RU2408363C2 - Biomarkers for estimating efficacy of aliskiren as hypertensive agent - Google Patents

Abstract

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

Description

FIELD OF THE INVENTION

The present invention mainly relates to analytical testing of 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 THE INVENTION

The renin-angiotensin system (ASD) plays an important role in the regulation of blood pressure and the volume of homeostasis. Renin is secreted by the kidneys in response to a decrease in the volume of circulating blood and a decrease in blood pressure and breaks down the substrate of angiotensinogen with the formation of inactive angiotensin I decapeptide I (Ang I). Ang I is converted to the active octapeptide Ang II by the angiotensin converting enzyme (ACF). Ang II interacts with cellular receptors, inducing vasoconstriction and the release of catecholamines from the adrenal medulla and presynaptic nerve endings. It also induces aldosterone secretion and sodium reabsorption. In addition, Ang II inhibits the release of renin, thereby providing feedback to the system. Consequently, Ang II acts at different levels (for example, at the level of the vasculature, sympathetic nervous system, adrenal cortex and medulla) to increase vascular resistance and increase blood pressure.

The renin-angiotensin system (RAS) can be blocked at different levels. Since renin inhibitors block PAC at a higher level than ACF inhibitors and Ang II antagonists, they have different effects on the components of PAC. After administration of the renin inhibitor, the formation of both Ang I and Ang II is blocked. Although ACE suppression only blocks Ang II formation, Ang I levels rise. Thus, Ang I is still able to convert to Ang II and other angiotensin peptides through other metabolic pathways, for example, through the chymase system.

Aliskiren (SPP100) is a low molecular weight nonpeptide antihypertensive agent (609.8). See Wood JM et al., Biochem. Biophys. Res. Commun. 308, 2003, pp. 698-705. Its mechanism of action is different from that of other commercial antihypertensive agents. Aliskiren blocks the renin-angiotensin system (ASD) at its first speed-limiting stage. In vitro, aliskiren is a potent inhibitor of human renin (IC ₅₀ = 0.6 nmol). In vivo, aliskiren administered orally (bp) or intravenously (iv) in several studies on marmoset monkeys depleted in sodium caused a complete suppression of plasma renin (ARP) activity, a steady decrease in mean arterial pressure (SBP), and a significant increase in plasma concentrations of active and total renin. In humans, plasma aliskiren concentrations rapidly increase after administration, reaching peak levels in 3-5 hours. Both C _max and AUC values increase with increasing dose, but not linearly. The half-life of aliskiren takes approximately 25 hours, and its bioavailability is approximately 2.7%.

Traditional medical approaches to the diagnosis and treatment of the disease are based only on clinical data, or in addition to them also on diagnostic testing. This generally accepted approach often leads to the choice of a treatment method that is optimal for the effectiveness of the prescribed drug therapy or to minimize the likelihood of side effects for a particular subject. A specific therapeutic diagnostic method (also known as theranostics) refers to an expanding field of medical technology that provides tests applicable for diagnosing a disease, selecting an adjusted treatment regimen, and monitoring a subject's response to treatment. That is, theranostics is applicable for predicting and evaluating the response of a particular subject to a drug, i.e. is an individualized medicine. Theranostic tests are also applicable to selecting subjects for treatment that are likely to receive a therapeutic effect as a result of treatment, or to elicit an early and objective indication of treatment effectiveness in specific subjects so that treatment can be changed with minimal delay.

Progress in pharmacogenetics, establishing correlations between responses to specific drugs and the genetic profiles of individual patients, is based on the development of new theranostic approaches. In this regard, there is a need in the art for evaluating variations in gene sequence and gene expression in different patients. 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 the drug. It follows that in this area there is a need for the identification and description of genetic mutations, for example SNPs, which can be used to identify subject genotypes associated with drug responses.

SUMMARY OF THE INVENTION

The present invention meets the needs of the art. Important correlations were established between polymorphisms in the angiotensin-converting enzyme (ACF) gene, polymorphisms in the angiotensin II receptor 2 gene type II (AGTR2) and a decrease in the clinical parameters of the mean values of diastolic and systolic blood pressure values, followed by treatment with aliskiren, used as an antihypertensive agent. These effects are not observed with irbesartan and placebo, but they are specific for treatment with aliskiren.

Thus, the present invention provides the use of aliskirn for the manufacture of a medicament for the treatment of hypertension in a group of specifically selected patients. Patients to be treated are selected into a group based on genetic polymorphisms in the biomarker genes available to the patients. Biomarker genes are the angiotensin converting enzyme (ACF) gene and the angiotensin type II receptor 2 gene (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 hypertensive individuals to treatment with aliskiren, based on the establishment of a nucleotide pair in one or more polymorphic genetic loci of the present invention.

The present invention also provides a theranostic method of treating hypertension in a particular individual. An antihypertensive agent is administered to an individual if a nucleotide pair at a polymorphic genetic locus according to the present invention indicates that the individual is responding to an antihypertensive agent. In one embodiment, the antihypertensive agent is aliskiren. Another therapeutic option for an individual is used if the nucleotide pair at the polymorphic genetic locus of the present invention indicates that the individual is not responding to an antihypertensive agent.

The present invention generally provides a method for lowering diastolic blood pressure during the daytime (DCDD) for outpatients. In a specific embodiment of the present invention, there is provided a theranostic method for lowering the average specific diastolic blood pressure (SODCD).

In addition, the present invention provides a method for lowering systolic blood pressure during the daytime for outpatients (SCDV). In a specific embodiment of the present invention, there is provided a theranostic method of lowering the average specific systolic blood pressure (SOSKD).

The present invention also provides a method for selecting an individual for inclusion in a clinical study to determine the effectiveness of an antihypertensive agent for treating hypertension. An individual may be included in the study if the genotype of the individual indicates the effectiveness of the antihypertensive agent in the treatment of hypertension of the individual. An individual may be excluded from the study if the individual’s genotype does not confirm the effectiveness of the antihypertensive agent for treating hypertension in that individual.

The present invention provides kits for practicing the methods of the present invention. The present invention also provides a method of using an angiotensin converting enzyme (ACF) gene product and an angiotensin II receptor gene type 2 gene product (AGTR2) as targets for finding new drugs.

Brief Description of the Drawing

The drawing represents histograms showing the percentage of individuals selected according to the ONP_4769 genotype that responded to treatment (responders) in all three tegaserod treatment groups combined, in the group with the highest dose of aliskiren (600 mg), in the irbesartan treatment group, and in the placebo group. In the histogram captions, the top row refers to the CT allele, and the bottom row refers to the TT allele.

DETAILED DESCRIPTION OF THE INVENTION

A retrospective pharmacogenetic analysis was performed to assess the possible relationship between genetic variation and the outcome of clinical treatment. During clinical treatment, aliskiren, administered in doses of 75, 150 or 300 mg once a day, proved to be an effective antihypertensive agent in the treatment of patients with mild to moderate hypertension, which led to a statistically significant decrease in systolic blood pressure in the daytime (SKDDV). All doses of active treatment were statistically more effective than placebo, which was reflected in a decrease in the average specific diastolic blood pressure (SODCD) at the endpoint of the clinical treatment in the group of patients selected for this treatment after 8 weeks, as well as in the treatment group according to the protocol at the end of the clinical study . Close results in reducing SDKD were achieved with the use of aliskiren at a dose of 150 mg and irbesartan at a dose of 150 mg. See example 1 below.

According to the parameter of mean specific systolic blood pressure (SOSKD) at the end point of a clinical trial, all doses of active treatment were statistically more effective than placebo. The efficacy of using aliskiren in doses of 300 and 600 mg is statistically significantly higher than the result of using placebo and irbesartan at the endpoint of a clinical trial. A similar decrease in SOSKD was achieved with the use of aliskirn at a dose of 150 mg and irbesartan at a dose of 150 mg. Doses of aliskiren 300 and 600 mg cause the most severe decrease in blood pressure. See example 1 below.

During the pharmacogenetic analysis, 48 polymorphisms were studied consisting of 12 genes of the renin-angiotensin-aldosterone (PAC) system or genes for which their participation in the regulation of blood pressure was previously established. Important relationships were observed between one polymorphism in the angiotensin-converting enzyme (ACF) gene (SNP_4769, SEQ ID NO: 1), two polymorphisms in the angiotensin II receptor gene, type 2, (AGTR2) (SNP_1445, SEQ ID NO: 2, and SNP_4795, SEQ ID NO: 3) and clinical parameters for decreasing mean diastolic and systolic blood pressure. These effects were not observed with irbesartan or placebo in the present study, presumably they are specific to aliskiren.

The nucleotide sequence of SNP_4769 (SEQ ID NO: 1) is as follows:

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

SNP_4769 is a coding SNP that changes the amino acid sequence from proline to serine in codon 32 of the ACF enzyme.

The nucleotide sequence of SNP_1445 (SEQ ID NO: 1) is as follows:

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 ATCTTTAGCT AATGTGTTTT TTTTTTTGGT TTTAAAATAA TGCTTCTAGT GAAAAAAATC ACAAAAACCT CAACACTGTA ACGTTTGAGA GCAACGGCTA TTCAGTTCGG TTAAACCGAA

SNP_1445 is an untranslated region of the mRNA of the AGTR2 gene (see Table 2B).

The nucleotide sequence of SNP_4795 (SEQ ID NO: 1) is as follows:

ssaasasaaa agsasagsag 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

SNP_4795 is present in the genomic region of the AGTR2 gene (see Table 2B). Certain objects, methods, implementations, options and properties of the present invention are described below with more or less detail for a real understanding of the present invention. In general terms, such a description provides for a new use of polynucleotide variations, SNPs, applicable for the diagnosis and treatment of subjects in need thereof. Thus, various objects of the present invention relate to polynucleotides encoding polynucleotide variations of the present invention of the ACF and AGTR2 genes. Various objects of the present invention also relate to diagnostic / theranostic methods and kits that use polynucleotide variations of the present invention to identify individuals predisposed to a disease, or to classify individuals according to their ability to respond to a drug, side effects or optimal dosage. In addition, the present invention provides methods for evaluating compounds and a computer system for collecting and analyzing data associated with polynucleotide variations of the present invention. The following are various specific embodiments of the present invention, which illustrate the above objects.

Definitions. The following are definitions of some of the terms used in the present invention. For definitions of other terms, see the U.S. Department of Energy dictionary, science department, human genome project <http://www.ornl.gov/sci/techresources/Human_Genome/glossary/>. Medical terms are presented by Chobanian et al. In Hypertension 42, 2003, pp. 1206-1252. The U.S. Cardiology Association’s definition of hypertension is available at <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 certain localization on the chromosome (at the locus) of the form 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 antibody fragments sufficient to bind an antibody fragment to a 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 (ie, side effects).

In the context of the present invention, the term "clinical study" means any study undertaken to collect clinical data on responses to a specific treatment, and includes, but is not limited to, stages I, II and III of clinical studies. Standard methods are used to determine the group of patients and include subjects in it.

In the context of the present invention, the term "effective amount" means an amount of a compound sufficient to achieve the desired therapeutic and / or prophylactic effect, for example, an amount that prevents or reduces the onset of symptoms associated with hypertension. In a preferred embodiment of the present invention, such a compound is aliskiren.

The amount of compound administered to a subject depends on the type and severity of the disease and on the condition of the individual, for example, on general health, age, gender, body weight, and drug resistance. It may also depend on the degree, severity and type of disease. The person skilled in the art is able to determine the necessary doses, depending on these and other factors. Typically, an effective amount of the compounds of the present invention, sufficient to achieve a therapeutic or prophylactic effect, ranges from about 0.000001 mg / kg body weight per day to about 10,000 mg / kg body weight per day. Preferably, the doses range from about 0.0001 mg / kg body weight per day to about 100 mg / kg body weight per day. The compounds of the present invention can be administered in combination with each other or with one or more additional drug compounds. In a preferred embodiment of the present invention, an effective amount of aliskiren is 75, 150, or 300 mg once daily.

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

In the context of the present invention, the term “gene” means a DNA segment containing all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

In the context of the present invention, the term “genotype” means a non-phase 5 ′ → 3 ′ sequence of nucleotide pairs found in one or more polymorphic sites of the locus of a pair of homologous chromosomes of an individual. In the context of the present invention, the term "genotype" refers to the full genotype or sub-genotype.

In the context of the present invention, the term "locus" means an arrangement on a chromosome or in a 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 (ie increases or decreases) the level of expression or level of biological activity of an ACF polypeptide or an AGTR2 polypeptide, respectively, in lack of modulating agent. The modulating agent may be a low molecular weight compound, a polypeptide, a hydrocarbon, a lipid, a nucleotide, or a combination thereof. The modulating agent may be an organic compound or an inorganic compound.

In the context of the present invention, the term “mutant” means any inherited wild-type change resulting from a mutation, for example a single nucleotide polymorphism. In the present description, the term "mutant" is used interchangeably with the concepts of "marker", "biomarker" and "target".

In the context of the present invention, the term "medical condition" means, but is not limited to, any condition or disease manifested as one or more physical and / or physiological symptoms for which treatment is indicated, and includes 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 "nucleotide pair" means nucleotides found in the polymorphic site of two copies of an individual's chromosome.

In the context of the present invention, the term “polymorphic site” means a locus position at which at least two different sequences are found in a group, and its highest frequency of occurrence is not more than 99%.

In the context of the present invention, the term "polymorphism" means any variant of the sequence that occurs in a population with a frequency of> 1%. A sequence variant may be present with a frequency substantially greater than 1%, for example 5%, 10% or more. Furthermore, this concept can be applied to a variant of a sequence found in an individual at a polymorphic site. Polymorphisms include nucleotide substitutions, insertions, deletions, and microsatellites, which may not necessarily lead to fixed differences in gene expression or protein function.

In the context of the present invention, the term "polynucleotide" means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without any limitation, single and double-stranded DNA, DNA representing a mixture of single and double-stranded regions, single and double-stranded RNA, RNA, representing a mixture of single and double-stranded regions, as well as hybrid molecules, DNA and RNA, which can be single-stranded or, more often, double-stranded, or a mixture of single and double-stranded areas. In addition, the term "polynucleotide" refers to three-strand regions, including RNA or DNA, or both RNA and DNA. The term "polynucleotide" also includes DNA or RNA molecules containing one or more modifications, the basis of which are DNA or RNA molecules, modified for 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 isosteres. The term polypeptide refers to both short-chain compounds, commonly called peptides, glycopeptides or oligomers, and long-chain compounds, commonly called proteins. Polypeptides may contain amino acids other than 20 amino acid encoded by the genes. Polypeptides include amino acid sequences modified either due to natural processes, for example, as a result of post-translational processing, or as a result of chemical modifications well known in the art. Such modifications are detailed in the manuals, in more detail in the monographs, as well as in a huge number of scientific publications.

In the context of the present invention, the term "SNP nucleic acid" means a nucleic acid sequence comprising a variable nucleotide, and in other respects the sequence is identical to the sequences of individuals and groups of individuals, so it exists as alleles. Such SNP nucleic acids preferably contain from about 15 to about 500 nucleotides in length. SNP nucleic acids can be part of the chromosome, or they can be an exact copy of part of the chromosome, for example, by amplification of this part of the chromosome as a result of PCR or cloning. Nucleic acids with SNPs are referred to herein simply as “SNPs”. SNP reflects the variability of the nucleotide at one position of the genome, in which two different bases are found in the human population with a perceptible frequency (i.e.> 1%). SNPs may be present in the gene or in intergenic regions of the genome. SNP probes according to the present invention are oligonucleotides complementary to SNP nucleic acid.

In the context of the present invention, the term “subject” preferably means a mammal, for example a human, and may also mean an animal, for example a domestic animal (for example, dogs, cats and others), a farm animal (for example, cows, sheep, pigs, horses and others) and laboratory animal (for example, monkeys, for example, cynomolgus monkeys, rats, mice, guinea pigs and others).

In the context of the present invention, the administration of an agent or drug to a subject or patient means an administration made either alone or by another person. It should also be borne in mind that various methods of treating or preventing diseases are of a “fundamental” nature, which means a general, but not only a general, treatment or warning in which some biological or medical significant results are achieved.

Identification and description of gene sequence variation. Due to the predominance and wide distribution of SNPs, they can be effective and important tools for the localization of genes involved in a person’s disease state. See, for example, Wang et al., Science 280, 1998, pp. 1077-1082. It is clear that the risk of developing many common diseases and the metabolism of drugs used to treat these diseases are largely dependent on the underlying genomic variations, although the effects caused by any of the options may be negligible.

SNP is called “allelic” because it contains polymorphism, and some representatives of the species may have a sequence without mutations (i.e., a natural allele), while others may have a 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 SNP is the cause of this phenotype. The association may exist solely due to the genomic proximity of SNPs and those genetic factors that are actually responsible for this phenotype, for example, SNPs and these genetic factors can be closely located. Thus, SNPs can be in non-equilibrium coupling (“NS”) with a “true” functional option. NS (also called allelic association) is observed in those cases when alleles at two different locations of the genome are associated to a greater extent than expected. Therefore, SNPs can serve as a marker that is close to the mutation causing a particular phenotype.

In the described polymorphic sites of the present invention, for convenience, the sense strand of the gene is considered. However, it will be apparent to one skilled in the art that nucleic acid molecules containing a gene can be complementary to double-stranded molecules and, therefore, a link to a specific site of the sense strand also refers to the corresponding site of the complementary antisense strand. Thus, a link can be made to the same polymorphic site in either of the two chains and the oligonucleotide can be designed to hybridize specifically any of the chains in the target region containing the polymorphic site. Therefore, the present invention also includes single-stranded polynucleotides that are complementary to the sense strand of the genomic variants described in the present invention.

Identification and description of SNPs. Many different methods can be used to identify and describe SNPs, including single-stranded conformational polymorphism (ONPP) analysis, heteroduplex analysis by denaturing high-performance liquid chromatography (HPLC), direct DNA sequencing, and computational methods. Shi et al., Clin. Chem. 47, 2001, pp. 164-172. There is a huge amount of sequence information available in the databases.

The most common current methods for typing SNPs include hybridization, extension of primers, and cleavage methods. Each of these methods should be combined with its corresponding detection system. Detection methods include fluorescence polarization (Chan et al., Genome Res. 9, 1999, pp. 492-499), luminometric detection of phosphate release (pyrosequencing) (Ahmadiian et al., Anal. Biochem. 280, 2000, pp. 103 -110), cleavage studies based on energy transfer of fluorescence resonance, HPLC and mass spectrometry (Shi, Clin. Chem. 47, 2001, pp. 164-172, US 6300076 B1). Other methods for detecting and describing SNPs are described in US patents 6297018 and 6300063.

Polymorphisms can also be detected using commercially available products, such as INVADER ™ technology (Third Wave Technologies Inc. Madison, Wisconsin, USA). In this study, the specific oligonucleotide upstream “invader” and partially overlapping downstream probe together form a specific structure upon binding to a complementary DNA template. This structure is recognized and cut at a specific site by specific cleavage enzymes, resulting in the release of the 5'-fragment of the oligonucleotide probe. This fragment then acts as an oligonucleotide “invader” with respect to synthetic secondary targets and secondary fluorescently labeled signal probes present in the reaction mixture. See also Ryan D. et al., Molecular Diagnosis 4, 1999, pp. 135-144, Lyamichev V. et al., Nature Biotechnology 17, 1999, pp. 292-296, US patents 5846717 and 6001567.

The identity of polymorphisms can also be determined by detecting base mismatches, including, but not limited to, RNase protection using ribosondes (Winter et al., Proc. Natl. Acad. Sci. USA 82. 1985, p. 7575, Meyers et al., Science 230, 1985, p. 122) and proteins that recognize erroneous mating of nucleotides, for example, E. coli mutS protein (Modrich P., Ann Rev Genet 25, 1991, pp. 229-253). In another method, variant alleles can be identified using single-strand polymorphism (CPC) conformation analysis (Orita et al., Genomics 5, 1989, pp. 874-879, Humphries et al. In the book: “Molecular Diagnosis of Genetic Diseases” under Ed. R. Elles, 1996, pp. 321-340) or denaturing gradient gel electrophoresis (DHGE) (Wartell et al., Nucl. Acids. Res. 18, 1990, pp. 2699-2706, Sheffield et al., Proc. Natl. Acad. Sci. USA 86, 1989, pp. 232-236). The polymerase-mediated primer extended method can also be used to identify polymorphisms. Several such methods have been described in the patent and scientific literature, including the genetic bit analysis method (WO 92/15712) and the ligase / polymerase-mediated genetic bit analysis method (US 5679524). Similar methods are described in WO 91/02087, WO 90/09455, WO 95/17676 and US 5302509 and 5945283. Extended primers containing polymorphism can be detected by mass spectrometry (MSS) as described in US 5605798. Another primer is an extended method is an allele-specific polymerase chain reaction (PCR) (Ruaflo et al., Nucl. Acids. Res. 17, 1989, p. 8392), Ruafio et al., Nucl. Acids Res. 19, 1991, pp. 6877-6882), WO 93/22456, Turki et al., J. Clin. Invest. 95, 1995, pp. 1635-1641). In addition, different polymorphic sites can be studied by simultaneous amplification of different regions of a nucleic acid using a set of allele-specific primers, as described in published PCT patent application WO 89/10414.

Haplotyping and genotyping of oligonucleotides. The present invention provides methods and compositions for haplotyping and / or genotyping individual genes. In the context of the present invention, the terms “genotype” and “haplotype” mean a genotype or haplotype containing a nucleotide pair or nucleotide, respectively, which are contained in one or more of the 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 of a gene. Additional polymorphic sites may be known polymorphic sites or sites subsequently discovered.

The compositions of the present invention contain nucleotide probes and primers designed for specific hybridization with one or more target regions containing or attached to a polymorphic site. The oligonucleotide compositions of the present invention are useful in methods for haplotyping and / or genotyping individual genes. The methods and compositions for locating an individual genotype or haplotype for new polymorphic sites described in the present invention are applicable for studying the effect of polymorphisms on the etiology of a disease associated with protein expression and function, studying drug targeting efficacy, predicting an individual's sensitivity to a disease associated with expression and the function of the protein, and predicting the individual's reactivity to drugs targeting the gene product.

Genotypic 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.

Genotypic oligonucleotides can hybridize to a target region located at a distance of one or more nucleotides down the chain of one or more new polymorphic sites identified in the present invention. Such nucleotides are useful in polymerase-mediated primer-extended methods for detecting one of the new polymorphisms found in the present invention and, therefore, such genotyped oligonucleotides are considered in the present invention as “extended oligonucleotide primers”.

The method of direct genotyping. described in the present invention. The genotyping method of the present invention may include isolating an individual nucleic acid molecule mixture comprising two copies of a test gene or fragment thereof, and determining the identity of the nucleic pair from one or more polymorphic sites in duplicate. It will be apparent to those skilled in the art that the two “copies” of an individual gene may be the same alleles or different. In a most preferred embodiment of the present invention, the genotyping method comprises determining the identity of a nucleotide pair at each polymorphic site. Typically, a mixture of nucleic acid molecules is isolated from a biological sample of an individual, for example, from a blood or tissue sample. Suitable tissue samples are whole blood, sperm, saliva, tears, urine, fecal matter, sweat, cheek scraping, skin and hair.

In Example 1 below, probe sets for studies of single nucleotide polymorphisms (SNPs) for the genotyping method were obtained for the ABI Assays-by-Design® platform. Livak K.J., Marmaro J., Todd J.A., Nature Genetics 9, 1995, pp. 341-342. For genotyping, according to the manufacturer's recommendation, 10 ng of genomic DNA is used.

The direct haplotype method described in the present invention. The genotyping method of the present invention may include isolating from a sample of an individual a nucleic acid molecule containing one or two copies of the test gene or its fragment, and determining the identity of the nucleotide at one or more polymorphic sites in this copy. Direct genotyping methods include, for example, CLASPER System ™ technology (US 5866404) or long-range allele-specific PCR (Michalotos-Beloin et al., Nucl. Acids. Res. 24, 1996, pp. 4841-4843). A nucleic acid can be isolated by a method that allows two copies of a gene or fragment to be separated. It will be apparent to those skilled in the art that any particular clone can only provide information on the haplotype of one of the two copies of a gene available to an individual. In one embodiment of the present invention, an individual haplotype pair is determined by identifying the phase sequence of the nucleotides at one or more polymorphic sites in each copy of the individual gene. In a preferred embodiment, the haplotype method involves identifying the phase sequence of the nucleotides at each polymorphic site of each gene copy.

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

The method of indirect genotyping using polymorphic sites in disequilibrium linkage with the target polymorphism. The identity of the alleles present in any of the new polymorphic sites of the present invention can also be determined indirectly by genotyping other polymorphic sites that are in disequilibrium linkage with such studied sites. As described above, two sites are in disequilibrium coupling if the presence of a particular variant in one site indicates the presence of another variant in the second site. Stevens JC, Mol. Diag. 4, 1999, pp. 309-317. Polymorphic sites in disequilibrium linkage with the polymorphic sites of the present invention can be localized in regions of the same gene, or in other genomic regions.

Amplification of the target gene region. Target regions can be amplified using any oligonucleotide-directed amplification method, including, but not limited to, polymerase chain reaction (PCR). (US 4965188), ligase chain reaction (LCR) (Varano et al., Proc.Natl. Acad. Sci. USA 88, 1991, pp. 189-193, published PCT patent application WO 90/01069) and oligonucleotide ligation (OL ) (Landegren et al., Science 241, 1988, pp. 1077-1080). The oligonucleotides used as primers or probes in such methods specifically hybridize to the nucleic acid region that contains or is attached to the polymorphic site. Typically, oligonucleotides contain 10-35 nucleotides, preferably 15-30 nucleotides. Most preferably, the nucleotide length is 20-25 nucleotides. The exact length of the oligonucleotide depends on many factors that are evaluated by a specialist.

Other known nucleic acid amplification methods can be used to amplify a target region, including transcription-based amplification systems (US 5130238, EP 329822, US 5169766, published PCT patent application WO 89/06700) and isothermal methods (Walker et al., Proc. Natl Acad. Sci. USA 89. 1992, pp. 392-396).

Hybridization of an allele-specific oligonucleotide with a target gene. Target region polymorphism can be assessed before or after amplification using one or more hybridization-based methods known in the art. Typically, allele-specific oligonucleotides are used in such methods. Allele-specific oligonucleotides can be used as differently labeled probe pairs, in which one component in a pair shows complete affinity for one variant of the target sequence, and the other component shows complete affinity for another variant. In some embodiments of the present invention, more than one polymorphic site can be detected using a set of allele-specific oligonucleotides or oligonucleotide pairs. Preferably, the components of such a kit have a melting point of not more than 5 ° C, more preferably not more than 2 ° C, when hybridized to detectable polymorphic sites.

Hybridization of the 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 target polynucleotide is covalently or non-covalently attached to a solid support. Attachment can be mediated, for example, by antibody-antigen, poly-L-lysine, streptavidin or avidin-biotin interactions, salt bridges, hydrophobic interactions, chemical bonds, UV crosslinking, annealing, etc. An allele-specific oligonucleotide can be synthesized directly, or on a solid support, or attached to a solid support after synthesis. Solid substrates suitable for use in the detection methods of the present invention include silicone, glass, plastic, etc., which can be designed, for example, in the form of holes (96-well plates), slides, plates, membranes, fibers, chips, cups and beads. A solid support may be processed, coated, or a derivative thereof may be prepared in order to facilitate the immobilization of an allele-specific oligonucleotide or target nucleic acid.

Determination of genotypes and haplotypes in a group and their correlation with a specific trait. The present invention provides a method for determining the frequency of a genotype or haplotype in a group. The method includes determining the genotype or haplotype of the gene present in each member of the group in which the genotype or haplotype includes a nucleotide pair or nucleotide detected at one or more polymorphic sites of the gene, and counting the frequency of occurrence of the genotype or haplotype in the group. The group may be a control group, a family group, a gender group, a population group, or a group with a specific trait (for example, a group of individuals with a particular trait being studied, for example, a medical condition or response to therapeutic treatment).

Another object of the present invention is associated with data on the frequency of occurrence of genotypes and / or haplotypes in the control population, which are used in the method of identifying associations between a trait and a haplotype or genotype. The symptom 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 studied ganotypes and haplotypes in the control group and comparing these data with the frequency of occurrence of genotypes and haplotypes in the group with a certain trait. The frequency of data for one or two groups (control and study) can be obtained by genotyping or haplotyping of each individual in groups carried out by the methods described above. Haplotypes of the studied group can be determined directly or, in another embodiment, by the method for predicting the genotype according to the haplotype described above.

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

When analyzing polymorphisms, calculations can be made to introduce corrections for a significant associative relationship, which can be detected by chance. Statistical methods applicable to the present invention are described in: Statistical Methods in Biology, 3rd ed., Bailey NTJ, 1997, Cambridge Univ. Press, in book: Waterman M.S. Introduction to Computational Biology, 2000, published by CRC Press and in the book: Bioinformatics, ed. Baxevanis A.D., Ouellette B.F.F., 2001, John Wiley & Sons, Inc.

In another embodiment of the present invention, the frequency of occurrence of the haplotype in different groups is determined in order to determine whether they are consistent with the Hardy-Weinberg equation. See book: Harti D.L. et al. Principles of Population Genomics, 3rd ed., 1997, Sinauer Associates, Sunderland, Mass.

In yet another embodiment of the present invention, statistical analysis is carried out using standard ANOVA tests with Bonferoni correction or using a boot program that repeatedly simulates the correlation of the genotype with the phenotype and calculates the significance level. The ANOVA test is used to test assumptions about whether a response is associated with or correlates with one or more characteristics or variables that are measurable. See book: Fisher L.D. & vanBelle G. “Biostatistics: A Methodology for the Health Sciences”, chapter 10, 1993, Wiley-lnterscience, New York.

In another embodiment of the present invention, for predicting a haplotype pair, the analysis comprises the following steps. First, each of the possible haplotype pairs is mapped to haplotype pairs in the control group. Typically, only one of the haplotype pairs in the control group mates with a possible haplotype pair, and this pair is present in the individual. Sometimes, only one haplotype present in control haplotype pairs is compatible with a possible haplotype pair of an individual, and in such cases, the individual is evaluated as containing the known haplotype pair of the haplotype and the new haplotype becomes derived by subtracting the known haplotype from the possible haplotype pair.

In another embodiment of the present invention, a detectable genotype or haplotype that is not in equilibrium with the haplotype or haplotype of interest can be used as a surrogate marker. A genotype that is in disequilibrium linkage with another genotype is detected if a particular genotype or haplotype of a given gene is found more often in a group that also shows a potential surrogate marker genotype, rather than in a control population. If the frequency is statistically significant, the marker genotype is an indicator of such a genotype or haplotype and can be used as a surrogate marker.

In another method for detecting a correlation between the presence of a haplotype and clinical responses, prediction models based on optimization algorithms for minimizing errors are used, one of which is a genetic algorithm. See book: “Reviews in Computational Chemistry,” ed. Lipkowitz K.B., Boyd DB, chapter 10, 1997, pp. 1-73, VCH Publishers, New York. False recall methods can be applied (book: “The Art of Scientific Computing”, chapter 10, 1992, Cambridge University Press, Cambridge), neural networks (book: Rich E., Knight K. “Artificial Intelligence”, 2nd ed., Chapter 10, 1991, McGraw-Hill, New York), standard gradient reduction methods (see Press et al., Chapter 10 above) or other global or local optimization approaches can also be applied (see . above discussion in Judson's work).

In the following examples, genotype-phenotype association studies and related analyzes were performed at SAS (Carey, North Carolina, USA) using Analyst® software. Association identification tests use well-defined genotypes as an independent variable, without assuming dominance, and various efficacy variables as dependent variables. Continuous dependent variable tests used in ANCOVA analysis and logistic regression are applied to specific dependent variables. Covariant derivatives of the analysis of the association of the genotype-phenotype are: treatment, the study area and the measurement of the baseline.

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

Correlation of a subject’s genotype or haplotype with treatment response. In preferred embodiments of the present invention, the symptoms are susceptibility to the disease, severity of the disease, stage of the disease, or response to the use of the drug. Such methods are applicable in the development of diagnostic tests and therapeutic treatment regimens for all pharmacogenetic applications, if it is possible to link the genotype and treatment results, including efficacy measurements, pharmacogenetic measurements and measurements of side effects.

In another preferred embodiment of the present invention, the sign of interest is the clinical response that is manifested in the patient, in some therapeutic treatment options, for example, a response to drug targeting or therapeutic treatment of a medical condition.

To detect a correlation between the clinical response to treatment and the genotype or haplotype, data on the genotype or haplotype are obtained based on the clinical responses of individuals in the treatment group, which in the present invention is called the "clinical group". These clinical data can be obtained by analyzing the results of a clinical trial that has already been conducted, and / or by developing and performing one or more new clinical trials.

Individuals included in the clinical group are usually divided by the presence of the studied medical condition. Such a separation of prospective patients may be based on standard medical examinations or on one or more laboratory tests. In another embodiment, the separation of patients can be made on the basis of haplotype for those situations in which there is a strong correlation between the haplotype and the susceptibility to the disease or the severity of the disease.

The therapeutic treatment under study is carried out for each individual in the study group, and the response of each individual to the treatment is measured based on one or more pre-selected criteria. It is suggested that in many cases the study group can exhibit a range of responses and that the researcher can choose the number of responder groups (for example, low, medium, high) that give different answers. In addition, the gene of each individual in the study population is genotyped and / or haplotyped, which can be done before or after treatment.

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

After obtaining clinical data and data on polymorphisms, correlations between the responses of individuals to treatment and the genotype or haplotype are revealed. Correlations can be obtained in several ways. In one method, individuals are grouped by genotype or haplotype (or haplotype pair) (this also applies to the polymorphic group) and then the mean and standard deviations of the clinical responses exhibited by the representatives of each polymorphic group are calculated.

From the analyzes described above, it follows that one skilled in the art, predicting a clinical response as a function of the available genotype or haplotype, can easily construct a mathematical model. The identification of the association between the clinical response and the genotype or haplotype (or haplotype pair) of the gene may be the basis for developing a diagnostic method for identifying those individuals who may or may not respond to treatment, or, in another embodiment, may respond at a lower level and, therefore, more intensive treatment, i.e. increased dose of the drug. The diagnostic method can be in one or several forms: for example, direct DNA testing (i.e., genotyping or haplotyping of one or more polymorphic sites in a gene), a serological test, or medical examination. The only requirement is that there should be a good correlation between the results of diagnostic testing and the underlying genotype or haplotype. In a preferred embodiment of the present invention, such a diagnostic method uses the predicted genotyping method described above.

Assessment of the subject's belonging to a specific genotypic group. It will be apparent to those skilled in the art that there may be some degree of inaccuracy in carrying out this determination of subject affiliation. Therefore, standard deviations from the levels of the control group can be applied to obtain probabilistic determination and the methods of the present invention can be applied to a wide range of possibilities based on the definitions of genotypic groups. Thus, by way of example, but not limitation, in one embodiment of the present invention, it is noted that if the measured level of the gene expression product falls to a value of 2.5 standard deviations from the average value of any of the control groups, then a particular individual may be assigned to a group of a given genotype. In another embodiment of the present invention, if the measured level of gene expression of the product falls within 2.0 standard deviations of the average value of any of the control groups, then a particular individual can be assigned to the group of this genotype. In yet another embodiment of the present invention, if the measured level of gene expression of a product falls within 1.5 standard deviations of the average value of any of the control groups, then a particular individual can be assigned to the group of this genotype. In another embodiment of the present invention, if the measured level of gene expression of the product is 1.0 or less than the average deviations of the average values of the levels of any of the control groups, then a particular individual can be assigned to the group of this genotype. Thus, this method allows you to determine, with varying degrees of probability, in which group of a particular subject should be placed and what assessment of belonging to the genotypic group, then this can help determine the risk category to which the individual can be assigned.

Correlation between clinical response and genotype or haplotype. In order to establish a correlation between the clinical response to treatment and the genotype or haplotype, it is necessary to obtain data on the clinical responses exhibited by the representatives of the treated group, which is called the “clinical group” below in the present invention. Clinical data can be obtained by analyzing the results of a clinical trial that has already been conducted, and / or clinical data can be obtained by developing and conducting one or more new clinical trials.

The standard control levels of the gene expression product obtained by the described method in different control groups can then be compared with the measured level of the gene expression product in a particular patient. Such a gene expression product may be a specific mRNA associated with such a specific genotypic group, or a gene expression product a polypeptide characteristic of a given genotypic group. Then the patient can be classified and assigned to a group of a certain genotype, based on how close the measured levels are to the control levels of this group.

A computer system for storing or displaying polymorphism data. The present invention also provides a computer system for storing and displaying polymorphism data specific for a given 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, a computer system is capable of screening haplotypes organized according to their inherent evolutionary relationships. A computer may provide any or all mathematical operations according to the methods of the present invention. In addition, the computer can execute a program that visually reflects on the screen (or on multiple screens) and on which the researcher can work to review and analyze large amounts of information regarding the gene and its genomic variations, including chromosome localization, gene structure, gene family, data gene expression, polymorphism data, genetic sequence data, as well as group clinical data (e.g., ethnographic data, clinical responses, genotypes, and haplotypes Type for one or more groups). The polymorphism data described in the present invention can be stored as a relational database (for example, an Oracle database or an ASCII set of flat files). Such polymorphism data may be stored on a computer hard disk or, for example, on a CD-ROM, or one or more information storage devices that can be installed on a computer. For example, data can be stored in one or more information storage devices that can be installed on a computer. For example, data may be stored in one or more databases connected to a computer through a network.

Nucleic acid diagnostics. Another object of the present invention provides SNP probes useful for classifying subjects by type of genetic variation. SNP probes according to the present invention are oligonucleotides that distinguish SNPs in conventional allele discrimination studies. In some preferred embodiments, the oligonucleotides of this aspect of the invention are complementary to one SNP nucleic acid allele, but not to any other SNP nucleic acid allele. The oligonucleotides of this embodiment of the present invention can distinguish SNPs in different ways. For example, under stringent hybridization conditions, an oligonucleotide of a certain length can hybridize with only one SNP, but not with others. A radioactive label or a label in the form of a fluorescent molecule may be applied to the oligonucleotide. In another embodiment, an oligonucleotide of a certain length can be used as a primer for PCR, while the 3'-terminal nucleotide is 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 SNP haplotype.

Genomic and cDNA fragments of the present invention include at least one new polymorphic site identified in the present invention, at least 10 nucleotides in length, and can reach the full length of the 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.

Kits of the present invention. The present invention provides nucleic acid and polypeptide identification kits useful for haplotyping and / or genotyping an individual gene. Such kits are useful for classifying individuals. Specifically, the present invention encompasses kits for detecting the presence of a polypeptide or nucleic acid corresponding to the marker of the present invention in a biological sample, for example, in any body fluid, including, but not limited to, serum, plasma, lymph, bile, urine, fecal matter, cerebrospinal fluid, ascites fluid or blood, as well as in samples for tissue biopsy. For example, a sample may include a labeled compound or agent capable of detecting a polypeptide or mRNA encoding a polypeptide corresponding to a marker of the present invention in a biological sample, and means for determining the amount of a polypeptide or mRNA in a sample, for example, an antibody that binds a polypeptide or oligonucleotide probe that binds with DNA or mRNA encoding a polypeptide. Kits may also include instructions for interpreting the results obtained using this kit.

In another embodiment of the present invention, there is provided a kit comprising at least two genotypic oligonucleotides packaged in separate containers. The kit may also contain other components, for example, a hybridization buffer (in which the oligonucleotides are used as probes), packaged in a separate container. In another embodiment, oligonucleotides are used to amplify the target region, and for this the kit may contain in separate containers a polymerase and a reaction buffer optimized for the primer-extended polymerase reaction, for example, in the case of PCR. In a preferred embodiment of the present invention, such a kit may further include means for selecting a DNA sample.

Antibody kits may include, for example, (1) a first antibody, for example, attached to a dense support that binds to a polypeptide corresponding to the marker of the present invention, and, optionally, (2) a second antibody that binds either to the polypeptide or to the first antibody and conjugates with a detectable label.

Kits based on oligonucleotides may include, for example, (1) an oligonucleotide, for example, a tag-detectable oligonucleotide that hybridizes to a nucleic acid sequence encoding a polypeptide corresponding to the marker of the present invention, or (2) a pair of primers suitable for amplification of a nucleic acid molecule corresponding to marker of the present invention.

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

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 thereof, that is, other natural forms of isolated polynucleotides that encode mutant polypeptides that are identical, homologous or related to those that encode polynucleotides. In another embodiment, non-natural variants can be obtained by mutagenesis or direct synthesis methods well known in the art.

Therefore, nucleic acid sequences capable of hybridizing with less stringency to any nucleic acid sequences encoding the mutant polypeptide of the present invention are within the scope of the present invention. Standard stringency conditions are described in detail in standard biological molecular cloning textbooks. See, for example, book: Sambrook, Fritsch, Maniatis "Molecular Cloning A Laboratory Manual" 2nd ed., 1989, Cold Spring Harbor Laboratory Press, ed .: DNA Cloning, volumes I and II, under the editorship of Glover D.N., 1985, book: "Oligonucleotide Synthesis", ed. Gait M.J., 1984, book: "Nucleic Acid Hybridization", ed. Hames B.D., Higgins S.J., 1984.

Characterization of the level of gene expression. Methods for detecting and measuring mRNA levels (i.e., levels of gene transcription) and levels of polypeptides - products of gene expression (i.e., levels of gene translation) are well known in the art and include the use of nucleotide microarrays and methods for detecting polypeptides, including mass spectrometers and / or methods for detecting and quantifying antibodies. See also: Strachan, T., Read A. Human Molecular Genetics, 2nd ed., 1999, John Wiley and Sons, Inc. Publication, New York.

Determination of transcription of the target gene. Determining the level of expression of a gene product in a biological sample, such as tissue or body fluid of an individual, can be done in various ways. The term "biological sample" refers to tissues, cells, biological fluids, as well as their components, obtained from the subject, as well as tissues, cells and fluids located in the subject. Many methods for detecting expression use isolated RNA. For in vitro methods, any RNA isolation methods that are not selective for mRNA isolation can be used to purify RNA from cells. See, for example, pr. "Curr. Prot. Mol. Biol. ", Ed. Ausubel et al., 1987-1999, John Wiley & Sons, New York.

In one embodiment, the expression level of the mRNA product of the target gene is determined. Methods for measuring specific mRNA level are well known in the art and include Northern blotting, reverse transcription PCR, or hybridization to an oligonucleotide sequence or microsequence. In other more preferred embodiments, the expression level can be determined by determining the expression level of the gene product, protein or polypeptide in tissue fluid or tissue samples, including, but not limited to, blood or serum. A large number of tissue samples can be easily processed using methods well known to those skilled in the art, for example, the one-step RNA isolation method described in US Pat. No. 4,843,155.

Isolated mRNA can be used for hybridization or amplification, which include, but are not limited to, southern blotting or norsen blotting, PCR, and probe sequences. One preferred diagnostic method for detecting mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize with the mRNA encoding the gene of interest. A nucleic acid probe can be, for example, a full-length cDNA or part thereof, for example an oligonucleotide whose length is at least 7, 15, 30, 50, 100, 250 or 500 nucleotides, and which is sufficient for specific hybridization under stringent conditions with mRNA or genomic DNA encoding a marker of the present invention. The present invention describes other methods suitable for diagnosis according to the present invention. Hybridization of mRNA with a probe indicates that this marker is expressed.

In one format, probes are immobilized on a solid surface and mRNA molecules are in contact with the probes, for example, this is a set of Affymetrix gene chips (Affymetrix, California, USA). One of skill in the art can easily adapt mRNA detection methods for use in detecting the level of mRNA encoded by markers of the present invention.

Other methods for determining the level of mRNA corresponding to the marker of the present invention in a sample are nucleic acid amplification, for example, using real-time PCR (PB-PCR) (experimental options are described in US Pat. No. 4,683,202), ligase chain reaction (Varana, etc., Proc. Natl. Acad. Sci. USA 88, 1991, pp. 189-193), self-sustaining sequence replication (Guatelli et al., Proc. Natl. Acad. Sci. USA 87, 1990, pp. 1874-1878), system transcriptional amplification, (Kwoh et al., Proc. Natl. Acad. Sci. USA 86, 1989, pp. 1173-1177), Q-beta replicase (Lizardi et al., Biol. Technology 6, 1988, p.1197), rolling ring type replication (US 5,854,033) or any other methods for amplifying a nucleic acid followed by detection of amplified molecules by methods known to those skilled in the art. Such detection schemes are particularly useful for detecting nucleic acid molecules if these molecules are present in a low copy number. In the context of the present invention, “amplification primers” refers to a pair of nucleic acid molecules that can anneal 5 'or 3' regions of a gene (plus and minus chains, respectively, or vice versa) and contain a short region between them. Typically, amplification primers are about 10-30 nucleotides in length and flank a region of about 50-200 nucleotides in length.

Real-time quantitative polymerase chain reaction (PB-PCR) is one way of assessing the levels of gene expression, for example, the genes of the present invention, for example, containing SNPs and the polymorphisms under study. RV-PCR uses RNA reverse transcriptase to catalyze DNA strand synthesis on RNA strands, including mRNA strands. Generated DNA can be specifically detected and quantified, and this process can be used to determine the levels of specific types of mRNA. One such method is the TAQMAN® method (PE Applied Biosystems, Foster City, California, USA), which uses the 5'nuclease activity of AMPLITAQ GOLD ™ DNA polymerase to cleave a specific probe form during PCR. This applies to the TAQMAN ™ probe. See Luthra et al., Am. J. Pathol. 153, 1998, pp. 63-68, Kuimelis et al., Nucl. Acids Symp.Ser. 37, 1997, pp. 255-256, and Mullah et al., Nucl. Acids Res. 26, 1998, pp. 1026-1031. During the reaction, probe cleavage separates the reporter dye and the dye dye, which leads to an increase in the fluorescence of the reporter. The accumulation of PCR products is detected directly by the appearance of reporter dye fluorescence. Held et al., Genome Res. 6, 1996, pp. 986-994. An increase in the number of starting copies of nucleic acid targets leads to a more rapid significant increase in the observed fluorescence. See Gibson, Heid, Williams et al., Genome Res. 6, 1996, pp. 995-1001.

Other technologies for measuring transcription status in a cell lead to the creation of pools of restriction fragmentation of limited complexity for analysis by electrophoresis, for example, by methods combining double restriction enzyme digestion with phasing of primers (see, for example, EP 0534858 A1), or by methods of selecting restriction fragments with sites, tightly adjacent to a particular end of mRNA (see, for example, Prashar, Weissman, Proc. Natl. Acad. Sci. USA 93, 1996, pp. 659-663).

Other methods statistically select cDNA pools, for example, by sequencing a sufficient number of bases, for example 20-50 bases, in each of the many cDNA molecules to identify each cDNA molecule, or by sequencing short labels, for example, 9-10 bases long, which are formed at known positions relatively specific mRNA end of the metabolic pathway. See, for example, Velculescu, Science 270, 1995, pp. 444-487. The cDNA levels in the samples are calculated and determined for each type of cDNA mean, arithmetic mean and standard deviation using standard statistical methods known to experts. Norman T.J. Bailey in: Biology, 3rd ed., 1995, Cambridge University Press.

Identification of polypeptides. Methods of immunological detection. The expression of the protein encoded by the genes of the present invention can be detected using a probe with a detectable label, or the label is subsequently applied. The term “labeled” with respect to a probe or antibody refers to direct labeling by combining with a probe or antibody, i.e. due to physical connection, as well as indirect labeling of the probe or antibody due to reaction with another reagent labeled directly. Examples of indirect labeling include detecting a primary antibody using a fluorescently labeled secondary antibody and a DNA probe labeled at the end with biotin so that it can be detected by fluorescently labeled streptavidin. Typically, a probe is an antibody that recognizes an expressed protein. A variety of formats can be used to determine if samples contain a target protein that binds to a given antibody. Immunoassay methods useful for identifying the target polypeptides of the present invention include, but are not limited to, dot blotting, western blotting, protein chips, competitive and non-competitive protein binding, enzyme-linked immunosorbent assay (ELISA). ,

immunohistochemistry methods, fluorescence activated cell sorting (FACS) separation, and other commonly used and widely described methods in the patent and scientific literature, including many commercial methods. An experienced professional can easily adapt known protein / antibody detection methods for use to determine if cells express the marker of the present invention and the approximate concentrations of a particular expressed polypeptide product in blood or other body tissues. Proteins of individuals can be isolated by methods well known to specialists in this field of technology. Applied protein isolation methods, for example, may be those described in: Harlow, Lane, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

In order to obtain antibodies to a protein encoded by one of the described genes, it is possible to immunize animal hosts by injection of a polypeptide or part thereof. Such host animals 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 host, they include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels, such as aluminum hydroxide, surfactants, such as lysolecithin, pluronic polyols , polyanions, peptides, oil emulsions, saucer mollusk hemocyanin and dinitrophenol, as well as potentially useful human adjuvants, for example, the Calmette Guerin vaccine strain (BCG) and Corynebacterium parvum.

Monoclonal antibodies (Mant) are homogeneous populations of antibodies to a particular antigen and can be obtained by a method involving the production of antibody molecules using cultured cell lines. Such methods include, but are not limited to, the hybridoma method of Kohler and Milstein, Nature, 256, 1975, pp. 495-497, US patent 4376110, the method of hybrid B-cell human Kosbor and others, Immunol. Today 4, 1983, p. 72, Cole et al., Proc. Natl. Acad. Sci. USA 80, 1983, pp. 2026-2030 and the EBV hybridoma method of Cole et al., In: Monoclonal Antibodies and Cancer Therapy, 1985, Alan R. Liss, Inc., cc.77-96.

In addition, methods developed to produce “chimeric antibodies” can be used (see Morrison et al., Proc. Natl. Acad. Sci. USA 81, 1984, cc.6851-6855, Neuberger et al., Nature 312, 1984 , cc.604-608 and Takeda et al., Nature 314, 1985, cc.452-454) based on splicing the genes of a mouse antibody molecule with a certain antigenic specificity with the genes of a human antibody molecule with a certain biological activity. A chimeric antibody is a molecule in which its various parts are derivatives of molecules of different animal species, for example, having a variable or supervariable fragment derived from a mouse Mant, and a stable fragment of a human immunoglobulin.

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

Methods applicable for the preparation of “humanized antibodies” can be adapted to produce antibodies to proteins, fragments thereof, or derivatives thereof. 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 methods, for example, Western blotting or immunofluorescence methods that detect protein expression. In such methods, it is usually desirable to immobilize either the antibody or proteins on a solid basis. Acceptable solid bases or carriers are any substrates capable of binding an antigen or antibody. Well-known bases or carriers include: glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, gabbro and magnetite.

To simplify the detection, the ELISA sandwich method is applicable, and many of its variants are known, each of which can be used in the present invention. In the context of the present invention, the “sandwich” method refers to all variations of the method based on the application of two sides. Both immunofluorescence and immunometric EIA methods are widely used in the art. However, other reporter molecules, for example radioisotopes, chemoluminescent or bioluminescent molecules, can also be used. It is obvious to a specialist how to vary the method, adapting it to specific conditions.

Monitoring of proteins of the whole genome, i.e. “Proteomics” can be accomplished by constructing a microarray in which the binding sites include immobilized, preferably monoclonal antibodies specific for the plurality of proteins encoded by the cellular genome. Preferably, antibodies are available against a significant fraction of the encoded proteins, or at least those proteins that are significant for testing or reinforcing the biological network model under study. It has been noted above that methods for producing monoclonal antibodies are well known. See, for example, Harlow, Lane in: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988, Cold Spring Harbor, New York, USA). In a preferred embodiment of the present invention, monoclonal antibodies are generated against synthetic peptide fragments designed based on the genomic sequence of a cell. Cell proteins are contacted 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 the art and typically involves isoelectric focusing along with the first measurement obtained by SDS-PAGE electrophoresis, along with the second measurement. See, for example, Hames et al. In: Gel Electrophoresis of Proteins: A Practical Approach, IRL Press, 1990, New York, USA), Shevchenko et al., Proc. Natl. Acad. Sci. USA 93, 1996, pp. 14440-14445, Sagliocco et al., Yeast 12, 1996, pp. 1519-1533, Lander, Science 274, 1996, pp. 536-539.

Identification of polypeptides. Mass spectroscopy. The identity and expression level of the target polypeptide can be determined by mass spectroscopy. The methodology based on mass spectroscopy is applicable for the analysis of the selected target polypeptide, as well as for the analysis of the target polypeptide in a biological sample. Mass spectrometry methods for analyzing a target polypeptide include: ionization methods, for example, but the methods are not limited to these methods, matrix assisted laser desorption / ionization (MALDI), continuous or pulse electrospray ionization (electrospray ionization (ESI) and related methods, such as ionospray or thermal spray, and massive cluster impact (MCI). Such ion sources can be selected by detection methods, which include the linear or nonlinear reflection method of measuring the time of flight of ions (TOF), the method of single or multiple quadrupole, the method of single or multiple magnetic sector, the ion-cyclotron resonance method with Fourier transform signal (Fourier transform ion cyclotron resonance - FTICR), ion traps and combinations thereof, e.g. ion trap / TOF. For ionization, various matrix / wavelength combinations (e.g., MALDI) or solvent combinations (e.g., ESI) can be used.

For mass spectroscopy, the target polypeptide can be dissolved in a particular solution or reagent system. The choice of a solution or system of reagents, for example, an organic or inorganic solvent, may depend on the properties of the target polypeptide and mass spectroscopy method and is based on methods well known in the art. See, for example, the description of the MALDI method by Vorm et al., Anal. Chem. 61, 1994, p. 3281 (1994), and the ESI method in Valaskovic et al., Anal. Chem. 67, 1995, p. 3802. Peptide mass spectroscopy is also described in PCT International Application No. 93/24834 and in US Pat. No. 5,792,664. The solvent is selected so as to minimize the risk of degradation of the target polypeptide due to the energy generated by evaporation. The risk of destruction can be reduced, for example, by immersing a sample in a matrix. For this, a matrix can be used, which is an organic compound, for example sugar, for example pentose or hexose, or a polysaccharide, for example cellulose. Such compounds are thermolytically destroyed to CO ₂ and H ₂ O in such a way that no residues of molecules capable of entering into chemical reactions arise. The matrix can also be an inorganic compound, for example ammonium nitrate, which is destroyed almost without residue. The use of these and other solvents is known to those skilled in the art. See, for example, US Pat. No. 5,062,935. A mass spectroscopy method has been described in J. Phys. Chem. 88, 1984, pp. 4451-4459 and PCT application WO 90/14148, and current methods of use are summarized in review articles. See Anal. Chem. 62, 1990, pp. 82-889 and Spectroscopy 4, 1992, pp. 10-18.

The mass of the target polypeptide determined by mass spectroscopy can be compared with the mass of the corresponding known polypeptide. For example, if the target peptide is a mutant protein, the corresponding known polypeptide may be the corresponding non-mutant protein, for example, a wild-type protein. Using ESI, the molecular weights of the sample in which they are contained in pentomolar amounts can be very accurately determined due to the presence of many ionic peaks that can be used to determine the mass. Submolecular protein levels are detected, for example, using ESI mass spectroscopy (Valaskovic et al., Science 273, 1996, pp. 1199-1202) and MALDI (Li et al., J. Am. Chem. Soc. 118, 1996, ss. 1662-1663).

Matrix assisted laser desorption ionization method (MALDI). The level of the target protein in a biological sample, such as body fluid or tissue sample, can be measured by mass spectroscopy methods, including, but not limited to, methods known in the art: matrix desorption laser ionization method (MALDI), measurement method time-of-flight (MALDI-TOF-MS), surface-enhanced time-of-flight laser mass spectroscopy (SELDI-TOF), which are described in detail below. Methods for implementing MALDI are well known to those skilled in the art. See Juhasz et al., Analysis, Anal. Chem. 68, 1996, pp. 941-946, as well as US patents 5777325, 5742049, 5654545, 5641959, 5654545 and 5760393 for descriptions of MALDI and delayed extraction protocols. Numerous methods for enhancing dissolution are also known. MALDI-TOF-MS have been described by Hillenkamp et al. In: Biological Mass Spectrometry, ed. Burlingame, McCloskey, 1990, Elsevier Science PubL, Amsterdam, pp. 49-60.

Various marker detection methods using mass spectroscopy can be applied. See book: “Bordeaux Mass Spectrometry Conference Report”, ed. Hillenkamp, 1988, pp. 354-362, book: Bordeaux Mass Spectrometry Conference Report, ed. Karas, Hillenkamp, 1988, cc. 416-417, Anal. Chem. 60, 1988, cc. 2299-2301, Biomed. Environ. Mass Spectrum 18, 1989, cc. 841-843. The use of laser beams in TOF-MS is shown, for example, in US Pat. Nos. 4,694,167, 4,686,666, 4,295,046 and 5,045,694, the references to which are incorporated by reference herein in their entirety. Other methods of mass spectroscopy allow the successful evaporation of large molecular weight biopolymers without fragmentation and allow the analysis of a large number of biological macromolecules using mass spectrometry.

Surface-enhanced time-of-flight laser mass spectroscopy (SELDI). Other techniques using new compositions of probe elements of mass spectroscopy with surfaces that allow the probe element to actively bind in a trap and accumulate specific analytes are described under the name Affinity Mass Spectrometry (AMS). See patents for SELDI method: US 5719060, 5894063, 6020208, 6027942, 6124137 and publication of patent applications US 2003/0003465. Several types of new mass spectroscopic probe elements have been described with a Surfaces Enhanced for Affinity Capture (SEAC) surface reinforced trap. See Rapid Commun. Mass Spectrom. 7, 1993, cc. 566-580. SEAC probe elements have been successfully used to reconstruct and bind biopolymers of different classes, especially proteins, using knowledge of surface protein structures and biospecific molecular recognition. Immobilized affinity trap devices on the surface of a mass spectrometric element 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 SELDI method, there are three different methodological options: (1) Surfaces Enhanced for Neat Desorption (SEND), in which the surfaces of the probe element, i.e. presenting the sample means, designed in such a way that they contain energy absorbing molecules (Energy Absorbing Molecules - EAM) instead of the "matrix" to facilitate the desorption / ionization of analytes added directly (purely) to the surface. (2) A variant of the SEAC method in which the surfaces of the probe elements, i.e. sample-presenting agents are designed in such a way that they contain a specific chemical and / or specific biological trap to facilitate either specific or non-specific attachment or adsorption (docking or binding) of analytes to the probe surface using various mechanisms (mainly non-covalent). (3) Surfaces Enhanced for Photolabile Attachment and Release (SEPAR) surface-enhanced for photolabile attachment and release, in which the surfaces of the probe element, i.e. sample presenting agents are designed to contain one or more types of cross-linked chemically defined molecules that serve as covalent binding agents. Chemical characteristics that determine the type and number of attachment points of photolabile molecules between SEPAR presenting agents (i.e., the surface of the probe element) and the analyte (e.g., protein) may include any or several of the various residues or chemical structures in the analyte ( for example, His, Lys, Arg, Tur, Phe, and Cys residues in the case of proteins and peptides).

Other objects of the present invention with biological activity. In various embodiments of the present invention, biological objects or mixtures of objects can be measured in order to obtain a drug and metabolic responses. The activities of proteins that are significant for describing cellular function can be measured, and embodiments of the present invention can be based on such measurements. Measurements of activity can be performed using any functional, biochemical or physical methods applicable to the particular activity described. If the activity is chemical transformation, the cellular protein can come into contact with natural substrates, and the rate of transformation is measured. If the manifestation of activity involves the association of multidimensional units, for example, the association of an activated DNA binding complex with DNA, the amount of associated protein or secondary association sequences, for example, the amount of transcribed mRNA, can be measured. In addition, if only functional activity is known, for example, cell cycle control, a manifestation of function may be recorded. If the function is known and measurable, changes in protein activity generate response data measured by the methods of the present invention. In other and non-limiting embodiments of the present invention, response data may be generated based on the assessment of various objects of the biological state of the cell. Response data can be extracted, for example, from changes in large quantities of certain proteins and changes in the activities of certain proteins.

The following example is provided to more fully describe preferred embodiments of the present invention. This example is not intended to limit the scope of the present invention set forth in the claims.

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

Introduction and summary. A retrospective pharmacogenetic analysis is performed to assess the possible relationship between genetic variation and the outcome of clinical treatment. Specifically, 49 polymorphisms are examined in 12 genes of the renin-angiotensin-aldesterone system (RAS) or in genes for which it was previously established that they are involved in the regulation of blood pressure. Significant associations are observed between one polymorphism in the angiotensin-converting enzyme (ACF) gene, two polymorphisms in the angiotensin II receptor gene, type 2 (AGTR2), and clinical parameters for reducing the average established diastolic and systolic blood pressure. These effects are not found with the introduction of irbesartan and placebo, in this analysis they are more likely to be specific for treatment with aliskiren.

Clinical study. A multicenter, randomized, double-blind, parallel-group clinical trial is conducted to examine the efficacy and safety of aliskiren compared to placebo in a mild to moderate hypertension patient group. The study is carried out for eight weeks, i.e. the end point of a clinical trial occurs after eight weeks of this particular study. The main objective of the study is to determine the effect of aliskiren in doses of 150 mg, 300 mg and 600 mg in lowering blood pressure and comparing this effect with the effect of irbesartan in a dose of 150 mg and placebo in a group of patients with mild to moderate hypertension. Demographic indicators are generally compared across all groups. Most patients are Caucasian, and most patients are under 65 years old with an average age of 50 years. The distribution of men and women in the study is approximately the same.

The first measurable performance indicator is the change in SODKD (a decrease in the value of SODKD relative to the base level). The second measurable performance indicators are the change in SOSKD (decrease in SOSKD relative to the baseline), the proportion of responders, the decrease in plasma renin (ARP) activity and the increase in active plasma renin (AKTR) relative to the baseline.

Regarding primary efficacy, paired comparisons show that all doses of active treatment were statistically higher compared with placebo regarding the reduction in mean specific diastolic blood pressure (SODCD) at the 8-week endpoint in the treatment group and in the control point of the control group. Similar results in reducing SODKD are achieved with a dose of 150 mg aliskiren and 150 mg irbersartan. Doses of aliskiren 300 and 600 mg cause the greatest decrease, but its value does not exceed the reduction values observed when using a dose of 600 mg.

Regarding secondary efficacy regarding mean specific systolic blood pressure (SOSCD), all doses of active treatment are statistically higher compared to placebo at the endpoint of the study. After 8 weeks, 300 and 600 mg aliskiren was statistically higher compared to placebo irbesartan at the endpoint. Similar results in reducing SOSKD reach a dose of 150 mg aliskiren and 150 mg irbersartan. Doses of aliskiren 300 and 600 mg cause the greatest reductions, but these reductions did not increase at a dose of 600 mg.

The proportion of successful responders (expressed as a decrease in SODCD <90 mm Hg or ≥ 10 mm Hg relative to the baseline or a decrease in SASCD <140 mm Hg or ≥ 20 mm Hg relative to the baseline) shows that all effective treatment regimens at the endpoint of the study for groups treatments show results that are statistically higher than placebo at the treatment endpoint. The results of SODCD at the treatment endpoint are 59-67% of respondents in the aliskiren treatment groups versus 56% for irbesartan and 38% for placebo. The results of SOSCD at the treatment endpoint are 57-68% of respondents in the Aliskiren treatment groups versus 59% for irbesartan and 36% for placebo. During treatment, dose-dependent increases in renin activity in aliskiren groups are observed (20.8, 29.9 and 93.6 U / ml for doses of aliskiren of 150, 300 and 600 mg, respectively). Aliskiren at a dose of 150 mg leads to a larger average increase in renin compared with irbesartan at a dose of 150 mg (11.2 U / ml). This is consistent with the mechanism of action of renin inhibitors.

Patients participating in a clinical trial are first asked for consent to participate in a pharmacogenetic analysis. Blood samples of all consenting patients are collected at different locations and then sent to Covance (Indianapolis, Indiana, USA). The genomic DNA of each patient was extracted from the blood by the Covance method using the PUREGENE ™ DNA Isolation Kit (D-50K) DNA isolation kit (Gentra, Minneapolis, Minnesota, USA). Ultimately, 511 patients were genotyped, which are approximately equal shares for each group.

Pharmacogenetic analysis of objects and research plan. A retrospective pharmacogenetic analysis is performed on all patients who agreed to provide samples for pharmacogenetic studies. The main goal is to identify the association between genetic variation and variations in the effectiveness of treatment with aliskiren, which is evaluated primarily by SODKD and secondly by SOSKD and the proportion of responders.

A methodological approach using candidate genes is used to select 48 polymorphisms in 12 genes of the renin-angiotensin system (RAS) or in genes previously associated with blood pressure regulation (Table 1). All available samples are genotyped to identify each SNP. The association is then detected by the method described below.

Table 1 Candidate genes selected for pharmacogenetic analysis Gene designation Gene name RENBP renin binding protein REN renin ACE angiotensin I converting enzyme (peptidyldipeptidase A) 1 ACE2 angiotensin I converting enzyme (peptidyldipeptidase A) 2 AGT angiotensinogen AGTR1 angiotensin II receptor type 1 AGTR2 angiotensin II receptor type 2 ABCC2 / MRP2 ATP-binding cassette, subfamily C (CFTR / MRP), representative 2 AGTRAP angiotensin receptor II-associated protein CYP11B2 cytochrome P450, family 11, subfamily B, polypeptide 2, aldosterone synthase TGFB1 transforming growth factor beta 1 NOS3 / eNOS nitric oxide synthase 3 (endothelial cells)

Genotyping. Studies of single nucleotide polymorphisms (SNPs) are carried out using information from the public dbSNP database and the patented Celera / ABI database. Genotyping probe sets are available for ABI's Assays-by-Design® platform. Livak KJ, Marmaro J, & Todd JA, Nature Genetics 9: 341-2 (1995). Genotyping is carried out using 10 ng of genomic DNA according to the manufacturer's recommendations.

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

Table 2A Genotyped polymorphisms pg locus id Gene Rs number Description 5284 ABCC2 hCV11305436 Intron 4786 ABCC2 rs2273697 A> G I417V 4783 ABCC2 rs2756104 Intron 4784 ABCC2 rs2756109 Intron 4785 ABCC2 rs3740066 T> C I1324I 4782 ABCC2 rs717620 untranslated 4797 ABCC2 rs8187710 A> G Y1515C 5286 ACE hCV1247681 Intron 4766 ACE rs4293 Intron 4769 ACE rs4317 C> T P32S 4767 ACE rs4329 Intron 4768 ACE rs4362 C> T F1129F 400 ACE rs4364 S712R 1345 ACE insertion / deletion 4746 ACE2 rs2285666 Intron 1669 ACE2 rs879922 Intron 4773 AGT rs1926723 Intron 1667 AGT rs4762 T> S M207T 2 AGT rs699 C> T T268M 4772 AGT rs943580 Genomic 4796 AGTR1 rs2638362 Intron 4780 AGTR1 rs3772616 Intron 411 AGTR1 rs5182 C> T L191L

Table 2B Genotyped polymorphisms pg locus id Gene Rs number Description 5287 AGTR2 hCV1841569 1445 AGTR2 rsl403543 untranslated 186 AGTR2 rs5193 untranslated 4795 AGTR2-LD rs4497127 Genomic 5288 AGTRAP hCV516817 Intron 4787 AGTRAP rs4073574 Genomic 4789 CYP11B2 rs4539 G> A R173K 575 CYP11B2 rs4544 C> T T339I 4788 CYP11B2 rs4545 A> G S435G 3541 CYP11B2 rs6431 Intron 4792 NOS3 rs1007311 Intron 482 NOS3 rs1799983 G> T E298D 4791 NOS3 rs1800779 Genomic 4464 NOS3 rs1800780 Intron 4793 NOS3 rs891512 Intron 5292 REN rs11571092 Intron 1513 REN rs1464816 Intron 4771 REN rs3730103 Intron 4794 REN rs6704321 C> G P403A 4770 RENBP rs2269371 G> A G274D 2407 RENBP rs2269372 Intron 1676 RENBP rs762656 Genomic 5285 TGFB1 hCV11707868 Intron 4790 TGFB1 rs2241717 Intron 4798 TGFB1 rs8105161 Intron

Statistical analysis. Genotype-phenotype relationship studies and other related assays are performed at SAS (Saga, North Carolina, USA) using Analyst®.

Association identification tests use well-defined genotypes as an independent variable, without assuming dominance, and various efficacy variables as dependent variables. Continuous dependent variable tests used in ANCOVA analysis and logistic regression are applied to specific dependent variables. Covariant derivatives of the analysis of the association of the genotype-phenotype are: treatment, the study area and the measurement of the baseline.

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

Angiotensin-converting enzyme (ACF). Angiotensin-converting enzyme (ACF) converts Ang I into the active Ang II octapeptide. ONP_4769 is genotyped in patients along with six other polymorphisms in the key RAS enzyme. A significant association is observed between SNP_4769 and the variable of primary efficiency of reducing SODKD relative to the base level (p = 0.0058) and the secondary variable - the share of responders (p = 0.001). For another secondary variable - a decrease in SOSKD relative to the base level, no significant association is observed, only a tendency is manifested (p = 0.0745).

When repeating the ANCOVA analysis for each treatment group, an association was observed only for patients in the aliskiren treatment groups, but not in the irbesartan or placebo treatment groups. There is no significant association between any SNP and secondary parameters: plasma renin activity (ARP) and active renin (AKTR).

The specific effects of aliskiren are shown in Table 3.

Table 3 The effect of variations in ACF and AGTR2 on SODKD, SOSKD and the proportion of responders in the treatment groups aliskiren ONP_4769 ONP_1445 ONP_4795 TT ST SS AA AG Gg Gg GA AA N 278 fourteen 0 106 69 118 106 72 115 SODKD decrease (mmHg) -11.1 -4.7 NA -11.2 -13.2 -9.3 -11.1 -13.3 -9.0 R 0.0058 * 0.0074 * 0.0022 * SASKD reduction -13.7 -6.2 NA -13.9 -15.7 -11.8 -13.6 -16.3 -11.5 R 0.0384 * 0.1360 0.0479 * TT ST SS AA AG Gg Gg GA AA Share of respondents 69% fourteen% NA 74% 71% 58% 74% 73% 56% R 0.0024 * 0.1399 0.0382 *

SNP_4769 is an encoding SNP that changes the composition of the amino acid sequence in codon 32, replacing proline with serine in the ACF enzyme. ACF I / D polymorphism (the presence or absence of a repeating sequence of 287 bp Alu polymorphism in intron 16) is associated with regulation of the level and activity of ACF and its branches in the PAC element. Rigat B. et al., J. Clin. Invest. 86, 1990, pp. 1343-1346.

Angiotensin II receptor type 2 (AGTR2). AGTR2 encodes an angiotensin II receptor, type 2. An angiotensin II receptor, type 2, is the target of several antihypertensive drugs. Ang II signals through binding to the type I receptor induce vascular contraction and increased blood pressure. It was found that receptor II, type 2, inhibits ACF activity and attenuates the action through type 1 receptor, causing vasodilation. Hunley T.E. et al., Kidney Int. 57, 2000, pp. 570-577 (2000), Ichiki, T. et al., Nature 377, 1995, pp. 748-750.

A significant association is observed between the SNP of the AGTR2 gene (SNP_1445 and SNP_4795) and the variable primary efficacy of reducing SODCD relative to the baseline (p = 0.0078 and 0.0004, respectively). SNP_4795 also shows a significant association with the secondary variable reducing SOSKD relative to the base level (p = 0.0245), but not with another secondary parameter - the proportion of responders. ONP_1445 meanwhile did not show a significant association with secondary parameters. There was no significant relationship between any SNP and secondary parameters, namely plasma renin activity (ARP) and active renin (AKTR).

In the ANCOVA analysis, an association was established for each group only in the treatment groups of aliskiren, and not in the treatment groups of irbesartan or placebo. When repeating the ANCOVA analysis for each treatment group, an association was observed only for patients in the aliskiren treatment groups, but not in the irbesartan or placebo treatment groups. The effect of the genotype in patients treated with aliskiren is shown above in Table 3.

These two SNPs are not encoding. SNP_1445 is located in the untranslated region of the AGTR2 gene, and SNP_4795 is in the genomic region with non-equilibrium linkage with the AGTR2 gene.

Discussion. In short, one variant in the ACF gene and two variants in the AGTR2 gene show significant association with the primary SODCD parameter. Option ACF and one option AGTR2 also shows significant association with secondary parameters - SOSKD and the proportion of responders. The second variant of AGTR2 does not show a significant association, but only shows such a tendency with respect to secondary parameters. This genotype effect is observed only in groups in which patients were treated with aliskiren, but not in the treatment groups with ibersantan or placebo. Therefore, the effect described above is a pharmacogenetic effect specific for aliskiren.

The effect of the ACF variant is especially interesting, since the difference between TT and CT genotypes seems to be very significant (decrease in SODCD from 11.1 to 4.7 mm Hg, decrease in SOSCD from 13.7 to 6.2 mm Hg, the proportion of responders increases from 14% up to 69%).

The homozygous SS genotype is absent in patients; therefore, the frequency of occurrence of this allele is much lower than the frequency for SNPs according to databases of 0.1.

When analyzing the response to treatment with aliskiren depending on the genotype, the proportion of the response to the TT genotype increases to about 70%. Therefore, this option can help place aliskiren on the mass market of antihypertensive drugs, which is facilitated by the spread of the genotype in the main part of the population (approximately 80% in the allelic frequency in the SNP database, but may even be higher, which follows from this analysis), having the best percentage responders (figure 1). It may also help to select possible “better” responders in further research.

It is very likely that the two SNPs in the AGTR2 gene are in disequilibrium linkage.

These reporter SNPs do not show associations with baseline SODKD and SOSKD values, once again confirming that the genetic effect is an aliskiren-associated pharmacogenetic effect.

Example 2. Clinical pharmacogenetic analysis of a clinical trial A2203

Introduction and summary. A retrospective pharmacogenetic analysis is performed to assess the potential relationship between genetic variation and the outcome of clinical treatment. See Example 1. Subsequently, the results of another clinical trial (A2203) are evaluated for replication analysis. Specifically, 48 polymorphisms were studied in 12 genes of the renin-angiotensin system (RAS) or in genes for which their participation in the regulation of blood pressure was previously established.

In Example 1, there is a significant association between one polymorphism in the angiotensin-converting enzyme (ACF) gene, two polymorphisms in the angiotensin II receptor gene, type 2 (AGTR2), and the clinical parameters for lowering the average specific diastolic and systolic blood pressure. Such effects were not found with irbesartan and placebo. In addition, it was found that the senseless variant of ACF (Pro32Ser), associated with a lower response, has a higher frequency of occurrence of the C (Pro) allele among representatives of the Negroid race, compared with representatives of the Caucasian race (19/154 versus 2/790).

In this example, all four patients with a serine residue (C allele) belong to the Negroid race. Due to the extremely small number of patients with C allele, it is impossible to conduct a study with these SNPs in the same model as in Example 1. In addition, the association of two SNPs in AGTR2, established in Example 1, was not reproduced.

The clinical trial in this example (A2203) was randomized and performed using a double-blind, multicenter, multicenter, placebo-controlled method and the use of parallel groups to evaluate the efficacy and safety of combinations of aliskiren and valsartan compared to monotherapy with individual components, as well as a combination of valsartan and hydrochlorothiazide (GCTZ), hypertensive patients.

The main objectives of this example are to evaluate the effect of lowering blood pressure when using a combination of aliskiren and valsartan (75/80 mg, 150/160 mg and 300/320 mg) compared with monotherapy with the individual components administered over a period of 6 weeks to patients with a clinical mean diastolic blood pressure ([SODCD] ≥ 95 mm Hg and <110 mm Hg), as well as an assessment of the effect on lowering blood pressure when using only one aliskiren 75 mg, 150 mg and 300 mg compared with placebo administered for 6 weeks. ntam clinically definite average diastolic blood pressure ([SODKD] ≥ 95 mm Hg and <110 mm Hg). Treatment groups are mainly compared with demographic and basic parameters, with an average age of 56 years, 56% of men and 6.8% of the black race.

The primary efficacy variable, a change relative to the baseline level of SODCD during aliskiren therapy compared with placebo, is statistically significant in the group of patients receiving 300 mg aliskiren. The average decrease in blood pressure for the two monotherapy options is the same as in previous studies. However, the placebo effect is higher than expected and higher than in previous studies of aliskiren. The decrease in blood pressure using a combination of aliskiren and valsartan was less than with hydrochlorothiazide (GCT) 12.5 mg / valsartan, and additivity was less with a maximum dose of aliskiren 300 mg / valsartan 320 mg than with less severe combinations.

Pharmacogenetic analysis of objects. A retrospective pharmacogenetic analysis includes all patients who agreed to provide samples for pharmacogenetic studies. The main task is to identify associations between genetic variation and variations in the effectiveness of treatment with aliskiren, which are mainly assessed by SODKD, as well as by SOSKD and the proportion of responders.

The “candidate” gene approach is used to select 48 polymorphisms in 12 PAC genes or in genes for which a relationship with blood pressure regulation has been established previously (Table 4). All available samples are genotyped for each SNP. Then perform associative studies as described in example 1.

Table 4 Selection of candidate genes for pharmacogenetic analysis Gene designation Gene name RENBP renin binding protein REN renin ACE angiotensin I converting enzyme (peptidyldipeptidase A) 1 ACE2 angiotensin I converting enzyme (peptidyldipeptidase A) 2 AGT angiotensinogen AGTR1 angiotensin II receptor type 1 AGTR2 angiotensin II receptor type 2 ABCC2 / MRP2 ATP-binding cassette, subfamily C (CFTR / MRP), representative 2 AGTRAP angiotensin II receptor - associated block CYP11B2 cytochrome P450, family 11, subfamily B, polypeptide 2, aldosterone synthase TGFB1 transforming growth factor beta 1 NOS3 / eNOS nitric oxide synthase 3 (endothelial cells)

Samples. Patients participating in clinical trial A2201 (see Example 1) and clinical trial A2203 are separately asked for consent to participate in a pharmacogenetic analysis. Blood samples of patients who consented to participate are collected at selected sites and then sent to Covance (Indianapolis, Indiana, USA). The genomic DNA of each patient was extracted from the blood using the Covance method using the PUREGENE ™ DNA isolation kit (D-50K) (Gentra, Minneapolis, Minnesota) and sent to the NIBR for genotyping. Ultimately, 511 patients participating in the clinical study in Example 1 (A2201) genotypically in approximately equal proportions for each of the groups. Six hundred eighty-four patients from A2203 genotypize in the ratio 3: 1 the number of patients in the placebo group and the aliskiren monotherapy groups in relation to other treatment groups.

The primary variables of effectiveness are changes in SODKD values (decrease in SODKD values at the end point compared with the initial level). Secondary efficacy variables are changes in the values of SOSKD (decrease in SOSKD at the endpoint compared to the initial level), the proportion of responders, a decrease in plasma renin (ARP) activity and an increase in active plasma renin (ARP) relative to the initial values.

Genotyping. SNP studies are planned using information from the public dbSNP database and the Celera / ABI private database. The developed sets of probes for studying haplotyping are obtained for the ABI Assays-by-Design® platform. Livak K.J. et al., Nat. Genet. 9, 1995, pp. 341-342. For genotyping using 10 ng of genomic DNA, which corresponds to the manufacturer's recommendation.

Genotyped polymorphisms. The list of polymorphisms identified in this study, including the locus code, gene, database link and effect, is given in Table 5.

Table 5 Genotyped polymorphisms pg_locus_id gene rs_number description 5284 ABCC2 hCV11305436 intron 4786 ABCC2 rs2273697 A> G I417V 4783 ABCC2 rs2756104 intron 4784 ABCC2 rs2756109 intron 4785 ABCC2 rs3740066 T> C I1324I 4782 ABCC2 rs717620 untranslated 4797 ABCC2 rs8187710 A> G Y1515C 5286 ACE hCV1247681 intron 4766 ACE rs4293 intron 4769 ACE rs4317 C> T P32S 4767 ACE rs4329 intron 4768 ACE rs4362 C> T F1129F 400 ACE rs4364 S712R 1345 ACE insertion / deletion 4746 ACE2 rs2285666 intron 1669 ACE2 rs879922 intron 4773 AGT rs1926723 intron 1667 AGT rs4762 T> C M207T 2 AGT rs699 C> T T268M 4772 AGT rs943580 genomic 4796 AGTR1 rs2638362 intron 4780 AGTR1 rs3772616 intron 411 AGTR1 rs5182 C> T L191L 5287 AGTR2 hCV1841569 1445 AGTR2 rs1403543 untranslated 186 AGTR2 rs5193 untranslated 4795 AGTR2-LD rs4497127 genomic 5288 AGTRAP hCV516817 intron 4787 AGTRAP rs4073574 genomic 4789 CYP11B2 rs4539 G> A R173K 575 CYP11B2 rs4544 C> T T339I 4788 CYP11B2 rs4545 A> G S435G 3541 CYP11B2 rs6431 intron 4792 NOS3 rs1007311 intron 482 NOS3 rs1799983 G> T E298D 4791 NOS3 rs1800779 genomic 4464 NOS3 rs1800780 intron 4793 NOS3 rs891512 intron 5292 REN rs11571092 intron 1513 REN rs1464816 intron 4771 REN rs3730103 intron 4794 REN rs6704321 C> G P403A 4770 RENBP rs2269371 G> A G274D 2407 RENBP rs2269372 intron 1676 RENBP rs762656 genomic 5285 TGFB1 hCV11707868 intron 4790 TGFB1 rs2241717 intron 4798 TGFB1 rs8105161 intron

Statistical analysis. Genotype-phenotype association studies and related assays are performed at SAS (Cary, North Carolina, USA) using Analyst® software.

Association identification tests use well-defined genotypes as an independent variable, without assuming dominance, and various efficacy variables as dependent variables. Continuous dependent variable tests used in ANCOVA analysis and logistic regression are used for clearly defined dependent variables. It is noted that not one of the results is suitable for testing on many hypotheses. A threshold p <0.05 is used to determine valid associations. The coincidence of random variables in the analysis of the genotype-phenotype association is: (1) the dose level for treatment, (2) the study area indicated by STU1A in A2201 (see example 1) or indicated by REGION in A2203, (3) measurement of the initial levels of SODKD and SOSKD and (4) race. All samples from all three groups in which patients were treated with aliskiren are analyzed. 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 of SODCD of genotyped patients (mean ± SEM) A2201 (see example 1) A2203 (this example) Treatment N = Body mass index Age Baseline SODKD Treatment N = Age Baseline SODKD Aliskiren 150 mg 99 31.01 ± 0.67 54.15s 1.30 98.76 ± 0.34 Aliskiren 75 mg 102 54.73 ± 1.37 98.69 ± 0.28 Aliskiren 300 mg 96 30.79 ± 0.58 55.93 ± 1.03 99.16 ± 0.36 Aliskiren 150 mg 115 55.83 ± 1.18 99.47 ± 0.35 Aliskiren 600 mg 101 30.53 ± 0.65 55.97 ± 1.08 98.90 ± 0.38 Aliskiren 300 mg 102 57.09 ± 1.22 99.26 ± 0.34 Irbesartan 150 mg 99 31.81 ± 0.72 56.66 ± 1.21 99.41 ± 0.40 Valsartan 80 mg 39 58.08 ± 1.77 99.30 ± 0.61 Placebo 103 30.65 ± 0.60 58.21 ± 1.15 98.89 ± 0.33 Valsartan 160 mg 33 53.45 ± 2.15 98.87 ± 0.69 Valsartan 320 mg 34 55.50 ± 1.65 99.16 ± 0.57

A2201 (see example I) A2203 (this example) Treatment N = Body mass index Age Baseline SODKD Treatment N = Age Baseline SODKD Aliskiren 75 mg and Valsartan 80 mg 35 55.43 ± 1.95 99.73 ± 0.64 Aliskiren 150 mg and valsartan 160 mg 34 56.26 ± 1.94 99.32 ± 0.61 Aliskiren 300 mg and valsartan 320 mg 41 57.10 ± 1.86 98.82 ± 0.46 Valsartan 160 mg and HCTZ 12.5 mg 41 55.41 ± 1.93 98.83 ± 0.55 Placebo 108 55.80 ± 1.24 98.86 ± 0.29

ACF. SNP 4769 is genotyped in patients along with six other polymorphisms in this crucial enzyme for ASD. A significant relationship is observed between SNP 4769 and the first variable of efficiency, a decrease in SODKD relative to the base level (p = 0.023), and with the second variable, the level of responders (p = 0.0048). For the other second variable, the decrease in the value of the SOKDDV relative to the initial level, there is no significant association, only a trend is noted (p = 0.14).

When repeating the ANCOVA assay with the same model for each treatment group, the association is detected only for the aliskiren treatment groups, but not for the irbesartan treatment group or placebo group. The specific effects of aliskiren are shown in Table 7. An insignificant relationship was established between SNPs and secondary parameters of plasma renin activity (ARP) and active renin (ARP).

Table 7 The effect of ACF and AGTR2 variations on SODKD, SOSKD and the proportion of responders in all groups of treatment with aliskiren in A2201 (* shown significance, p <0.05) ONP_4769 ONP_1445 ONP_4795 TT ST SS AA AG Gg Gg GA AA n 278 fourteen 0 106 69 118 106 72 115

LSMEAN analysis of reducing SODKD (mmHg) -11.4 -5.6 NA -11.0 -13.3 -9.4 -10.8 -13.4 -9.2 R 0.023 * 0.0071 * 0.0026 * LSMEAN analysis of reducing SASKD (mmHg) -11.7 -5.9 NA -11.2 -13.8 -9.8 -10.8 -14.2 -9.5 R 0.14 0.13 0.046 * Share of respondents 0.14 0.13 0.046 * 75% 71% 58% 75% 74% 57% R 0.0048 * 0.17 0,060

SNP_4769 is a coding SNP that changes the amino acid sequence from proline to serine at codon 32 in ACF isoforms 2 and 3. ACE I / D polymorphism (the presence or absence of 287 bp Alu repeats the sequence in intron 16) is associated with the level of ACF regulation and activity and its ramifications in ASD. Rigat B. et al., J. Clin. Invest. 86, 1990, pp. 1343-1346. In this case, ACF I / D is tested and associative relationships with the response to aliskiren are observed. ONP_47b9 is located in the 12th intron of the ACF gene encoding the somatic isoform and is localized in the first exon of the gene encoding the second and third isoforms. ACF I / D is located in the 16th intron, which is in the same non-equilibrium coupling as ONP_4769, in the groups of Caucasians, African Americans, and Chinese (SNP viewer, Applied Biosystems, Foster City, California, USA).

AGTR2. It was found that the angiotensin II receptor, type 2, inhibits ACF activity and weakens receptor-mediated actions, type 1, causing vasodilation as a result. Ichiki T et al., Nature 377, 1995, pp. 748-750, Hunley TE et al., Kidney Int. 57, 2000, pp. 570-577.

A significant association is observed between SNP 1445 and SNP 4795 and primary variable efficiency, a decrease in SODKD relative to the baseline (p = 0.0071 and 0.0026, respectively). SNP 4795 also shows a significant relationship with the second variable, a decrease in SOSKD relative to the base level (p = 0.046), but not with the second parameter, the share of responders. Meanwhile, SNP 1445 does not show a significant relationship with the second parameters. They do not find a significant relationship between SNPs and secondary parameters, ARPs and AKTR.

When repeating the ANCOVA analysis with the same model for each treatment group, an association was found only for the aliskiren treatment groups, but not for the irbesartan treatment group or placebo group. The effects of the genotype in patients treated with aliskiren are presented above in Table 7.

When trying to repeat the associations observed with these two SNPs in the A2203 study in the aliskiren treatment groups, the results are not repeated (p> 0.05). Also, no manifestations of an association tendency are observed. Based on the results of preliminary clinical studies, because of a higher than previously observed response to placebo (8.6 mm Hg), the dose response and placebo-subtracted effects of treatment with aliskiren are lower than expected. Such inconsistency may be caused, at least in part, by the loss of copying of the AGTR2 association.

Both of these SNPs are not coding. SNP 1445 is an untranslated region of the AGTR2 gene, and SNP_4795 are in the genomic region in non-equilibrium linkage with the AGTR2 gene. At least in the Chinese, these two SNPs are in the same non-equilibrium linkage block (SNPbrowser, Applied Biosystems, Foster City, California, USA).

Discussion. This analysis reveals significant associations between polymorphisms in two genes with variable clinical outcomes in study A2201 (see example 1). One variant in the ACF gene and two variants in the AGTR2 gene show significant association with the primary parameter, SODKD. Option ACF and one option AGTR2 also show significant association with secondary parameters, SOSKD and the proportion of responders. The second variant of AGTR2 does not show significant association, but only shows the same tendency with respect to secondary parameters. This effect of the genotype is observed only in groups of patients who were treated with aliskiren, but not in the treatment groups with irbesartan or placebo. Therefore, the effect described above means that the pharmacogenetic effect is specific to aliskiren.

The action of the Pro32Ser variant of ACF is of particular interest, since the difference between TT and CT genotypes is very significant (decrease in SODKD 11.4 versus 5.6 mm Hg, decrease in SOSKD 11.7 against 5.9 mm Hg and the responder ratio is 70% against 14% ) However, a homozygous SS genotype is absent in all patients. Consequently, the frequency of occurrence of this allele is much lower than the frequency for SNPs according to databases of 0.1, and the average number of patients with CT genotype is very small. It was found that the frequency of occurrence of the C allele is much higher in representatives of the Negroid race compared with representatives of the Caucasoid race (19/154 versus 2/790). In experiment A2203, all 4 patients with a serine residue (C allele) belong to the Negroid race. Due to the extremely small number of patients with C allele, A2203 analysis cannot be performed with the same model.

ACF polymorphisms and their effect on plasma ACF concentration and blood pressure are mainly detected in African and African American groups. Bouzekri N. et al., Eur. J. Hum. Genet. 12, 2004, pp. 460-468, Cox R. et al., Hum. Mol. Genet. 11, 2002, pp. 2969-2677, Zhu X. et al., Am. J. Hum. Genet. 68, 2001, pp. 1139-1148. The observation of a higher allelic frequency of ONP_4769 in the ACF gene in negroid patients in two SPP100 studies confirms the subsequent study of the functional effect of this polymorphism on the level of ACF or its activity. In addition, the amino acid change in codon 32 is presumably an isoform of this enzyme in the testes.

When analyzing the response to aliskiren depending on the genotype, the proportion of responders with TT genotype increases to about 70%. This result may contribute to the placement of aliskiren in the mass market of antihypertensive drugs, since a significant part of the human community (approximately 80% according to the frequency of occurrence of alleles with SNPs in the databases, and may even be larger according to the present study) has a TT genotype associated with a larger share of responders (figure 2). It may also help in further research to select the supposedly “best” aliskiren responders.

It is highly probable that the two SNPs of the AGTR2 gene are in non-equilibrium linkage, since the chromosome region around this locus is located to a large extent in one block of non-equilibrium linkage. The function of Ang II signal transmission through the type 2 receptor has been less studied compared to the type 1 receptor. This discovery may also mean the involvement of AGTR2 in the function of Ang II in the form of downstream regulation of blood pressure with respect to inhibition of renin.

These described SNPs do not show an associative relationship with measurements of the initial levels of SODKD and SOSKD. This means that the genetic effect is an aliskiren-associated pharmacogenetic effect.

Equivalents

Details of one or more embodiments of the present invention are summarized in the claims below and are as described above. Although any methods and materials similar or equivalent to those described in the present invention can be used to implement the present invention or for testing, preferred methods and materials are described in the present description. Other properties, objects and advantages of the present invention can be gleaned from the description and claims of the present invention. In the description and the appended claims, the singular include references to plural objects, although the contents obviously indicate otherwise. Despite the obvious contradiction, all the methods and scientific terms used in the present invention have the same purpose, which is usually understood by specialists in the field to which the present invention belongs. All publications, patents and patent applications cited in the present invention are 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 has been specifically and separately shown for inclusion in the form references to their essence.

The present invention is not limited to the use of the terminology given in specific embodiments of its implementation, since this terminology is given to explain individual objects of the present invention. Many variations and modifications of the present invention can be obtained without deviating from the spirit and scope of the present invention, which, of course, is obvious to experts in this field. It will also be apparent to those skilled in the art that the methods and equipment listed in the present invention can be replaced with functionally equivalent, relevant areas of the scope of the present invention. Such modifications and variations are consistent with the claims of the present invention. The present invention is limited by the terms given in the claims, however, the use of their equivalents is fully legitimate along with them.

Claims (6)

1. The use of aliskiren to obtain a drug for the treatment of hypertension in a group of patients selected on the basis of genetic polymorphisms in biomarker genes available to patients, and genetic polymorphisms are indicators of the effectiveness of aliskiren in the treatment of hypertension and are selected from the group including: ONP_4769, as indicated in SEQ ID NO: 1 in the angiotensin converting enzyme (ACF) gene, SNP_1445, as indicated in SEQ ID NO: 2 in the angiotensin II receptor gene, type 2 (AGTR2), SNP_4795, as indicated in SEQ ID NO: 3 in the AGTR2 gene, and their combinations. 2. A method for determining the sensitivity of an individual with hypertension to treatment with aliskiren, comprising the steps of:
(a) establishing in two copies of the genes that the individual has, the identity of the nucleotide pairs in one or more polymorphic genetic loci, the genetic loci selected from the group comprising the angiotensin converting enzyme (ACF) gene, angiotensin II receptor gene, type 2 (AGTR2), moreover, the genetic polymorphisms are selected from the group consisting of: SNP_4769, as indicated in SEQ ID NO: 1 in the angiotensin converting enzyme (ACF) gene, SNP_1445, as indicated in SEQ ID NO: 2 in the angiotensin II receptor gene, type 2 (AGTR2), SNP_4795 as indicated in SEQ ID NO: 3 in the AGTR gene 2, and combinations thereof, and
(b) assessing whether an individual belongs to the “high-resonance” group if nucleotide pairs at polymorphic loci indicate that the individual is responding to treatment with aliskiren. 3. The use of aliskiren to obtain a medicament for the treatment of hypertension in an individual, comprising the steps of:
(a) establishing in two copies of the genes that the individual has, the identity of the nucleotide pairs in one or more polymorphic genetic loci, the genetic loci selected from the group comprising the angiotensin converting enzyme (ACF) gene, angiotensin II receptor gene, type 2 (AGTR2), moreover, the genetic polymorphisms are selected from the group consisting of: SNP_4769, as indicated in SEQ ID NO: 1 in the angiotensin converting enzyme (ACF) gene, SNP_1445, as indicated in SEQ ID NO: 2 in the angiotensin II receptor gene, type 2 (AGTR2), SNP_4795 as indicated in SEQ ID NO: 3 in the AGTR gene 2, and combinations thereof, and
(b) administering aliskiren to an individual if a nucleotide pair at a polymorphic locus indicates that the individual is responding to treatment with aliskiren. 4. The use of aliskiren to obtain a medicinal product to lower the average detectable systolic blood pressure (SOSKD) in an individual, including the stages:
(a) establishing in two copies of the genes that the individual has, the identity of the nucleotide pairs in SNP_4795, as indicated in SEQ ID NO: 3 in the angiotensin II receptor gene, type 2A (GTR2), and
(b) administering aliskiren to an individual if the nucleotide pair of SNP_4795 as indicated in SEQ ID NO: 3 indicates that the individual is responding to treatment with aliskiren. 5. The use of aliskiren to obtain a medicine for lowering diastolic blood pressure in an individual, comprising the steps of:
(a) establishing in two copies of the genes that the individual has, the identity of the nucleotide pairs in one or more polymorphic genetic loci, the genetic loci selected from the group comprising the angiotensin converting enzyme (ACF) gene, angiotensin II receptor gene, type 2 (AGTR2), moreover, the genetic polymorphisms are selected from the group consisting of: SNP_4769, as indicated in SEQ ID NO: 1 in the angiotensin converting enzyme (ACF) gene, SNP_1445, as indicated in SEQ ID NO: 2 in the angiotensin II receptor gene, type 2 (AGTR2), SNP_4795 as indicated in SEQ ID NO: 3 in the AGTR gene 2, and combinations thereof, and
(b) administering aliskiren to an individual if the identity of the nucleotide pairs at one or more polymorphic loci indicates that the individual is responding to treatment with aliskiren. 6. The use of a gene product of a gene selected from the group comprising the angiotensin converting enzyme gene (ACF) and the angiotensin II receptor gene, type 2 (AGTR2), as a target for the action of the drug, which includes the following stages:
(a) the contact of the drug with the first gene product encoded by a polynucleotide with a nucleotide pair in a polymorphic site in the region of the selected gene, indicating a high reactivity in the treatment of hypertension with aliskiren,
(b) identifying the effect of the drug on said first gene product,
(c) contact of the drug with a second gene product encoded by a polynucleotide with a nucleotide pair in a polymorphic site in the region of the selected gene, indicating low reactivity in the treatment of hypertension with aliskiren,
(g) identifying the effect of the drug on said second gene product,
(e) identification of similarities and differences between the activity identified in stage (b) and the activity identified in stage (d);
moreover, the genetic polymorphisms are selected from the group consisting of: SNP_4769, as indicated in SEQ ID NO: 1 in the angiotensin converting enzyme (ACF) gene, SNP_1445, as indicated in SEQ ID NO: 2 in the angiotensin II receptor gene, type 2 (AGTR2), SNP_4795 as indicated in SEQ ID NO: 3 in the AGTR2 gene, and combinations thereof.

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