskiren in A (* shows significance, p<0,05)
ONPONPONP
TTARTICLESSAAAGGGGGGAAA
n2781401066911810672115

Analysis LSMEAN reduce SOCKD (MND)-11,4-5,6NA-11,0margin of 13.3-9,4-10,8-13,4to-9.2
p0,023*0,0071*0,0026*
Analysis LSMEAN reduce SOCKD (MND)-11,7-5,9NA-11,2and 13.8-9,8-10,8-14,2-9,5
p0,140,130,046*
The percentage of responders0,140,130,046*75%71%58%75%74%57%
p0,0048*0,17to 0.060

ONP is coding SNPS, which changes the amino acid sequence of the Proline to serine at codon 32 isoforms 2 and 3 of ACF. Polymorphism of the ACE I/D (the presence or absence of a 287 BP Alu repeat sequence in intron 16) associated with the level of regulation of the ACF and activity and its ramificatiei in RACES. Rigat Century. etc., J. Clin. Invest. 86, 1990, SS-1346. In this case, testing the ACF I/D and see an associative connection with the response to aliskiren. Anpb is located in the 12th intron of the gene ACF encoding somatic isoforms, and localized in the first exon of the gene encoding the second and third isoform. The ACE I/D is located in the 16th intron, which is located in the same non-equilibrium coupling that ONP, in groups of Europeans, African Americans, and Chinese (viewer SNPS, the company Applied Biosystems, foster city, CA, USA).

AGTR2. It is established that the angiotensin II receptor, type 2, inhibits the activity of ACF and weakens the actions mediated by the receptor, type 1, causing the blood vessels to dilate. Ichiki T, and others, Nature 377, 1995, s-750, Hunley TE, etc., Kidney Int. 57, 2000, s-577.

See significant Association between SNPS 1445 and SNPS 4795 and primary variable EF is aktivnosti, reduction SOCKD relative to baseline (p=0,0071 and 0,0026, respectively). SNPS 4795 also shows a significant relationship with the second variable, the reduction SOCKD relative to baseline (p=0,046), but not with the second option, the proportion of responders. Meanwhile, the UNP 1445 shows no communication with the second parameter. Not find a significant relationship between SNPS and secondary settings, the BDA and ACTR.

When repeating the analysis ANCOVA with the same model for each group of treatment Association found for group treatment aliskiren, but not for a group of treatment with irbesartan or placebo group. Impact of genotype in patients undergoing treatment aliskiren presented above in table 7.

When you try to repeat the Association observed with these two SNPS in the study A in the treatment groups by aliskiren, the results are not repeated (p>0,05). Also not see the manifestation of the trend toward Association. Based on the results of preliminary clinical trials due to higher than was observed previously, the response to placebo (8,6 mm Hg), dose-response and the magnitude of the placebo-deductible effects when treating aliskiren lower than expected. Such inconsistency may be caused, at least partially, the loss of a copy of the Association AGTR2.

Both of these SNPS are not coded. SNPS 1445 is netransliruemoi area AGTR2 gene, and ONP are located in the genomic region in nonequilibrium coupling with AGTR2 gene. At least the Chinese, these two SNPS are in the same block of nonequilibrium clutch (SNPbrowser, the company Applied Biosystems, Foster City, CA, USA).

Discussion. This analysis reveals a significant Association between polymorphisms in two genes with variable clinical outcomes in the study A (see example 1). One variant in the gene ACF and two options in the AGTR2 gene show a significant Association with the primary parameter, ADKD. Option ACF and one of the options AGTR2 also show a significant Association with the secondary parameters, SOCKD and the proportion of responders. The second option AGTR2 does not show a significant Association, but only shows the same trend with respect to the secondary parameters. Such action genotype observed only in the groups of patients who were treated by aliskiren, but not in groups of treatment with irbesartan or placebo. Therefore, the above effect means that pharmacogenetic effect specific to aliskiren.

Action Pro32Ser option ACF is of particular interest, because the difference between the genotypes TT and ST is considerable (decrease SOCKD of 11.4 5.6 mm Hg, a decrease SOCKD 11,7 against 5.9 mm Hg and the proportion of responders 70% vs. 14%). However, homozygous CC genotype is not present in all p. the patients. Therefore, the occurrence frequency of this allele is much less than the frequency for SNPS according to databases, equal to 0.1, and the average number of patients with genotype ARTICLE very slightly. Found that the occurrence frequency of the allele With much higher among representatives of blacks compared to Caucasian (19/154 against 2/790). In the experiment A all 4 patients with a serine residue (allele C) belong to the Negroid race. Due to the extremely small number of patients with the allele With the analysis A cannot be conducted with the same model.

Polymorphisms ACF and their influence on the concentration of the ACF in plasma and blood pressure revealed mainly in groups of Africans and African Americans. Bouzekri, N., and others, Eur. J. Hum. Genet. 12, 2004, SS-468, SOH R., and others, Hum. Mol. Genet. 11, 2002, SS-2677, X. Zhu and others, Am. J. Hum. Genet. 68, 2001, SS-1148. The observation of a higher allelic frequency ONP in the gene ACF in black patients in the two studies SPP100 confirms further study the functional effects of this polymorphism on the level of ACF or its activity. In addition, the amino acid change at codon 32 is assumed to be the isoform of this enzyme in the testes.

When analyzing the response to aliskiren depending on the genotype, the proportion of responders with genotype TT increases to approximately 70%. This result may contribute to the accommodation and escorena on the mass market of antihypertensive agents, as a significant part of the human community (about 80% according to the frequency of alleles of the SNPS database, and can even be more according to this study) has the TT genotype associated with a higher proportion of responders (figure 2). It can also help in further research to select the supposedly "best" of responders answering aliskiren.

It is very likely that two SNPS AGTR2 gene are in nonequilibrium coupling, because the chromosomal region around the given locus is substantially in the same block of nonequilibrium clutch. The function of signal transmission Ang II via receptor type 2 has been studied to a lesser extent compared to receptor type 1. This discovery may also indicate participation in AGTR2 functions of Ang II in the regulation of blood pressure lower in the chain in relation to inhibition of renin.

These described SNPS do not show associations with dimensions of the original levels ADKD and SOSD. This means that the genetic effect is aliskiren-related pharmacogenetic effect.

Equivalents

The details of one or more embodiments of the present invention are summarized below in the claims and correspond to the above description. Although any methods and materials similar or equivalent to OPI the data in the present invention, can be used to implement the present invention or for testing, the preferred methods and materials are described in the present description. Other characteristics, objects and advantages of the present invention can be gleaned from the descriptions and claims of the present invention. In the description and the attached claims forms in the singular include references to multiple objects, despite the fact that the content clearly indicates otherwise. Despite the obvious contradiction, all techniques and scientific terms used in the present invention, have the same purpose, which is usually understood by specialists in the area belongs to the present invention. All cited in the present invention, publications, patents and patent applications included in it by reference to their essence, and in full for all the same purposes for which each individual publication, patent or patent application were specifically and individually indicated to be included in reference to their essence.

The present invention is not limited to the use of the terminology found in a specific variants of its implementation, since this terminology is given to explain the individual objects of the present invention. Many variations and modifications of the present invention can be obtained without the bias is of the spirit and the scope of the present invention, which, of course, obvious to specialists in this field. Experts also apparent that the present invention methods and equipment can be replaced with functionally equivalent, corresponding to the scope of the present invention. Such modifications and variations correspond to the formula of the present invention. The present invention is limited to the terms set forth in the claims, however, along with them to the full extent lawful use cash equivalents.

1. The use of aliskiren to obtain drugs for the treatment of hypertension in patients selected on the basis of genetic polymorphisms in biomarker genes present in patients with genetic polymorphisms are indicators of the effectiveness of aliskiren in the treatment of hypertension and selected from the group comprising: ONP, as indicated in SEQ ID NO: 1 in the gene angiotenzinkonvertiruyuschego enzyme (ACE), ONP, as indicated in SEQ ID NO: 2 in the gene for the angiotensin II receptor, type 2 (AGTR2), ONP, as indicated in SEQ ID NO: 3 in the AGTR2 gene, and combinations thereof,.

2. The method of determining the sensitivity of an individual with hypertension treatment aliskiren, which includes stages:
(a) the establishment of two copies of genes present in an individual, the identity of the nucleotide pairs in one or more polymorphic genetic lock the sah, moreover, genetic loci selected from the group comprising a gene angiotenzinkonvertiruyuschego enzyme (ACE)gene of angiotensin II receptor, type 2 (AGTR2), and genetic polymorphisms selected from the group comprising: ONP, as indicated in SEQ ID NO: 1 in the gene angiotenzinkonvertiruyuschego enzyme (ACE), ONP, as indicated in SEQ ID NO: 2 in the gene for the angiotensin II receptor, type 2 (AGTR2), ONP, as indicated in SEQ ID NO: 3 in the AGTR2 gene, and combinations thereof, and
(b) evaluation facilities of the individual to "viacorresponding" group, if the nucleotide pair at the polymorphic loci indicate that the individual responds to the treatment aliskiren.

3. The use of aliskiren to obtain drugs for treating hypertension in an individual, comprising the stage of:
(a) the establishment of two copies of genes present in an individual, the identity of the nucleotide pairs in one or more polymorphic genetic loci, and genetic loci selected from the group comprising a gene angiotenzinkonvertiruyuschego enzyme (ACE)gene of angiotensin II receptor, type 2 (AGTR2), and genetic polymorphisms selected from the group comprising: ONP, as indicated in SEQ ID NO: 1 in the gene angiotenzinkonvertiruyuschego enzyme (ACE), ONP, as indicated in SEQ ID NO: 2 in the gene for the angiotensin II receptor, type 2 (AGTR2), ONP as indicated in SEQ ID NO: 3 in the AGTR2 gene, and combinations thereof, and
(b) introducing aliskiren is and individual, if the nucleotide pair at the polymorphic locus indicates that the individual responds to the treatment aliskiren.

4. The use of aliskiren to obtain medicines for lowering the average determined systolic blood pressure (SOCKD) in an individual, comprising the stage of:
(a) the establishment of two copies of genes present in an individual, the identity of the nucleotide pairs in ONP, as indicated in SEQ ID NO: 3 in the gene for the angiotensin II receptor, type 2A(gtr2 are), and
(b) the introduction of aliskiren individual, if the nucleotide pair at ONP, as indicated in SEQ ID NO: 3, indicates that the individual responds to the treatment aliskiren.

5. The use of aliskiren to obtain medicines for lowering diastolic blood pressure in an individual, comprising the stage of:
(a) the establishment of two copies of genes present in an individual, the identity of the nucleotide pairs in one or more polymorphic genetic loci, and genetic loci selected from the group comprising a gene angiotenzinkonvertiruyuschego enzyme (ACE)gene of angiotensin II receptor, type 2 (AGTR2), and genetic polymorphisms selected from the group comprising: ONP, as indicated in SEQ ID NO: 1 in the gene angiotenzinkonvertiruyuschego enzyme (ACE), ONP, as indicated in SEQ ID NO: 2 in the gene for the angiotensin II receptor, type 2 (AGTR2), ONP as indicated in SEQ ID NO: 3 in the gene AGT2, and combinations thereof, and
(b) the introduction of aliskiren individual, if the identity of the nucleotide pairs in one or more polymorphic loci is indicative that the individual responds to the treatment aliskiren.

6. The use of a gene product of a gene selected from the group containing the gene angiotenzinkonvertiruyuschego enzyme (ACE) gene and the receptor of angiotensin II type 2 (AGTR2), as targets for drug action, which includes the following stages:
(a) contact of the drug with the first gene product encoded by polynucleotides with the nucleotide pair at the polymorphic site in the region of the selected gene, indicating high reactivity in the treatment of hypertension by aliskiren,
(b) identification of the drug action on said first gene product,
(in) the contact of the drug with the second gene product encoded by polynucleotides with the nucleotide pair at the polymorphic site in the region of the selected gene, indicating a low reactivity in the treatment of hypertension by aliskiren,
(g) identification of the drug action on the specified second gene product,
(d) identification of similarities and differences between the activity identified at stage (b), and activity identified at stage (g);
moreover, genetic polymorphisms in the gap group, includes: ONP, as indicated in SEQ ID NO: 1 in the gene angiotenzinkonvertiruyuschego enzyme (ACE), ONP, as indicated in SEQ ID NO: 2 in the gene for the angiotensin II receptor, type 2 (AGTR2), ONP, as indicated in SEQ ID NO: 3 in the AGTR2 gene, and combinations thereof.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: algorithm of comparing mass spectra with a genome database is applied repeatedly after updating the database with new records or after deleting records from the database or after replacing the database with a database composed of new records. Additional records are generated by making changes in the sequence of identified biopolymers which correspond to replacements, deletions, insertions and modifications of one or more amino acid residues. Present invention also relates to a computer system whose operation is based on the disclosed method.

EFFECT: high accuracy of identifying sequence of amino acid residues of a biopolymer.

5 cl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method involves biomaterial sampling, DNA recovery and PCR-based genomic Ornithobacterium rhinotracheale DNA detection with using specially selected primers and reaction products detection. For genomic DNA detection, the PCR procedure uses two primers: upstream 5'-TGGGATTACCGCAAAATACC-3' and downstream 5'-AAACGGAGTGGTTACGCTCA-3. If observing an amplification DNA fragment sized 142 base pairs, the genomic Ornithobacterium rhinotracheale DNA is concluded to be present in the analysed sample.

EFFECT: method provides high sensitivity and specificity to Ornithobacterium rhinotracheale microorganisms that allows reliable detection of the presence of the latter in the analysed sample.

2 cl, 2 tbl, 3 ex

FIELD: measurement equipment.

SUBSTANCE: device is intended to scan results of diagnostics in medicine, veterinary medicine, in control of food products, in forensic science. Device comprises optical reception system, registration and control systems, the first and second illuminators, source of supply, thermal control unit with control system of temperature regulation and reaction reservoir. Thermal control unit additionally includes metal substrate with light-reflecting or retro-reflecting layer applied on it, which is fixed on face surface of thermal control unit with the help of heat conducting adhesive coating. Reaction reservoir is installed so that the distance between its working surface and substrate makes 0.3-10 mm. Thermal control unit is fixed perpendicular to optical axis of reception system and together with elements of optical system is fixed on common platform, which may be installed at various angles from 0 to 90° to horizontal plane to provide for the possibility to fill reaction reservoir and do PCR both in horizontal and vertical position of reaction reservoir. Thermal control unit is equipped with detachable cover for its removal from optical trajectory of optical system in process of fluorescence registration.

EFFECT: device provides for performance of hybridisation and PCR reactions in online mode.

10 cl, 7 dwg

FIELD: chemistry; biochemistry.

SUBSTANCE: invention relates to biotechnology, particularly to a set of two primers for amplifying nucleic acid of hepatitis A virus, a method of detecting hepatitis A virus in a biological sample, as well as a kit for detecting hepatitis A virus in a biological sample. The method of detecting hepatitis A virus (HAV) in a biological sample which contains antibodies produced by an individual, is realised by bringing a solid substrate into contact with capturing nucleic acids containing one or more oligonucleotides. Further, the solid substrate is brought into contact with the biological sample under hybridisation conditions. The solid substrate is then separated from the sample. Further, the isolated nucleic acids are amplified with a set of two primers. Presence of amplified nucleic acids as an indicator of the presence or absence of HAV in the sample is then determined. The method can also involve determination of the presence of an amplified internal control sequence.

EFFECT: invention enables provides a sensitive, reliable diagnostic test for detecting hepatitis A virus (HAV) in biological samples from potentially infected individuals.

43 cl, 5 dwg, 1 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely phthisiology, and can be used for prediction of development of respiratory tuberculosis.

EFFECT: method provides more effective genetic prediction of respiratory tuberculosis in adults ensured by PCR-based molecular HLA - DQB1* gene typing, and if detecting HLA - DQB1*05 allele in the genotype, the risk of development of respiratory tuberculosis and unfavourable course are predicted.

1 tbl, 1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: device for liquid sample introduction into the fluid carrier flow running through a continuous flow tube having an outlet and a common inlet wherein there are introduced both a carrier flow, and a liquid sample; the device comprises a tank for continuous fluid carrier supply into the inlet; the tank is designed to maintain the constant fluid carrier level above the inlet and fluid connected with the continuous flow tube in such a manner that when used, the fluid carrier flow and liquid sample are absorbed through the continuous flow tube when the tank is under atmospheric pressure and when the fluid carrier represents a hydrophobic liquid, while the liquid samples represents water test. The method involves the use of the device described above, creation of fluid connection of the continuous flow tube inlet and the tank, introduction of the fluid carrier in the tank and introduction of the liquid sample in the fluid carrier; while the fluid carrier flow and liquid sample are absorbed through the continuous flow tube when the tank is under atmospheric pressure. Also the method can involve immersion of a liquid sample distributor in the fluid carrier which is contained in the tank, liquid sample distribution in the inlet and running of the distributed dosed liquid sample by the liquid sample distributor so to supply the distributed liquid sample into the inlet and to absorb it through the continuous flow tube. The fluid carrier flow and liquid sample are absorbed through the continuous flow tube when the tank is under atmospheric pressure. Also the group of inventions includes a continuous flow system which contains the device specified above, and a method of nucleic acid amplification in PCR or LCR format, with the use of specified system.

EFFECT: method improvement.

21 dwg, 4 ex, 8 cl

FIELD: medicine.

SUBSTANCE: DNA is recovered from patient's peripheral venous blood to detect TNFα gene polymorphism. If observing -308GA and -308AA genotypes, a severe course of chronic calculous cholecystitis with frequent alternating exacerbations is predicted. -308GG genotype detected enables predicting moderate and mild courses of chronic calculous cholecystitis of recurrent or monotonous pattern.

EFFECT: use of the method allows to predict the clinical course and severity of chronic calculous cholecystitis in a relatively short time.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: blood serum is analysed for promoter region methylation of GSTP, p16 and N33 genes by methyl-sensitive PCR method. If observing abnormal methylation at least in two of said genes, the presence of a disease is concluded.

EFFECT: higher accuracy of early DNA-diagnosis of prostate cancer ensured by the use blood serum DNA to be analysed, technique exhibits higher specificity due to the correct gene specification in a test system and allows applying profit-proved detection methods.

1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: conventional method for Mycobacterium tuberculosis Beijing genotype strain is spoligotyping based on the structural analysis of M tuberculosis chromosome in the direct repeats divided by variable spacers. A method for length polymorphism heteroduplex analysis of DNA restriction fragments containing an insertion element IS6110 (method IS6110-RFLP), applied to mycobacterium tuberculosis strain differentiation, including Beijing genotype strain, cannot be used for Beijing genotype characterisation as a single gene family because of variety of the produced restriction profiles. The invention detects PCR amplified DNA specific fragments (genome regions between proximal copies of the element IS6110) of size 290-pn and 470-pn for "ancient" (atypical) Beijing strains or of size 260-pn, 290-pn and 470-pn for "modern" (typical) Beijing M. tuberculosis strains. The results are analysed by PCR product electrophoresis in the horizontal agarose mini-gel.

EFFECT: fast and reliable mycobacterium tuberculosis Beijing genotype detection.

6 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, namely to method of predicting preventive and/or therapeutic effect of RAR-α agonist in oncological patients. In order to realise the method, level of expression of molecules of family p160, such as AIB1, SRC-1, TIF2, and level of expression of molecules of family SP110b in sample, taken from patient's malignant tumour, are determined and ratio between said levels of expression is determined. In case if ratio "level of AIB1 expression/ level of SP110b expression" is higher than 0.240, it is considered that RAR-α agonist is efficient in therapeutic treatment of patient's malignant tumour. Similar conclusion is made if value of ratio "level of SRC-1 expression/ level of SP110b expression" exceeds 0.662, as well as in case if value of ratio "level of TIF2 expression/ level of SP110b expression" exceeds 0.141. In case if said values of ratio of expression levels is detected, patient is introduced RAR-α agonist for treatment of malignant tumour, such as cancer of liver.

EFFECT: application of claimed method allows to predict efficiency of treatment with RAR-α agonists quite accurately.

10 cl, 5 tbl, 4 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology, endocrinology, and can be used for normalising the blood microvesicle level in impaired glucose tolerance. That is ensured by graduated physical activity, and administration of metformin 500 mg twice a day. The therapeutic course is at least 5 weeks.

EFFECT: offered combination of therapeutic modalities enables normalising the blood microvesicle level in a relatively short time that promotes prevention of thrombotic complications in the case patients.

1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to gerontology, endocrinology, and can be used for pathologically raised biological age reduction in the patients with abdominal obesity. That is ensured by prescription of efficient graduated static and dynamic physical activity, daily swimming in a pool for not less than 30 minutes a day, and also administration of metformin in dosage 850 mg twice a day. The therapeutic course is 7 weeks.

EFFECT: offered combination of the modalities enables to adjust the biological and chronological ages that improves quality of life in the case patients.

2 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haematology, and can be used for normalisation of adenosine diphosphate and adenosine triphosphate thrombocyte secretion activity in the patients with arterial hypertension and impaired glucose tolerance. That is ensured by graduated physical activities, daily swimming in a pool for at least 20 minutes a day, and also prescribed metformin 500 mg twice a day and lisinopril 10 mg once a day in the morning.

EFFECT: combination of therapeutic modalities enables rapid normalisation of adenosine diphosphate and adenosine triphosphate thrombocyte secretion activity that promotes prevention of thrombotic complications in the case patients.

9 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology, endocrinology, and can be used for normalising the blood α2 antiplasmin concentration in arterial hypertension and impaired glucose tolerance. That is ensured by graduated physical activities, daily swimming in a pool for at least 20 minutes a day, and also prescribed metformin 500 mg twice a day and lisinopril 10 mg once a day at regular hours in the morning. The therapeutic course is 1 months.

EFFECT: offered combination of therapeutic modalities enables normalising the blood α2 antiplasmin level in a relatively short time that promotes prevention of thrombotic complications in the case patients.

1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to cardiology, endocrinology, and can be used for normalising the blood plasminogen activator inhibitor concentration in arterial hypertension and impaired glucose tolerance. That is ensured by graduated physical activities, daily swimming in a pool for at least 20 minutes a day, and also prescribed metformin 500 mg twice a day and lisinopril 10 mg once a day in the morning. The therapeutic course is 1 months.

EFFECT: offered combination of therapeutic modalities enables normalising the blood plasminogen activator inhibitor in a relatively short time that promotes prevention thrombotic complications in the case patients.

1 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably to haematology and cardiology and refers to reduction of spontaneous erythrocytes aggregation in patients with arterial hypertension and dislipidemy. To achieve this, combination of hypolipidemic diet with dosed different physical exercises, daily swimming during at lest 40 minutes in the middle of the day is used. Additionally is administered 10 mg of lisinopril in the morning once daily and simvastatin 20 mg after supper once daily. Course of treatment is 2 months.

EFFECT: method ensures correction of spontaneous erythrocyte aggregation in this group of patents and results risk decrement of such thrombotic complications as myocardial infarction, insult and thromboses of different localisation.

1 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: invention belongs to medicine, notably, to gerontology, cardiology and can be used for pathologically raised biologic age in patients with arterial hypertension. To achieve this, rationally dosed static and dynamic physical exercises are administered, daily swimming in swimming pool at least 30 minutes daily and 10 mg of lisinopril daily in the morning. Course of treatment is 7 weeks.

EFFECT: combination of treatment methods ensures equalisation of biologic and chronologic ages, thus improving quality of life in this group of patients.

2 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: there is described a new compound - 1-(β-phenylethyl)-4-amino-1,2,4-triazolium bromide exhibiting cardioprotective, anti-ischemic, antihypertensive, antioxidant, protein synthetic and energotropic action.

EFFECT: improved clinical effectiveness.

1 cl, 5 ex, 4 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicinal agents and method for increasing biological availability of renin inhibitor, consisting in the fact that to mammal in need of such treatment, combination containing renin inhibitor and exhaustive protein inhibitor is jointly administered, in which inhibitor of exhaustive protein represents inhibitor MDR1, such as PSC833, and renin inhibitor represents derivative of amide δ-amino-γ-hydroxy-ω-arylalkanoic acid, with formula of (I), where R1, R2, R3 and R4 have values specified in the formula.

EFFECT: production of pharmaceutical composition, having hypotensive effect, containing renin inhibitor in therapeutically efficient amount in combination with inhibitor of exhaustive protein, and application of exhaustive protein inhibitor for preparation of medicinal agent intended to increase biological availability of renin inhibitor.

21 cl, 5 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to hematology, cardiology, endocrinology, and can be used for optimisation of functional reactivity of cardiovascular system (CVS) in patients with arterial hypertension with metabolic syndrome and heart failure of I-IIA stage. For this purpose, first, state of CVS functional reactivity at physicoemotional load is estimated. Systolic, diastolic, average dynamic (APavdyn.), heart rate (HR) are registered. On the basis of obtained parametres index of functional reactivity (IFR) before and after load is determined by formula IFR=(APavdyn.x HR)/100 (SU). Treatment is administered if IFR value increment is more than 20 SU. Treatment combines individually selected hypocaloric diet, daily swimming in swimming pool for not less than 15 minutes per day, morning hygienic gymnastics, therapeutic and preventive gymnastics and doing subdivided physical exercise during the day, and intake of valsartan 80 mg in the morning one time per day and pioglitazone 30 mg in the morning one time. Treatment duration is not less than 3 months.

EFFECT: method allows to diagnose failure of functional CVS reactivity, and combination of claimed methods of treatment allows to correct it efficiently, which favours prevention of complications of arterial hypertension with metabolic syndrome and heart failure of I-IIa stage.

2 ex

Sugar-coated drug // 2407515

FIELD: medicine, pharmaceutics.

SUBSTANCE: declared invention refers to chemical-pharmaceutical industry and concerns a drug which contains a portion containing an oxygen-intolerant active ingredient wherein the oxygen-intolerant active ingredient is (R)-5,6-dimethoxy-2-[2,2,4,6,7-pentamethyl-3-(4-methylphenyl)-2,3-dihydro-1-benzofuran-5-yl]isoindoline and a sugar coating containing (1) sugar alcohol as a base material of the sugar coating where sugar alcohol is erythrite and (2) a binding agent where the binding agent is gum arabic wherein a portion is sugar-coated. Also, the invention concerns a method for preparing the declared drug.

EFFECT: agent exhibits high storage stability and oxygen tolerance.

8 cl, 18 ex, 15 tbl, 1 dwg

Up!