CN1858243A - Method for predicting angiotonin II receptor agonist hypotensor medicine function and its use - Google Patents
Method for predicting angiotonin II receptor agonist hypotensor medicine function and its use Download PDFInfo
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Abstract
The present invention provides a kind of reagent kit including reagent for detecting the polymorphic site genotype of bradykinin receptor gene of nucleic acid template. By means of PCR-RFLP method or Taqman method, the detection is fast, safe, convenient, sensitive and reliable. The detection can predict the hypotensive effect of angiotonin II receptor 1 agonist hypotensor and predict the blood fat state of the primary hypertension patient, so that the medicine may be selected based on the individual difference to raise clinical treating efficiency and safety and lower the toxic side effect risk and treating cost.
Description
Technical field
The present invention relates to a kind of method and application thereof of predicting angiotonin II receptor agonist hypotensor medicine function, by measuring biological sample specific site genotype, the drug effect of prediction angiotensin-ii receptor 1 agonist hypotensor.
Background technology
Essential hypertension is one of modal chronic disease, is the substantial risk factor of cerebral apoplexy, coronary heart disease, diabetes etc., and therefore effectively controlling blood pressure has important clinical and public hygienics meaning.Clinically be used for the treatment of hypertensive medicine six classes are arranged, comprising: angiotensin-ii receptor (AT1-R) antagonist, beta-blocker, angiotensin converting enzyme inhibitor (ACEI), calcium antagonist, alpha adrenergic receptor retarding agent and diuretic(s) etc.But hypertensive so far inverse amplification factor is lower, and the data of the China and the U.S. shows that inverse amplification factor only is respectively 8.1% and 34%.This low inverse amplification factor not only with antihypertensive drugs itself render a service relevant, and with to lack effective outcome prediction guidance system clinically relevant.
The existing clinical method that is used to predict depressor curative effect, choose reasonable antihypertensive drugs mainly is doctors experience prediction and monitoring of blood pressure.These methods are too rough, and the doctor is difficult to carry out according to the patient individual difference selection, the compatibility and quantitative of medicine, to obtain better curative effect, to reduce toxic side effect, has increased the weight of patient's body and mind misery and economical load indirectly.Studies show that in a large number the individual difference of drug effect effect is relevant with environment, inherited genetic factors and interaction thereof.Pharmacogenomics is the difference of probing into drug effect from genomic level, discloses the front subject and the hi-tech means of the hereditary feature of these differences.Its achievement in research can be the doctor and drafts the personalized medicine scheme, and accurately predicting and raising curative effect of medication reduce the poisonous side effect of medicine risk, thereby reduce medical expense, improve patients ' life quality and make significant contribution.
According to the literature, pharmacogenomics has been illustrated the gene pleiomorphism of some drug targetings and the incidence relation between the certain drug effect, and that the product that these achievements cause has begun to be used for is clinical.For example, breast cancer related gene detection kit (the Oncotype DX that has gone on the market in the California, USA approval
TM).This product is to be used to predict reactivity and the chemical sproof test kit (Science 2004 303:1754-1755s) of patient to chemotherapeutics.Because the specificity and the susceptibility of this test kit are better, its test item has been classified the preceding essential items for inspection of metastatic breast cancer chemotherapy as, thereby estimates the recurrence rate of mammary cancer.
Primary hypertension relative gene has been found that so far and surpasses 70 that antihypertensive drugs and Study on gene polymorphism majority concentrate on both interactions to [Drugs, 2004 in the influence of cardiovascular event generation; 64 (16): 1801-1816].For example existing the report carried the hyperpietic at α-seats such as adducin gene 460W, adopts diuretic therapy than adopting other step-down treatments, more helps to reduce the occurrence probability of patient's myocardial infarction and apoplexy.There are interaction in the polymorphism of ACE insertion/disappearance and Angiotensin proenzyme inhibitor (ACEI) for another example, show as the difference of different genotype at aspects such as the mRNA expression of angiotensin receptor albumen (AT1), left ventricular hypertrophy, arteriosclerosis.
But, with the research of hypotensive effect genes involved very limited [Drugs, 2004; 64 (16): 1801-1816].Only report comprises that there are the incidence relation of gene pleiomorphism and depressor curative effect in nitricoxide synthase (NOE) gene pleiomorphism and diuretic(s), α-adducin gene pleiomorphism and diuretic(s), G protein alpha-subtype gene polymorphism and beta-2 adrenoceptor retarding agent and ACE gene pleiomorphism and angiotensin-ii receptor (AT1-R) antagonist etc.
Angiotensin-ii receptor 1 (AT1-R) antagonist (sartans) is a clinical the most frequently used line depressor.The mechanism of action of such medicine is optionally to combine with AT1-R, suppresses the contracting blood vessel function of Angiotensin II and makes blood pressure drops.Sartans is also protected the potassium natriuresis because of it and is specially adapted to the treatment that hypertension is followed congestive heart failure and renal failure patient except being used for the treatment of essential hypertension.Chang Yong AT1-R class drug main will comprise clinically: losartan (Losartan), valsartan (Valsartan), irbesartan (Irbesartan), Kang Deshatan (Candesartan), eprosartan (Eprosartan), Tasosartan (Tasosartan) and telmisartan (Telmisartan) etc.
Although sartans as a clinical new line antihypertensive drugs product of releasing, though efficacy of antihypertensive treatment is reliable and side effect is less, also is difficult to overcome many deficiencies, as still has the part patient insensitive to this type of medicine, show as the step-down onset slowly, weak curative effect; Drug side effect has appearred in a few patients, comprises cough and angioedema (similar to angiotensin-convertion enzyme inhibitor class medicine, but incidence is relatively low), liver toxicity and hyperglycemia and anaemia reaction etc.For example Reeves etc. discovers that to 2955 adult hyperpietic's irbesartan step-downs treatments about 44% patient does not reach satisfied clinical efficacy of antihypertensive treatment (Hypertension.1998; 31 (6): 1311-6).The research of Guthrie R etc. also shows, after irbesartan treatment course of treatment of continuous 12 weeks, 150-300mg/d, 53% the patient's efficacy of antihypertensive treatment of only having an appointment is remarkable.
Existing pharmacogenomics research report at irbesartan is the large-scale clinical study of a Sweden, i.e. SILVHIA test.This research finds successively, has the mononucleotide polymorphism site of a plurality of genes may be relevant with the improvement effect of the efficacy of antihypertensive treatment of irbesartan or left ventricular hypertrophy in the renin-angiotensin-aldosterone system path.The gene that this research has been reported comprises, angiotensinogen gene, angiotensin-converting enzyme gene, angiotensin-ii receptor 1 gene and aldosterone synthase gene (Am J Hypertens.2004; 17 (1): 8-13.J Hypertens.2001; 19 (10): 1783-7.J Hypertens.2002; 20 (4): 657-63.Am J Hypertens.2002; 15 (5): 389-93.).In addition, may some relevant other genetic polymorphisms comprise the main metabolic enzyme-Cytochrome P450 of apolipoprotein B gene (APOB), ldl receptor gene (LDL-R), transforming growth factor gene (TGF β 1), preproendothelin gene (preproendothelin-1) and irbesartan-2C9 gene (CYP2C9) (BMC Cardiovasc Disord.2004 28 with the drug reaction of irbesartan; 4 (1): 16.Clin Cardiol.2004; 27 (5): 287-90.Clin Cardiol.2004; 27 (3): 169-73.J Hypertens.2002; 20 (10): 2089-93.).The association study of kinin receptor gene and the irbesartan efficacy of antihypertensive treatment of not slowing is not so far seen the test kit product that research causes at the irbesartan pharmacogenomics yet.
Summary of the invention
The technical problem that solves
Because there is individual difference in drug reaction, the clinician is selecting medicine to lack enough personalized medicine foundations so far, be difficult to accomplish to provide the compatibility of drugs and the dosage of " formula of cutting the garment according to the figure " for patient, can not be immediately, the risk that takes place of controlling blood pressure and toxic side effect effectively, even may delay treatment opportunity, cause patient health and dual economically loss.
In view of above-mentioned deficiency, blindness when the present invention is intended to overcome selection of clinical angiotensin-ii receptor 1 (AT1-R) antagonist or develops the novel composition of combination with it, for personalized medicine provides method, test kit and the application thereof of the effect of a kind of prediction angiotensin-ii receptor 1 (AT1-R) antagonist class medication medication, be convenient to instruct clinical application.
Technical scheme
For achieving the above object, take following technical scheme:
A kind of pharmaceutically-active method of predicting angiotensin-ii receptor 1 agonist hypotensor, this method is by measuring biological sample bradykinin receptor gene polymorphism sites genotype, the hypotensive effect of prediction angiotensin-ii receptor 1 agonist hypotensor, wherein the bradykinin receptor gene polymorphism sites is bradykinin b 2 receptor gene C-58T polymorphism:
A) bradykinin receptor gene genotype be-the 58C/T heterozygote or-during 58T/T homozygous mutation type, the hypotensive effect of prediction angiotensin-ii receptor 1 antagonist is good;
B) bradykinin receptor gene genotype be-during the 58C/C mutant, the hypotensive effect of prediction angiotensin-ii receptor 1 antagonist is poor.
The method that said determination bradykinin receptor gene polymorphism sites genotype adopts is selected from a kind of in the biological detecting methods such as PCR, gene chip, gene sequencing, genescan, Taqman.PCR, gene chip, gene sequencing, genescan genotype detection method are the conventional methods of using of those skilled in the art, and the Taqman technology is a kind of method of using the fluorescence technique real-time quantitative PCR, and specific operation process is seen the embodiment of the invention 1.Wherein preferred detection method is PCR or Taqman technology.
Above-mentioned biological sample is a kind of in blood sample, oral mucosa examination, saliva sample, the urine sample.Wherein, preferred biological specimen is periphery whole blood or blood cell sample.
Above-mentioned angiotensin-ii receptor 1 agonist hypotensor is selected from irbesartan (Irbesartan), losartan (Losartan), valsartan (Valsartan), telmisartan (Telmisartan/Micardis, telmisartan), Candesartan (Candesartan, Kang Deshatan), eprosartan (Eprosartan, Eprosartan), a kind of in Olmesartan (OlmesartanMedoxomil, olmesartan medoxomill), Tasosartan (Tasosartan/Verdia) and their active metabolite, salt or the ester class.Wherein preferred irbesartan or losartan, they are medicines commonly used, representative in the angiotensin-ii receptor 1 agonist hypotensor thing.
The pharmaceutically-active method of a kind of prediction angiotensin-ii receptor 1 agonist hypotensor, this method is by measuring biological sample bradykinin receptor gene pleiomorphism, the target organ functional status of prediction primary hypertension patient, wherein the bradykinin receptor gene polymorphism sites is bradykinin b 2 receptor gene C-58T polymorphism, and the target organ functional status is the blood fat state:
A) bradykinin receptor gene genotype be-the 58C/T heterozygote or-during 58T/T homozygous mutation type, prediction primary hypertension patient, its plasma triglyceride (TG) level height;
B) bradykinin receptor gene genotype be-during the 58C/C mutant, the prediction primary hypertension patient, its plasma triglyceride (TG) level is low.
The method that said determination bradykinin receptor gene polymorphism sites genotype is adopted is selected from a kind of in the biological detecting methods such as PCR, gene chip, gene sequencing, genescan, Taqman.PCR, gene chip, gene sequencing, genescan genotype detection method are the conventional methods of using of those skilled in the art, and the Taqman technology is a kind of method of using the fluorescence technique real-time quantitative PCR, and specific operation process is seen the embodiment of the invention 1.Wherein preferred detection method is PCR or Taqman technology.
Above-mentioned biological sample is a kind of in blood sample, oral mucosa examination, saliva sample, the urine sample.Wherein, preferred biological specimen is periphery whole blood or blood cell sample.
Above-mentioned angiotensin-ii receptor 1 agonist hypotensor is selected from irbesartan (Irbesartan), losartan (Losartan), valsartan (Valsartan), telmisartan (Telmisartan/Micardis, telmisartan), Candesartan (Candesartan, Kang Deshatan), eprosartan (Eprosartan, Eprosartan), a kind of in Olmesartan (OlmesartanMedoxomil, olmesartan medoxomill), Tasosartan (Tasosartan/Verdia) and their active metabolite, salt or the ester class.Wherein preferred irbesartan or losartan, they are medicines commonly used, representative in the angiotensin-ii receptor 1 agonist hypotensor thing.
The pharmaceutically-active test kit of a kind of prediction angiotensin-ii receptor 1 agonist hypotensor, comprise the nucleic acid-templated genotypic reagent of bradykinin receptor gene polymorphism sites of measuring biological sample, PCR reaction reagent, digestion with restriction enzyme reaction reagent or Taqman reaction reagent, specific probe are wherein arranged.
Wherein specific probe is for detecting the wild-type probe and the mutant probe of bradykinin receptor gene polymorphism sites.
The reagent of biological sample amplifying nucleic acid template wherein, PCR reaction reagent, the reagent that does not specialize in digestion with restriction enzyme reaction reagent or the Taqman reaction reagent are the conventional reagent that use of those skilled in the art.
The biological sample kind that is wherein detected is one or more in blood sample, oral mucosa examination, saliva sample, the urine sample.This is periphery whole blood or blood cell sample for the blood sample of wherein said detection.
Above-mentioned angiotensin-ii receptor 1 agonist hypotensor is selected from a kind of in irbesartan, losartan, valsartan, telmisartan, Candesartan, eprosartan, Olmesartan, Tasosartan and their active metabolite, salt or the ester class.
Above-mentioned prediction angiotensin-ii receptor 1 agonist hypotensor drug effect, for the prediction angiotensin-ii receptor 1 agonist hypotensor hypotensive effect or/and the prediction primary hypertension patient the blood fat state.
Test kit provided by the invention prediction angiotensin-ii receptor 1 agonist hypotensor drug effect, its process comprise (a) from take from examined extract the periphery Trace Blood sample nucleic acid-templated; (b) with the special segment on the PCR method amplification PRCP gene; (c) with restriction enzyme the PCR product being carried out enzyme cuts; (d) carry out (b) (c) step with the positive control template simultaneously; (e) the electrophoretic separation enzyme is cut product, identifies and judged result according to the banding pattern characteristics of positive template.
Test kit provided by the invention prediction angiotensin-ii receptor 1 agonist hypotensor drug effect, its process comprise (a) from take from examined extract the periphery Trace Blood sample nucleic acid-templated; (b) with the special segment on the Taqman method amplification PRCP gene; (c) identify also judged result.
A kind of pharmaceutically-active method of predicting angiotensin-ii receptor 1 agonist hypotensor, this method are by measuring biological sample bradykinin receptor gene polymorphism sites genotype, the hypotensive effect of prediction angiotensin-ii receptor 1 agonist hypotensor.Method of the present invention also can be in medicament research and development application.
Above-mentioned application is meant according to the influence of bradykinin receptor gene pleiomorphism genotype to angiotensin-ii receptor 1 antagonist class medication medication effect, design the compound medicines that adjusting bradykinin receptor expression level or its activity can strengthen angiotensin-ii receptor 1 antagonist class medicine hypotensive effect, described compound medicines comprises selectivity or nonselective angiotensin II receptor antagonists and bradykinin, the bradykinin analogue, bradykinin receptor part or bradykinin function enhancers, described bradykinin function enhancers comprises the bradykinin receptor activating transcription factor, the agent of polypeptide function activation, the metabolic enzyme of bradykinin metabolic pathway, cofactor.
The pharmaceutically-active method of a kind of prediction angiotensin-ii receptor 1 agonist hypotensor, this method is by measuring biological sample bradykinin receptor gene polymorphism sites genotype, the target organ functional status of prediction primary hypertension patient.Method of the present invention also can be in medicament research and development application.
Above-mentioned application is meant according to the influence of bradykinin receptor gene pleiomorphism genotype to angiotensin-ii receptor 1 antagonist class medication medication effect, design the useful influence of intensifier target organ dysfunction or reduce the compound medicines that the target organ dysfunction influences, described compound medicines comprises selectivity or nonselective angiotensin II receptor antagonists and target organ function-improving agent.
The present invention studies based on pharmacogenomics for many years.Carry out the step-down treatment by take irbesartan to the hyperpietic, investigate the relation of inherited genetic factors and curative effect of medication.Irbesartan is a kind of in the angiotensin-ii receptor 1 agonist hypotensor thing, optionally combine with AT1-R, thereby the vasoconstriction effect that suppresses Ang II makes blood pressure drops, clinically is used for the step-down treatment.The transformation period of irbesartan is grown (being about 19 hours), therefore is suitable for taking medicine every day once, and common dose is 150-300mg/ days, and clinical being widely used in treated light, moderate essential hypertension.
The gene pleiomorphism of studying among the present invention is the bradykinin b 2 receptor gene pleiomorphism.Bradykinin is a kind of important biological active agents, plays an important role in physiology, pathologic processes such as vascular function adjusting, inflammatory reaction.It is important neurohumour regulation system in the body that bradykinin participates in kallikrein kinin system, and most effects are mediated by the bradykinin beta 2 receptor.Bradykinin combines with the bradykinin beta 2 receptor stimulates the synthetic nitrogen protoxide of nitric oxide synthetase, and the effect of the nervous plain II of antagonizing vessel makes vasorelaxation, and blood pressure drops plays the effect of blood pressure regulation.Studies show that vein uses bradykinin can cause arteries diastole, the peripheral vascular resistance of rat to reduce and short water/biological effects (Hypertension 21:961-965) such as sodium discharge through kidney.After bradykinin discharges mainly by combining its effect of bringing into play with bradykinin b 2 receptor (BDKRB2).BDKRB2 is a kind of G protein coupling receptor, by the BDKRB2 genes encoding, is basic model and expresses in human body.The pressure value of the transgenic mice of the human BDKRB2 of overexpression significantly is lower than the normal control group, uses the pressure value of specific BDKRB2 antagonist energy render transgenic mouse to recover normal (Hypertension 29:488-493).Discover that there are 4 kinds of polymorphisms in three exons of bradykinin beta 2 receptor gene and a promoter region, wherein promoter region-58 exists T to be replaced into the point mutation (Immunopharma-cology.33:32-35 of C; Hypertension.1996,28:1081-1084.), (Jpn Circ is J.1999 for the primary hypertension incidence in this sudden change and Japanese population, the Chinese han population; 63 (10): 759-62; Hypertens Res.2001,24:299 302.) and the relevant (Hypertension.2000Jul of cough side effect that causes of ACEI; 36 (1): 127-31).Discover, bradykinin beta 2 receptor gene promoter area-58T/C polymorphism is relevant with primary hypertension patient ACEI efficacy of antihypertensive treatment, carrying the allelic primary hypertension patient blood pressure drops of T amplitude, big (Chen Gailing is etc. hypertension magazine .2004.12 (3): 229-232).
What the present invention relates to discovers:
After the 1 antagonist class pharmacological agent of hypertensive patient's menses angiotensin II acceptor, taking medicine back the 28th day, the hypotensive effect that carries the primary hypertension patient at seats such as bradykinin b 2 receptor T significantly is worse than the wild homozygous genotype patient of C/C, and the plasma triglyceride level of primary hypertension patient is higher.The target organ functional status of the C-58T polymorphism genetic polymorphism of prompting bradykinin b 2 receptor gene and the hypotensive effect of angiotensin-ii receptor 1 antagonist class medicine and primary hypertension patient is relevant.
The baseline blood pressure level of the mutant mice of shortage BDKRB2 gene is higher, under the high salt diet condition, and severe hypertension (Circulation 96:3570-3578) in easily suffering from.Have experimental evidence to show, angiotensin-ii receptor 1 antagonist can strengthen the vasorelaxation function that arterial vascular endothelium relies on, and this agency part is mediated (Circulation.1993 by bradykinin; 87:931-938).And the T allelotrope of BDKRB2 gene C-58T polymorphism is associated with higher bradykinin b 2 receptor genetic transcription activity (Immunopharmacology 33:32-35), thereby T allelotrope carrier is when receiving the treatment of AT1 receptor antagonist, hypotensive effect through bradykinin-bradykinin b 2 receptor approach strengthens, and causes its efficacy of antihypertensive treatment to be better than C/C homozygous wildtype patient.
Bradykinin b 2 receptor gene genotype among the present invention, can be used as one of indication mechanism of prediction angiotensin-ii receptor 1 antagonist class antihypertensive drug, can indicate and qualitative and predict the drug effect of angiotensin-ii receptor 1 agonist hypotensor and the target organ functional status of primary hypertension patient quantitatively, instruct clinician's personalized medicine.
The genotype of bradykinin b 2 receptor, as one of indication mechanism of prediction angiotensin-ii receptor 1 antagonist class antihypertensive drug: receptor active/functional status or the target organ functional status that can indicate bradykinin b 2 receptor gene pleiomorphism correspondence, function target spot as new drug instructs the research and development of compound antihypertensive drug; And can indicate compound antihypertensive drug in conjunction with development, the more individual dose,optimum of choose reasonable angiotensin-ii receptor 1 antagonist class medicine and the compatibility of suitable combination drug.
Beneficial effect
The invention provides a kind of method of utilizing functional gene prediction angiotensin-ii receptor 1 antagonist class curative effect of antihypertensive drug, can be used as the pharmaceutically-active indication mechanism of angiotensin-ii receptor 1 agonist hypotensor, by measuring bradykinin b 2 receptor gene polymorphic loci gene type, the drug effect of prediction angiotensin-ii receptor 1 agonist hypotensor and the target organ functional status of primary hypertension patient.According to of the influence of bradykinin b 2 receptor gene pleiomorphism genotype, design and regulate the compound medicines that bradykinin receptor expression level or its activity or target organ functional status can strengthen angiotensin-ii receptor 1 antagonist class medicine hypotensive effect angiotensin-ii receptor 1 antagonist class medication medication effect.Be convenient to the doctor and when medication, select, improved the efficient and the security of clinical application and treatment, reduced risk and economical load that toxic side effect takes place according to individual difference.Use the invention achievement of this class functional gene polymorphism, select medicine, prediction medication curative effect and instruct new drug development to have the using value of industry and service instructing the clinical of antihypertensive drug from now on more economically effectively.
Embodiment
Embodiment 1: measure the C-58T pleomorphism site of bradykinin b 2 receptor gene and predict the hypotensive effect of angiotensin-ii receptor 1 antagonist class antihypertensive drug irbesartan
(1) the C-58T polymorphic site genotype of mensuration bradykinin b 2 receptor gene:
(1) genomic dna of extraction host cell:
(a) add the 30ml erythrocyte cracked liquid in whole blood, slowly shake up, room temperature left standstill 10 minutes, during, shake for several times, thoroughly splitting erythrocyte;
(b) in 4 ℃, 2000 leave the heart/minute, 10 minutes, remove supernatant, the white corpuscle that will precipitate is broken up on the oscillator in rotation, adds proteolytic enzyme 40ul, RNA enzyme 50ul, shakes up, and adds write cell lysis buffer and puts 15ml, 37 ℃ of water-baths of mixing were taken out after 20 minutes, put in the cold water;
(c) add cold albumen precipitation liquid 4ml, be placed on-20 ℃ of refrigerators 5 minutes behind the mixing, take out in 4 ℃, 3000 rev/mins centrifugal 10 minutes.Supernatant liquor poured into slowly shake in the 50ml centrifuge tube that has added the 15ml Virahol for several times, separate out to the DNA floss;
(d) the DNA floss of separating out is moved to another 1.5ml and has packed on the 75% alcoholic acid filter paper, make evaporate dried.
(e) add DNA hydrating fluid 1.5ml, put shaking table, shaken over night, standby;
(f) mensuration of DNA concentration adopts ultraviolet spectrophotometry, measures the OD value under two wavelength of 260nm and 280nm respectively, is DNA concentration with OD260nm * 50 income values.And with OD260nm/OD280nm ratio estimation DNA purity;
(2) use the Taqman method to detect the C-58T polymorphic site genotype of bradykinin b 2 receptor gene
C-58T functional gene polymorphic site and flanking sequence thereof with PCR instrument amplification bradykinin b 2 receptor gene, in 5ul PCR reaction system, contain genomic dna 10ng, 2.5ul Taqman 2X Universal PCR MasterMix No AmpErase UNG (composition comprises: AmpliTaq Gold DNA Polymerase, dNTPs withDutp, Passive Reference, the damping fluid of having optimized), and the forward primer of 0.72uM, each 0.16uM of allele-specific probe of the reverse primer of 0.72uM and two sections band fluorescence report groups.
The PCR reaction conditions:
95 ℃ of 10min, 1 circulation;
92℃ 15s，
60℃ 1min，
50 circulations.
(b) on 7900 type quantitative real time PCR Instruments, detect fluorescence information
The PCR plate of finishing the PCR reaction is put on the 7900 type quantitative real time PCR Instruments, is selected for use " AllelicDiscrimination " program, scan judgement with the result:
The genotype of sending FAM fluorescence person is the T/T homozygote;
The genotype of sending VIC fluorescence person is the C/C homozygote;
The genotype of sending two kinds of fluorescence persons is the T/C heterozygote.
(2) prediction drug effect:
Genotype is-during the 58C/C homozygous wildtype, angiotensin-ii receptor 1 agonist hypotensor is relatively poor to the contraction hypotensive effect of primary hypertension patient;
Genotype is-the 58C/T heterozygote or-during the 58T/T homozygous mutation, angiotensin-ii receptor 1 agonist hypotensor is better to the contraction hypotensive effect of primary hypertension patient.
Above method is pressed bradykinin b 2 receptor genotype grouping (homozygous wildtype group, heterozygosis subtype group+homozygous mutation type group) by clinical verification with primary hypertension patient, gives irbesartan respectively 28 days, observes its hypotensive effect, and the result is as follows:
Primary hypertension patient take after the irbesartan the 28th day systolic pressure (SBP) antihypertensive effect-seats such as 58T are carried in the individuality and are significantly higher than-58C/C homozygote (P＜0.05), the hypotensive effect relevant (seeing Table 1) of prompting bradykinin b 2 receptor gene C-58T polymorphism and irbesartan.
The relation of the hypotensive effect of table 1 irbesartan and bradykinin b 2 receptor gene C-58T polymorphic site
| Hypotensive effect | Genotype | The example number | Means standard deviation | The P value |
| SBP | C/C C/T+T/T | 230 611 | 16.4±17.4 19.6±19.4 | - 0.030 |
Embodiment 2: measure the C-58T pleomorphism site of bradykinin b 2 receptor gene, the target organ influence of prediction angiotensin-ii receptor 1 antagonist class antihypertensive drug irbesartan
(1) the C-58T polymorphic site genotype (with embodiment 1) of mensuration bradykinin b 2 receptor gene
(2) danger of Microalbuminuria takes place in prediction:
To primary hypertension patient, genotype-58C/T heterozygote or-the plasma triglyceride level of 58T/T homozygous mutation type individuality is significantly higher than the individuality of genotype for-58C/C homozygous wildtype.
Above method is divided into groups primary hypertension patient by the research checking; Bradykinin b 2 receptor genotype grouping (homozygous wildtype group, heterozygosis subtype group+homozygous mutation type group) is observed it and the proteinuria situation occurred, and the result is as follows:
In primary hypertension patient, seats such as T are carried individual plasma triglyceride level and are significantly higher than-58C/C homozygote (P＜0.05) (seeing Table 2).
The relation of the C-58T pleomorphism site of table 2 bradykinin b 2 receptor gene and the blood fat state of primary hypertension patient
| Genotype | N | Mean±SD | β(SE) | The P value |
| C/C C/T+T/T | 229 607 | 1.28±0.59 ±0.90 | Ref 0.11(0.05) | - 0.035 |
Embodiment 3: test kit is formed (Taqman method) and is used
1, reagent constituents comprises:
A liquid: Taqman 2X Universal PCR Master Mix No AmpErase UNG (composition comprises: AmpliTaqGold DNA Polymerase, dNTPs with Dutp, Passive Reference, the damping fluid of having optimized);
B liquid: forward primer, reverse primer;
C liquid: the allele-specific probe of two sections band fluorescence report groups,
The probe that carries VIC fluorescence report group is corresponding to " C " allelotrope;
The probe that carries FAM fluorescence report group is corresponding to " T " allelotrope.
2, use this test kit amplification functional gene polymorphic site and flanking sequence thereof and the genotypic step of detection pleomorphism site as follows:
The amplifying target genes fragment: in 5ul PCR reaction system, contain genomic dna 10ng, the A liquid of 2.5ul, and 0.72uM B liquid and 0.16uM C liquid, carry out the PCR reaction by following condition:
On 7900 type quantitative real time PCR Instruments, detect fluorescence information
The PCR plate of finishing the PCR reaction is put on the 7900 type quantitative real time PCR Instruments, is selected for use " AllelicDiscrimination " program, scan judgement with the result:
The genotype of sending FAM fluorescence person is the T/T homozygote;
The genotype of sending VIC fluorescence person is the C/C homozygote;
The genotype of sending two kinds of fluorescence persons is the T/C heterozygote.
Claims (21)
1. pharmaceutically-active method of predicting angiotensin-ii receptor 1 agonist hypotensor, this method is by measuring biological sample bradykinin receptor gene polymorphism sites genotype, the hypotensive effect of prediction angiotensin-ii receptor 1 agonist hypotensor.
2. the method for claim 1 is characterized in that, is by measuring biological sample bradykinin b 2 receptor gene C-58T polymorphism, the hypotensive effect of prediction angiotensin-ii receptor 1 agonist hypotensor.
3. method as claimed in claim 2 is characterized in that, the method that described mensuration bradykinin b 2 receptor gene C-58T pleomorphism site genotype adopts is selected from a kind of in the biological detecting methods such as PCR, gene chip, gene sequencing, genescan, Taqman.
4. as arbitrary described method in the claim 1～3, it is characterized in that described biological sample is a kind of in blood sample, oral mucosa examination, saliva sample, the urine sample.
5. as arbitrary described method in the claim 1～3, it is characterized in that: described angiotensin-ii receptor 1 agonist hypotensor is selected from a kind of in irbesartan, losartan, valsartan, telmisartan, Candesartan, eprosartan, Olmesartan, Tasosartan and their active metabolite, salt or the ester class.
6. predict the pharmaceutically-active method of angiotensin-ii receptor 1 agonist hypotensor for one kind, this method is by measuring biological sample bradykinin b 2 receptor gene polymorphism sites genotype, the target organ functional status of prediction primary hypertension patient.
7. method as claimed in claim 6 is characterized in that, is by measuring biological sample bradykinin b 2 receptor gene C-58T polymorphism, predicting former generation hyperpietic's blood fat state.
8. method as claimed in claim 7 is characterized in that, the method that described mensuration bradykinin b 2 receptor gene C-58T pleomorphism site genotype adopts is selected from a kind of in the biological detecting methods such as PCR, gene chip, gene sequencing, genescan, Taqman.
9. method as claimed in claim 6 is characterized in that, described biological sample is a kind of in blood sample, oral mucosa examination, saliva sample, the urine sample.
10. method as claimed in claim 6 is characterized in that: described angiotensin-ii receptor 1 agonist hypotensor is selected from a kind of in irbesartan, losartan, valsartan, telmisartan, Candesartan, eprosartan, Olmesartan, Tasosartan and their active metabolite, salt or the ester class.
11. pharmaceutically-active test kit of prediction angiotensin-ii receptor 1 agonist hypotensor, comprise the nucleic acid-templated genotypic reagent of bradykinin receptor gene polymorphism sites of measuring biological sample, PCR reaction reagent, digestion with restriction enzyme reaction reagent or Taqman reaction reagent, specific probe are wherein arranged.
12. test kit as claimed in claim 11, wherein specific probe is for detecting the wild-type probe and the mutant probe of bradykinin receptor gene polymorphism sites.
13. test kit as claimed in claim 11, the biological sample kind that is wherein detected are in blood sample, oral mucosa examination, saliva sample, the urine sample one or more.
14. test kit as claimed in claim 13, this is periphery whole blood or blood cell sample for the blood sample of wherein said detection.
15. test kit as claimed in claim 11 is characterized in that: described angiotensin-ii receptor 1 agonist hypotensor is selected from a kind of in irbesartan, losartan, valsartan, telmisartan, Candesartan, eprosartan, Olmesartan, Tasosartan and active metabolite thereof, salt or the ester class.
16. test kit as claimed in claim 11, it is characterized in that: described prediction angiotensin-ii receptor 1 agonist hypotensor drug effect, for the prediction angiotensin-ii receptor 1 agonist hypotensor hypotensive effect or/and the prediction primary hypertension patient the target organ functional status.
17. the application of the described method of claim 1 in medicament research and development.
18. application as claimed in claim 17, it is characterized in that, described application is meant according to the influence of bradykinin receptor gene pleiomorphism genotype to angiotensin-ii receptor 1 antagonist class medication medication effect, design the compound medicines that adjusting bradykinin receptor expression level or its activity can strengthen angiotensin-ii receptor 1 antagonist class medicine hypotensive effect, described compound medicines comprises selectivity or nonselective angiotensin II receptor antagonists and bradykinin, bradykinin analogue, bradykinin receptor part or bradykinin function enhancers.
19. application as claimed in claim 18 is characterized in that, described bradykinin function enhancers comprises bradykinin receptor activating transcription factor, the agent of polypeptide function activation, the metabolic enzyme of bradykinin metabolic pathway, cofactor.
20. the application of the described method of claim 6 in medicament research and development.
21. application as claimed in claim 20, it is characterized in that, described application is meant according to the influence of bradykinin receptor gene pleiomorphism genotype to angiotensin-ii receptor 1 antagonist class medication medication effect, design the useful influence of intensifier target organ dysfunction or reduce the compound medicines that the target organ dysfunction influences, described compound medicines comprises selectivity or nonselective angiotensin II receptor antagonists and target organ function-improving agent.
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| CN 200510011682 CN1858243A (en) | 2005-04-30 | 2005-04-30 | Method for predicting angiotonin II receptor agonist hypotensor medicine function and its use |
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| CN 200510011682 CN1858243A (en) | 2005-04-30 | 2005-04-30 | Method for predicting angiotonin II receptor agonist hypotensor medicine function and its use |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109207586A (en) * | 2018-11-06 | 2019-01-15 | 宁波艾捷康宁生物科技有限公司 | Gene SNP site is applied in hypertension individuation drug therapy genotype detection and kit |
| US11668718B2 (en) | 2015-12-15 | 2023-06-06 | Takeda Pharmaceutical Company Limited | Peptide quantitation assay for differentiating full-length high molecular weight kininogen (HMWK) and cleaved HMWK |
-
2005
- 2005-04-30 CN CN 200510011682 patent/CN1858243A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US11668718B2 (en) | 2015-12-15 | 2023-06-06 | Takeda Pharmaceutical Company Limited | Peptide quantitation assay for differentiating full-length high molecular weight kininogen (HMWK) and cleaved HMWK |
| CN109207586A (en) * | 2018-11-06 | 2019-01-15 | 宁波艾捷康宁生物科技有限公司 | Gene SNP site is applied in hypertension individuation drug therapy genotype detection and kit |
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