CN101087891A - Method for treatment with bucindolol based on genetic targeting - Google Patents

Abstract

The present invention concerns the use of methods for evaluating bucindolol treatment for a patient, particularly one with heart failure. It concerns methods for determining whether to administer or prescribe bucindolol to a patient based on whether the patient is homozygous for the Arg 389 polymorphism in the beta1-adrenergic receptor (AR).

Description

Use the methods of treatment of bucindolol based on gene target

Background of invention

The application requires the U.S. Provisional Patent Application 60/609,689 submitted on September 14th, 2004 and the right of priority of the U.S. Provisional Patent Application 60/610,706 submitted on September 17th, 2004, they by reference integral body be incorporated herein.

According to subsidy HL052318, HL07071609, ES06096, HL071118 and the HL48013 of NIH (the National Institutes of Health), government may have the right among the present invention.

1. technical field

The present invention relates to pharmacogenetics and Cardiology.More particularly, the present invention relates to use based on the genotype of polymorphism in patient's adrenergic receptor gene the method for the individuation heart failure therapy of bucindolol, wherein said adrenergic receptor gene comprises β ₁-adrenergic receptor (β ₁AR) gene and α _2c-adrenergic receptor (α _2cAR) gene.

2. association area is described

According to (the American Heart Association of American Heart Association, AHA), about 62,000,000 Americans have the cardiovascular diseases of certain form, it can comprise the inborn defect and the congestive heart failure of hypertension, coronary heart disease (heart attack and pectoralgia), apoplexy, heart and blood vessel, and has every year approaching people up to a million to die from these illnesss.Next American's death toll that the cardiovascular diseases of also saying the annual report of AHA causes comprises more than the summation of 7 kinds of causes of death of cancer.Be that the female patient of cardiovascular diseases is slightly more than the male patient on the whole unexpectedly.The U.S. was in 1999, and heart trouble accounts for 40% of all death.Although the existing development of methods of treatment recently, mortality ratio in heart failure in 5 years is still near 50%.

Singly just there are nearly six million peoples to suffer from chronic heart failure (" HF "), account for 1.5% of population greatly, and make a definite diagnosis 550,000 new patients every year in the U.S..Although medical therapy makes much progress M ﹠ M still very high (Mann et al., 2005) on treatment HF.Current standard care method to HF relates to the inhibitor (ACE inhibitor, ARB and/or aldosterone receptor antagonist) of application renin-angiotensin-aldosterone system (RAAS) and the beta blocker that the competitive inhibition myocardial cell goes up B-adrenergic receptor.Beta blocker is being effectively aspect the reduction mortality ratio and is being considered to the most effective generally HF drug type, but still a treatment patient to 50-60% has effect.In addition, U.S. patient clinical data about approved beta blocker curative effect in the mortality ratio test is not very attracting, show that from the public data in unique extensive, purpose treatment mortality ratio test the mortality ratio in U.S. patient has been compared increase with placebo.

Therefore, treatment has substantive needs for improved HF, and these improved HF treatments have the subpopulation that higher curative effect and the speed of response, quilt tolerate and are suitable for having special requirement such as diabetes better better.Yet, face great challenge at the agent of this treatment background developing new drug is verified.Since calendar year 2001, in 13 III clinical trial phases of HF, had only three to be positive.Use ARB (CARDESARTAN, candesartan) (McMurray et al., 2003 for two in these positive tests; Granger et al., 2003).The 3rd positive test, the A-HeFT test of using BiDil (combination of Dilatrate-SR and hydralazine) is a subgroup test (non-descendants American), wherein only comprises 12% U.S. HF crowd (Taylor et al., 2004).Be clear that, need to continue Development of New Generation HF medicine.

Though use β ₁The depleted patient of the ventricular function of agonist treatment acute exacerbation, but contacting or causing worsening heart failure more of heart and using agonist prolonged by the rising of health generation catecholamine.By contrast, B-adrenergic receptor antagonist (being called as beta blocker) is used in chronic heart failure as main treatment pattern.

Early stage in nineteen nineties, one group of U.S. researchist in heart failure who uses beta blocker in heart failure judges, in order to confirm this still disputable therapy, need carry out the mortality ratio test.A scheme and fund application have been drafted by a U.S. group, and this application is subsidized by VA cooperation clinical study plan and NHLBI approval subsequently.Scheme through approval does not have the concrete regulation medicine, but regulation is selected best beta blocker based on the II issue according to the potentiality of achievement and intensity.The medicine of being considered is carvedilol, tartrate metoprolol, succsinic acid metoprolol CR/XL and bucindolol.The tartrate metoprolol is excluded, because at MDC test unresolvable tartaric acid metoprolol the effect of mortality ratio is lower than expectation (Waagstein et al., 1993); Do not select succsinic acid metoprolol CR/XL, because it lacks the data of curative effect and tolerance in heart failure; Do not select carvedilol, in part because its tolerance poor (Krum et al., 1995) in the heart failure late.Excellent tolerance (Eichliorri et al., 1997 based on bucindolol; Gilbert et al., 1990; Bristow et al., 1994; Pollock et al., 1990), curative effect (Gilbert et al., 1990; Bristowet al., 1994; Pollock et al., 1990; Eichhorn et al., 1990) and promoter's concern level, bucindolol is that the consistent of the selection council selected.Therefore bucindolol becomes the object of beta blocker survival rate evaluation test (" BEST "), and this is first HF mortality ratio test of plan and startup.

The BEST test finished in 1999 in nineteen ninety-five.After starting BEST, plan has also started other three mortality ratio test, use MERIT-HF (the MERIT-HF Study Group of succsinic acid metoprolol CR/XL, 1999), use CIBIS-II (the CIBIS-II Investigators of bisoprolol, 1999) and use the COPERNICUS (Packer et al., 2002) of carvedilol.Since these test into group quicker and the restriction still less, CIBIS-II and MERIT-HF finished before BEST, and these two tests have all obtained positive result.

The BEST test is before finishing, finished prematurely in 1999, this part is because the researchist has lost equilibrium, and based on other two the positive understandings of testing are continued to increase (BEST Trial Investigators, 2001 to the ratio of opening the beta blocker label; Domanski etal., 2003).The promoter does not have to select to proceed the business development of bucindolol based on the known results when test stops.Although BEST researchist observes useful result in the 3rd class (Class III) non--non-descendants American, this is similar to the positive result reported among CIBIS II and the MERIT-HF before six months, and the researchist observes relatively poor result in the 4th class (Class IV) and non-descendants American patient.In addition, when test stopped, the main research terminal point that BEST does not satisfy its whole reason mortality ratio (reduced by 10%, p=0.10) (BEST TrialInvestigators, 2001).The researchist infers that perhaps the difference between other beta blocker and bucindolol be attributable to " pharmacological characteristics of bucindolol uniqueness " (BEST Trial Investigators, 2001), this has given prominence to the chemistry and the middle viewed difference of functional performance of this diversity type compound.

In addition, even most of beta blocker has demonstrated the useful effect of grouping in heart failure, but in the result that can not explain, very big interindividual variation (CIBIS-II Investigators, 1999) is arranged by baseline clinical characteristics.In fact all found to the interindividual variation in the reaction of pharmacotherapy for nearly all medicine.In some situation such as in all high chronic heart failure of beta blocker treatment M ﹠ M, the titration algorithm is complicated, interindividual variation is big, and have other treatment option, so assessment has significant effects to the possibility of favourable (or unfavorable) long reaction of pharmacotherapy to decision-making.5 annual death rates of heart failure patient nearly 50% have impelled the big quantity research that treatment is selected and have produced multiple medicines object space case, wherein generally include beta blocker, angiotensin-convertion enzyme inhibitor (or angiotensin receptor antagonist), diuretic(s) and digoxin.In relatively stable patient,, and during some months, slowly be increased to target dosage or tolerable dose with the initial beta blocker treatment of low dosage (promptly about 10mg).During the titration that makes progress, the initial of the dose titration of other medicines or extra drug is not uncommon.Therefore must be with beta blocker treatment method in heart failure by individuation.In fact, the statement of " administration must by individuation and tightly monitored " can be found in U.S.'s approval is used for the treatment of the prescription information of two kinds of beta blocker preparations in heart failure.In addition, the promoted reverse of beta blocker of cell that studies show that failure heart in animal model and people and whole body reconstruction may need the stable treatment (Lowes et al., 2002) of some months.Have been noted that substantial differences, comprise that left ventricular ejection fraction (LVEF) changes (van Campen et al., 1998), exercise tolerance (Bolger, 2003) and survival rate (Packe et al., 2001) the beta blocker reaction.Yet, based on data importance, should consider to treat most of Patients with Chronic Heart Failure with beta blocker, wherein hypothesis does not have contraindication to transship (volumn overload), need variable force infusion (inotropic infusions), bradyrhythmia, Hemodynamics unstable and asthma such as volume.

Therefore, not only between various beta blockers, especially bucindolol is compared with other beta blocker, can feel difference, and also observes difference aspect the ability that specific beta blocker treatment is reacted in different patients' deflections.Need especially explain the evidence of these interindividual variations to the evidence of bucindolol therapeutic value.

Summary of the invention

Thereby the invention provides based on to influencing the method for individual evaluation to polymorphism in the adrenergic receptor of bucindolol reaction at the individuation treating cardiovascular disease.In certain embodiments, it relates to the individualized treatment at heart failure.

In certain embodiments, provide assessment bucindolol treatment patient's method, it comprises the sequence information that obtains relevant at least one polymorphism in patient's adrenergic receptor gene, and wherein said information can indicate the curative effect of bucindolol in the patient.Sequence information can be nucleic acid sequence information and/or amino acid sequence information.In specific embodiments, the adrenergic receptor gene is β ₁AR or α _2cAR.In some cases, obtain the sequence information of relevant polymorphism in two genes.

In addition, described polymorphism is included in β ₁-adrenergic receptor (β ₁AR) on 1165 Nucleotide in the gene, 389 amino acids in its corresponding coded protein, another is at α _2cOn the 964-975 position Nucleotide of AR gene, 322-325 amino acids in its corresponding coded protein.

The present invention is based on the contriver and determines, if 389 arginic β in the encoding gene product ₁The AR gene isozygotys, and just provides the physiological condition that can obey the bucindolol treatment to the patient.In addition, the present invention is based on definite, causes the α of gene product 322-325 amino acids disappearance _2cDisappearance in the AR gene is deleterious for treating the heart failure in late period with bucindolol.Term " treatment " is interpreted as the treatment that is diagnosed as the patient of cardiovascular disorder or cardiovascular patient with sympotoms is arranged relevant, and is relative with preventive measure.

Normally understanding is that polymorphism appears under the genetic background; Yet, influencing in the situation of coded gene product in polymorphism, the variation in this gene product also can be called as polymorphism.

According to the present invention, described method comprises whether assessment is prescribed or used bucindolol and give cardiovascular patient, it comprise from the patient obtain relevant his/her adrenergic receptor gene and/or influence to the information of polymorphism in its encoding gene product of bucindolol reaction.

Therefore, the present invention relates to directly or by determining β ₁1165 and/or α on the AR gene _2c964-975 position nucleotide sequence is derived and is obtained relevant β on the AR gene ₁AR and/or α _2cThe information of polymorphism and prescribe or use bucindolol in the AR protein based on the information of being obtained.It should be understood that for β ₁AR or α _2cTwo chains of any cDNA that the proteinic nucleic acid of the same clan of AR comprises the mRNA transcript of code for said proteins, produce from described mRNA transcript and about β ₁AR or α _2cTwo chains of the genomic dna of AR gene.

To cardiovascular patient at β ₁389 and/or the α of AR _2cThe knowledge which kind of polymorphism the 322-325 position of AR has provides the assessment basis of bucindolol to the patient of whether using or prescribe.

The present invention also differentiates will the heart failure patient of active responding to the application beta blocker especially treatment of bucindolol.

The present invention also offers needs the individuality of this treatment to send the beta blocker especially device and the composition of bucindolol.

Method of the present invention comprises determines the β of heart failure patient at individuality ₁Genotype on the AR gene is not if wherein the patient is β ₁The carrier of Gly389 (that is, one or two Gly389 allelotrope being arranged), the patient may show positive reaction to the bucindolol of standard dose so.In one embodiment, prescribe bucindolol to β ₁The heart failure patient that Arg389 isozygotys.Method of the present invention it is also conceivable that, is knowing that the patient is that " β isozygotys ₁Arg389 " the basis on, promptly mean two β of patient ₁The AR gene is 389 arginine in the encoding gene product all, prescribe on this basis or the bucindolol of using standard dose is given the patient who needs this treatment.Method of the present invention relates to prescribes or uses bucindolol and give the β that isozygotys ₁Arg389 patient, in any case and this be to determine, the patient has this genotype.

In other embodiments, method of the present invention comprises determines that heart failure patient is at individual α _2cGenotype on the AR gene is not if wherein the patient is α _2cThe carrier of Del322-325, the patient may demonstrate positive reaction to the bucindolol of standard dose so.In certain embodiments, bucindolol is write out a prescription to α _2cMiddle 322-325 amino acids is the heart failure patient of homozygous wildtype (that is, amino acid does not have disappearance).Method of the present invention it is also conceivable that, is " homozygous wildtype α understanding the patient _2cAR " the basis on, promptly the patient is at coding for alpha _2cThe α of 322-325 amino acids among the AR _2cDisappearance not in the AR gene order is prescribed on this basis or the bucindolol of using standard dose is given the patient who needs this treatment.Method of the present invention relates to prescribe or use that bucindolol gives for disappearance be that patient's (not being the disappearance carrier) of homozygous wildtype and this do not consider how to determine patient's genotype.

Consideration in some cases is that the patient can carry out gene type assay to one of these polymorphisms, then determines other polymorphism subsequently; In this case, two different samples of assessment.As an alternative, can obtain single sample and assess two kinds of polymorphisms of separating.In another embodiment, bucindolol is write out a prescription to having the β of isozygotying ₁Arg389 and homozygous wildtype α _2cThe heart failure patient of the dual-gene type of AR (diplotype).Method of the present invention it is also conceivable that, is understanding the patient for β ₁Arg389 or for wild-type α _2cThe combination of AR or dual-gene type is on the basis of isozygotying, and prescribes or the bucindolol of using standard dose is given and needed the patient of treatment like this.

Method of the present invention also comprises, according to individuality with respect to β ₁Whether Arg389 is (and not being the 389Gly carrier) of isozygotying and/or for α on the AR gene _2cWhether AR gene individuality is homozygous wildtype and is not α _2cOn Del322-325 carrier's the basis, determine whether individuality has similar pathological and physiological condition, be such as but not limited to DCM (dilated cardiomyopathy), ischemic cardiomyopathy, ischemic heart disease (stenocardia (angina), myocardial infarction), pheochromocytoma, migraine, irregular pulse, hypertension and various anxiety disorder (anxiety disorder) may have positive reaction to the standard dose bucindolol.

In certain embodiments, the present invention relates to be used to assess bucindolol treatment patient's method, it comprises that understanding (i) is at patient β ₁Sequence on 1165 Nucleotide of one or two encoding sequence of AR gene or (ii) at patient β ₁Proteinic 389 amino acids of AR, wherein said individuality is being considered to treat with bucindolol.

In other embodiments, the present invention relates to be used to assess bucindolol treatment patient's method, it comprises understanding (i) one or two α the patient _2cIn the sequence of the allelic 964-975 of AR position Nucleotide or (ii) at patient α _2cIn the proteinic 322-325 amino acids of AR whether disappearance is arranged, wherein said individuality is being considered to treat with bucindolol.

Can consider not to be that the protein to all patients in any embodiment of the present invention all assesses, but consider to obtain sample and some protein in the sample are assessed its protein sequence.

The present invention it is also conceivable that according to the common and usual meaning of term " understanding " and represents to have customizing messages.Can consider generally that the medical science practitioner will assess and whether prescribe or use that bucindolol is given the patient and the medical science practitioner will arrange one or more about one or two β of patient described in this assessment implementing ₁AR allelotrope or its coded protein and/or about one or two α of patient _2cThe check of AR allelotrope or its coded protein.Under the polymorphism background of being discussed in this article, term " allelotrope " and " gene " but mutual alternative when using.

Other aspects of the present invention comprise the method that is used for the treatment of the patient with heart patient's condition, and it can comprise that the bucindolol of prescribing or using significant quantity gives the patient, are the β of glycine but wherein the patient do not have detection level in 389 positions ₁AR protein or wherein at two β ₁1165 cytosine(Cyt) patients are isozygotied in the allelic nucleotide coding sequence of AR.In any situation, if doctor or medical science practitioner by the patient at β ₁Have the Arg389/Arg389 polymorphism in the AR gene and know that bucindolol is suitable medicine for the patient, they just can prescribe and use bucindolol so.

As an alternative or in addition, the method that treatment has a patient of the heart patient's condition can comprise that the bucindolol of prescribing or using significant quantity gives the patient, but wherein the patient does not have the α with amino acid 322-325 disappearance of detection level _2cAR protein or wherein at two α _2cHaving Nucleotide 964-975 (" not having disappearance ") patient in the allelic encoding sequence of AR isozygotys.In any situation, if doctor or medical science practitioner are not α by the patient _2cThe carrier of Del322-325 polymorphism in the AR gene, promptly be not that heterozygosis neither be isozygotied and know that bucindolol is suitable medicine for the patient for described disappearance, they just can prescribe and use bucindolol so.

Other method comprises assessing whether heart failure patient will produce positive reaction to bucindolol, it comprises: a) obtain indication i) one or two β of patient ₁There is polymorphism on 1165 coding sites in the encoding sequence of AR gene or ii) at β ₁The information that has polymorphism on the proteinic 389 amino acids positions of AR; And b) prescribes or use bucindolol.

In addition, other methods that the present invention comprised relate to bucindolol treats the patient, and it comprises: a) obtain indication i) one or two β of patient ₁There is polymorphism on 1165 coding sites in the allelic encoding sequence of AR or ii) at β ₁The information that has polymorphism on the proteinic 389 amino acids positions of AR; And b) or open the bucindolol treatment prescription and give the patient, wherein patient's genotype shows that the patient is for β ₁Arg389 in the AR protein isozygotys, and does not perhaps open the bucindolol prescription to the patient, and wherein patient's genotype shows that the patient is for β ₁Arg389 in the AR protein does not isozygoty.

It is also conceivable that the present invention relates to bucindolol has β in the preparation treatment ₁Purposes in the AR gene in the medicine of patient's cardiac patient's condition of Arg389/Arg389 polymorphism.The embodiment of discussion method can be used bucindolol to prepare medicine and implement.

The invention still further relates to, from just consider patient, obtain biological specimen with the bucindolol treatment, and by determining (i) patient β ₁The sequence of 1165 Nucleotide or (ii) patient β in one or two encoding sequence of AR gene ₁389 position amino acid determine that thereby the Arg389 polymorphism assesses it in the AR protein.If the present invention considers assessment β ₁Whether AR protein can seek so that to contain any in the sample be the β of glycine in 389 positions ₁AR protein.

Other method comprises whether the assess heart failure patient has positive reaction to bucindolol, and it comprises: a) obtain indicate whether i) at one or two α of patient _2c964-975 position nucleotide sequence has lacked or ii) at patient's α in the AR allelotrope _2cThe information that 322-325 amino acids sequence has lacked in the AR protein; And b) prescribes or use bucindolol.If the present invention considers α _2cWhether AR protein can be sought so and contain any α with relevant disappearance in the sample _2cAR protein.

In addition, other methods that the present invention comprises relate to bucindolol treats the patient, and it comprises: a) obtain indicate whether i) at one or two α of patient _2c964-975 position nucleotide sequence has lacked or ii) at patient's α in the AR allelotrope _2cThe information that 322-325 amino acids sequence has lacked in the AR protein; And b) or to the patient open the prescription of bucindolol treatment, wherein patient's genotype indication patient is for α _2cAR allelotrope is homozygous wildtype, does not perhaps open the prescription of bucindolol treatment to the patient, and wherein patient's genotype indication patient is for α _2cAR protein is not homozygous wildtype.

It is also conceivable that the present invention relates to bucindolol treats at α in preparation _2cIt in the AR allelotrope purposes in the medicine of patient's cardiac patient's condition of homozygous wildtype.The embodiment of discussion method can be used bucindolol to prepare medicine and implement.

The invention still further relates to, from the patient who just considers the bucindolol treatment, obtain biological specimen, and by determining (i) patient β ₁The sequence of 1165 Nucleotide in allelic one or two encoding sequence of AR, (ii) patient β ₁389 amino acids in the AR protein, (iii) at one or two α _2cWhether 964-975 position Nucleotide has disappearance and/or (iv) at patient's α in the allelic encoding sequence of AR _2cWhether the 322-325 amino acids has disappearance in the AR protein, determines β ₁Arg389 polymorphism and/or α in the AR protein _2cThereby the Del322-325 polymorphism is assessed it in the AR protein.

For realizing these methods, doctor, medical science practitioner or their colleague can obtain the biological sample that is used to assess.Described practitioner or their colleague can analyze described sample, perhaps it can give the outside or laboratory independently.Medical science practitioner can know described detection whether just providing with from the same relevant patient β of coded protein identification ₁AR gene or allelic information, perhaps the medical science practitioner can only understand described detection and directly or indirectly indicates patient's genotype reflection Gly389/Gly389 phenotype (" Gly isozygotys " sequence), Arg389/Gly389 phenotype (" heterozygosis " sequence) or Arg389/Arg389 phenotype (" Arg isozygotys " or " homozygous wildtype " sequence).

Similarly, the medical science practitioner can know described detection whether just providing with from the same relevant patient α of coded protein identification _2cAR gene or allelic information, perhaps the medical science practitioner can only understand described detection and directly or indirectly indicates patient's genotype reflection homozygous wildtype sequence (in the equipotential gene in office not disappearance), heterozygosis Del322-325 phenotype (" heterozygosis " sequence) or Del322-325/Del322-325 phenotype (" homozygous deletion " sequence).

In some embodiments of being discussed in an embodiment, for β ₁AR has or the patient of the heterozygosis sequence or the Gly sequence of isozygotying is known as " Gly carrier ".Similarly, for α _2cAR has or the patient of heterozygosis sequence or homozygous deletion sequence is called as " Del322-325 carrier " or " disappearance carrier ".

In these any situations, medical science practitioner " knows " and will allow him or she to determine whether that bucindolol is the relevant information that suitably medical treatment is selected.Can consider that for example, the described detection of laboratory implementation is to determine patient's genotype, so its staff also knows suitable information.They the concrete outcome of implementing to detect can be reported to described professional or the laboratory can report simply that based on the experimental result bucindolol be suitable medicine.

In further embodiment, the patient is at one or two β ₁Genotype in the AR allelotrope encoding sequence on 1165 in the Nucleotide is known.In the present invention, whether 1165 positions of encoding sequence comprise guanine or cytosine(Cyt) is important in one or two allelotrope.This indication can be at β ₁389 positions are found any amino acid in the AR protein.1165 cytosine(Cyt) coding arginine in encoding sequence, and the coding of the guanine in encoding sequence glycine.In specific embodiments, at two β of patient ₁1165 bit sequences are known in the AR allelotrope.The result can be to be to be cytosine(Cyt) in guanine, two allelotrope or to be guanine in an allelotrope and to be cytosine(Cyt) in another allelotrope in two allelotrope.

In certain embodiments, the patient is at one or two α _2cGenotype in the AR allelotrope encoding sequence on the Nucleotide 964-975 position is known.In the present invention, at α _2cWhether disappearance is arranged in one or two allelotrope of AR gene is important.This indication is at α _2cIn the 322-325 amino acids of AR protein sequence (amino acid 322,323,324 and 325) whether aminoacid deletion is arranged.In specific embodiments, at two β of corresponding patient ₁Whether disappearance is arranged in the nucleotide sequence of 964-975 position in the AR allelotrope is known.

Those skilled in the art are readily appreciated that the encoding sequence of gene represents to be used to transcribe the gene strand of messenger RNA(mRNA).The sequence of encoding sequence is complementary to the transcript sequence of being transcribed.Owing to the sequence complementarity between encoding sequence and the non-coding sequence, can determine the sequence of any encoding sequence by the sequence of understanding transcript, noncoding strand or coded protein.The known nucleotide sequence that has a lot of modes can determine position described in one or two allelotrope of those skilled in the art.Such mode comprises, but be not limited to chain termination sequencing, restriction enzyme digestion, allele-specific polymeric enzyme reaction, single-strand conformation polymorphism analysis, genetic analysis (genetic bit analysis) in a small amount, temperature gradient gel elec-trophoresis (TGGE) or ligase chain reaction (LCR).

As an alternative, can assess β ₁The AR protein sequence.In certain embodiments, at one or more β of patient ₁389 amino acids are known in the AR protein.Can consider that any sample from the patient of being assessed will comprise multiple β ₁AR protein can be analyzed.These proteinic analyses can determine whether the patient have on 389 just arginine, on 389 just glycine or two types of blended β ₁AR protein.Similarly, can assess α _2cThe AR protein sequence.In specific embodiments, at one or more α of patient _2cIn the AR protein 322-325 amino acids whether have the disappearance be known.

Can consider that any sample from the patient of being assessed will comprise multiple β ₁AR and α _2cAR protein can be analyzed.These proteinic analyses can determine whether the patient have on 389 just arginine, on 389 just glycine or two types of blended β ₁AR protein.Similarly, it can determine whether that the patient only has wild-type sequence (do not have disappearance), only has disappearance or two types of blended α of corresponding 322-325 amino acids in the 322-325 amino acids _2cAR protein.

The method of determining specific position sequence in the protein is well-known to those skilled in the art.They can relate to uses antibody, high pressure liquid chromatography or mass spectroscopy.

As mentioned above, β ₁AR gene or protein and/or α _2cThe sequence of specific position can be known in AR gene or the protein.Certain methods of the present invention relates to determines β ₁AR gene or protein sequence and/or α _2cSequence in AR gene or the protein sequence.

Therefore, can consider that described embodiment can relate to from the patient obtains biological specimen.Biological specimen is to comprise the sample of biomaterial such as all or part organ, tissue, cell, nucleic acid, protein or other such macromole and material.Described sample can comprise saliva, serum, blood, blood plasma, spinal fluid, seminal fluid, lymph liquid, urine, excrement, pleural effusion, ascites, tissue samples, biopsy sample, cell swab or their combination.In other embodiments of the present invention, described sample can comprise the cell from lung, skin, muscle, liver, kidney, colon, prostate gland, breast, brain, bladder, small intestine, large intestine, uterine neck, stomach, pancreas, testis, ovary, bone, marrow or backbone.In some embodiments, described sample is whole blood, blood plasma or serum sample, however in other embodiments, by lavation, be coated with or wipe away on the patient or among the zone obtain described sample.In certain embodiments, biological specimen is the blood sample basis.

In some embodiments of the present invention, patient's β ₁AR gene and/or proteinic sequence and/or patient's α _2cAR gene and/or proteinic sequence can be evaluated mistakes.The intent of the present invention is to finish this analysis before the patient is just considering the bucindolol treatment or as the part of Integrated Checkout.For example, patient's β ₁AR gene and/or proteinic sequence and his α _2cAR gene and/or protein can be determined and input database or the input patient case history.In this case, the medical science practitioner can pass through to obtain the relevant i of patient) one or two β ₁1165 positions or ii) β in the AR allelotrope encoding sequence ₁389 positions in the proteinic aminoacid sequence of AR, iii) one or two α _2c964-975 position and/or iv) α in the AR allelotrope encoding sequence _2cThe historical data of the sequence of 322-325 position has come what solution sequence is in the proteinic aminoacid sequence of AR.

The invention still further relates to and be reported in β ₁AR allelotrope or protein and/or α _2cThe measurement result of nucleic acid or protein sequence on the relevant position in AR allelotrope or the protein.In certain embodiments, described method comprises that preparation comprises (i), result's (ii), (iii) and/or (iv) the report described in the mensuration leading portion.This report will be discerned the patient by name, SSN (social security number) and/or other identification number or qualification.It also can comprise the real data as measurement result or the summary of its data.

In some embodiments, described method comprises that evaluation may need the patient of bucindolol treatment.Can have some symptoms to bucindolol as the patient who treats option or made a definite diagnosis and suffered from some medical condition for just considering, such as heart failure, DCM (dilated cardiomyopathy), ischemic heart disease, pheochromocytoma, migraine, irregular pulse, hypertension or anxiety disorder.In certain embodiments, the patient has the symptom of ischemic heart disease or has been diagnosed as ischemic heart disease, and what it can be concrete is angina and/or myocardial infarction.In some particular cases, the patient has symptom in heart failure or has been diagnosed as heart failure.Described heart failure can be considered the heart failure in late period, although the present invention can be not limited to this patient.Use term " heart failure in late period " according to the meaning common and usual in the Cardiology field.In some embodiments, the patient who opens bucindolol prescription can have III level or the heart failure of IV level according to the NYHA hierarchy system.The NYHA hierarchy system is a kind of evaluating system, yet the intent of the present invention is not limited to this mode and this meaning that just illustrates rather than limit.The patient can classify according to another such system.The present invention is other is intended that the patient and can classifies according to diverse ways, but the present invention will implement in a similar manner.

Yet in other embodiments, the patient can have heart failure but not be the sign or the symptom of heart failure in late period.In this case, according to the NYHA categorizing system, the patient maybe can be characterized as I or II level heart failure patient.In these embodiments, can measure the relevant Arg389 β of patient ₁The genotype of polymorphism, the people who has the Arg/Arg phenotype in the case is the candidate of bucindolol treatment.Therefore, method of the present invention can comprise, if by determining whether the patient has Arg389/Arg389 polymorphism and they and have and just use the heart failure that bucindolol prevents the patient.Perhaps, specific patient is specially adapted to this, but is not limited to have heart failure symptoms, has the risk of heart failure factor or has family history in heart failure or the patient of the history of formerly falling ill.

In addition, described method can relate to other therapeutical agent or implement surgical intervention or other gets involved strategy used or prescribe in order to treat the patient.

According to the present invention, described method can also relate to understand the genotype of patient about 389 polymorphisms be Arg389/Arg389, also known be to isozygoty to prescribe after the arginine genotype or use bucindolol to the patient.In addition or as an alternative, can have the wild-type α that isozygotys understanding the patient _2cThe AR polymorphism is (at α _2cArbitrary allelotrope of AR gene does not carry the disappearance of 964-975 position Nucleotide) afterwards, prescribe or use bucindolol and give the patient.In addition, can be this situation, do not show arbitrary or two kinds of genotypic patients will or use bucindolol not to its prescription.Can write out a prescription or use especially to the patient be the beta blocker of bucindolol.

In other embodiments, described method can comprise that understanding is at (iii) one or two α of patient _2cIn the nucleotide sequence of 964-975 position or (iv) one or more α of patient in the AR gene coded sequence _2cIn the proteinic 322-325 amino acids of the AR sequence whether disappearance is arranged.Can also understand the patient at β in addition or independently ₁The genotype of AR gene 11 65 positions (proteinic 389 positions).

If heart failure in late period (that is, NYHA III level or IV level) patient shows the α that isozygotys _2cARDel322-325 genotype and patient do not show the Arg389/Arg389 genotype, consider so will not prescribe to the patient or use bucindolol.In certain embodiments, the patient is accredited as the patient of black race.As an alternative, if the patient does not show the α that isozygotys _2cAR Del322-325 genotype can be prescribed or uses bucindolol to the patient.

When determining whether bucindolol is suitable drug for the patient, it is also conceivable that out of Memory.This can comprise risk, drug allergy, the drug toxicity of diagnosis, other disease or the patient's condition of history, other disease or the patient's condition of ethnic group, sex, age, former surgical intervention, stage in heart failure, the relevant cardiovascular disorder of patient and/or the other medicines of just taking.

Therefore, the intent of the present invention also relates to the result who implements dual-gene type analysis or obtain dual-gene type analysis.In specific embodiments, dual-gene type analysis relates to direct or indirect assessment (1) β ₁Thereby 389 polymorphisms determine whether that the patient has Arg389/Arg389 genotype and (2) α among the AR _2cThereby 322-325 position polymorphism determines whether that the patient has the Del322-325/Del322-325 genotype among the AR.Other polymorphism can be included in the haplotype, and especially those are affected or influence the patient to make the polymorphism of favourable response capacity as the bucindolol of therapeutical agent.

Any embodiment of discussing about one aspect of the invention will be applied to others of the present invention equally.This comprises about β ₁AR and α _2cEach embodiment of discussing among the AR.Especially, relevant β ₁Any embodiment that AR gene, allelotrope or protein are discussed can be implemented on α _2cAR gene, allelotrope or protein, and vice versa.

Embodiment in the embodiment part should be understood that can be applicable to the embodiment of the present invention of all aspects of the invention.

Term " or " in the claims application be used to represent " and/or " the meaning, unless spell out just one of them or these options are to repel mutually, although disclosed content support only represent one of them and " and/or " definition.

In this application, term " approximately " is used for representing that numerical value comprises standard error or the deviation that is used for determining this value equipment therefor or method.

According to long-standing patent law, when word " " and " a kind of " and word " comprise " when being used in combination in claim or specification sheets, unless specifically indicate the meaning that its expression is one or more.

From the following detailed description, other purpose, feature and benefit of the present invention will be conspicuous.Yet, it should be understood that, although detailed description and specific embodiment illustrate specific embodiments of the present invention, but they just are used for illustrating, because the various changes and modifications in spirit and scope of the invention, from these describe in detail, will be conspicuous to those skilled in the art.

Description of drawings

The following drawings constitutes the part of this specification sheets and is used for further specifying some aspect of the present invention.By can better understand the present invention with reference to the specific embodiments one or more and that associating is described in detail herein in these accompanying drawings.

Fig. 1. describe several beta blockers, comprise bucindolol, chemical structure.

Fig. 2. based on II or III phase clinical testing data in heart failure or the different antiadrenergic agents of other data and the comparison diagram of treatment.

Fig. 3. this figure illustrates bucindolol at stably expressing beta ₁Arg389-or β ₁Allele-specific effect in the cell of Arg389.The result is the mean value ± SE of 4 tests.

Fig. 4. bar graph illustrates having target and crosses expression Gly389 and Arg389 β ₁The transgenic mice cardiac of AR is to the reaction of β-blocking-up.Shown is from β ₁-Arg389 and β ₁The mean value that obtains in the indicated proteinic Western trace in-Gly389 mouse (n=3-4 in the every group) heart (± SE) result.Data are with respect to contrasting (being untreated) numerical value by stdn.Only from β ₁Find overall therapeutic response (ANOVA, p＜0.002) in the heart of-Arg389 mouse to Proprasylyte (Proprasylyte).

Fig. 5. this figure illustrates according to β ₁The result's in heart failure of AR genotype classification 95% fiducial interval and risk ratio.

Fig. 6. this figure illustrate according to the treatment and β ₁Patient's survival rate in the bucindolol of AR genotype classification-placebo research.

Fig. 7. this figure illustrate according to the treatment and β ₁Reach the possibility of hospital care associating terminal point dead or in heart failure in the bucindolol of AR genotype classification-placebo research.

Fig. 8. 3 months and the variation from baseline noradrenaline levels ± SEM in 12 months according to the treatment group.Numeral in A and B under bar shaped is the number of patient in every group, and described patient has on the baseline and the interval measurement on each time point; The p value is used to the variation of comparison in each treatment group.

Fig. 9. with respect to full reason mortality ratio one or four/part, the risk ratio of baseline norepinephrine four/part.The norepinephrine point of contact of determining four/part is: the first, and≤304pg/ml; The second, 305pg/ml to 436pg/ml; The 3rd, 437pg/ml to 635pg/ml; The 4th, 〉=636pg/ml.Patient's number of each four/part is: in the placebo the one 294, the 2 255, the 3 248, the 4 264; In the bucindolol group the one 239, the 2 274, the 3 284, the 4 267.

Figure 10. with respect to the associating terminal point of full reason mortality ratio+CHF hospital care, according to the 1/1st risk ratio of baseline norepinephrine quartile value.

Figure 11. norepinephrine is in contrast in 3 months probability analysis of full reason mortality ratio subsequently in placebo and bucindolol treatment group.

Figure 12. stripped contractile response of non-depletion and depleted people's ventricle and β ₁The AR genotype is relevant.According to the right ventricle girder that is used to described in the method from non-depletion and depleted human heart.There is the girder of 11 hearts to study in every group in four groups.All Arg bands (strps) are all from the object that isozygotys.The Gly carrier is made up of 9 heterozygotes in 10 heterozygotes and the depleted group in the non-depleted group, and remaining is the Gly homozygote.The maximum that derives from dose effect curve is reflected at non-depleted group (P=0.01) and depleted the group in (P=0.008) for Arg389 all is bigger.

The dosage that Figure 13 A-D. bucindolol or xamoterol progressively increase is to isolating right ventricle girder peak value cardiac contractile force (mN/mm from depleted human heart ²) influence.Not have (bucindolol, group A; Xamoterol, group C) and (bucindolol, group B are arranged; Xamoterol, group D) by 10 ^-5Thereby M Forskolin pre-treatment enhancing β-AR signal transduction is finished dose effect curve.In the Forskolin experimental pretreatment, allow during whole treatment, to cultivate the girder of only using Forskolin, and from the girder of bucindolol or xamoterol processing, deduct any influence power. ^*,=p＜0.05 pair baseline tension force; , p＜0.10 pair baseline tension force; §, p＜0.05 pair entire curve is 0 slope;

, p＜0.05 pairs 10 ^-9M to 10 ^-60 slope of M dosage; The p value that bracket is relevant is to interactional check between the rate of curve, and wherein a p value is at 10 ^-9M to 10 ^-6The dosage of M and the 2nd p value are at entire curve.

Figure 14. chronic myocardium β ₁The opposition result that adrenergic stimulates.

Figure 15. treat the feature that norepinephrine changes among the patient in risk group with bucindolol.Standard deviation (SD) comprises in the drawings.

Figure 16. treat the other feature that norepinephrine changes among the patient in risk group with bucindolol.Standard deviation (SD) comprises in the drawings.

Figure 17. α in BEST patient _2CDependency illustrates between AR genotype and the whole body noradrenaline levels.

Figure 18. after three months, α in BEST patient _2C(dependency illustrates the reaction of AR genotype and whole body norepinephrine between the pg ± SEM).

Figure 19. after three months, α in BEST patient _2CThe reaction of-AR genotype, whole body norepinephrine (dependency between the pg ± SEM), and the survival rate of treatment group among the BEST patient.

Figure 20 A-D. β ₁The influence of-AR Arg/Gly 389 genetic mutations in isolating people RV girder, Racemic isoproterenol (isoproterenol) being reacted.A. β isozygotys ₁The Iso reaction of-AR Arg 389 depletion.B. β ₁The Iso reaction of-AR Gly 389 carrier's depletion.C. β isozygotys ₁The Iso reaction of-ARArg 389 non-depletion.D. β ₁The Iso reaction of the non-depletion of-AR 389 Gly carrier.

Figure 21. α _2C-AR and β ₁-AR genetic mutation: the therapeutic action in BEST (mortality ratio).

Figure 22. the beta blocker mortality ratio test in American population, mortality ratio risk ratio ± 95%C.I.s.

Figure 23. beta blocker in NF hRV girder to the influence of cardiac systolic function.

Figure 24. beta blocker enlarges the influence of the NF hRV girder cardiac contractile function of signal transduction handling through 10 μ M Forskolins (F).

Figure 25. beta blocker in the hRV of depletion girder to the influence of cardiac systolic function.

Figure 26. beta blocker enlarges in the depleted hRV girder of signal transduction the influence to cardiac systolic function handling through 10 μ M Forskolins (F).

Figure 27. research and design I.

Figure 28. research and design II.

Figure 29. research and design III.

Exemplary is described

As if the present inventor faces such data, and bucindolol provides still less favourable therapeutic response in some patient's subgroup, and compare still less favourable reaction according to some standard with other beta blockers.Change between the individuality that they propose to suppose in (HF) in heart failure bucindolol to be reacted is because the hereditary basis that changes and determined this hypothesis.In this process, the contriver can obtain the remarkable example to appropriateness in bucindolol therapeutic value and the treatment people heart failure thereof.

Assessed β ₁The hereditary variability of AR 389 amino acids (1165 Nucleotide in the gene) in protein.This is based on this characteristic that is called two kinds of acceptors of Arg389 and Gly389, and this confirms in transfectional cell that wherein the adenylyl cyclase activity of basal level and agonist stimulation is about 3 times higher (Mason et al., 1999) for the Arg acceptor.Yet before this research, unclear is that the patient who has Arg389, still has a Gly389 can be benefited from the bucindolol treatment more more.For example, the increased functionality of Arg389 may make bucindolol impossible as effective antagonist.Ultimate check to this hypothesis does not also realize, wherein assessment death, heart transplantation or HF hospital care (in the deterioration of the actual patient central force depletion for the treatment of with placebo or bucindolol).

Therefore the present invention uses three level methods whether to address Arg (or Gly) 389 β ₁The AR allelotrope representative prediction pharmacogenetics locus to the beta blocker reaction in heart failure is comprising studying in the clinical trial (seeing the following embodiment that goes through 3) of transfectional cell (seeing the following embodiment that goes through 1), transgenic mice (seeing the following embodiment that goes through 2) and extensive multicenter placebo.Clinical trial is represented as BEST (beta blocker assessment existence test).In described transfectional cell, estimated bucindolol stimulates cAMP to norepinephrine functional antagonistic action.The cAMP that even the Arg389 acceptor demonstrates obviously higher norepinephrine to stimulate generates, but this reaction of bucindolol antagonism.Compare with the Gly389 express cell, the absolute reduction that cAMP generates in the Arg389 express cell is obviously bigger, and this is because the height norepinephrine hormesis of Arg389 and the effect of the described reaction of the complete antagonism of bucindolol.At 0.1 μ M bucindolol, to observe about 80% of Arg389 cAMP reaction and suppress, this is equivalent to the drug plasma concentration (undisclosed data) of used dosage in BEST.These results suggest are with β ₁-Gly389 genotype is compared, owing to bucindolol treatment causing heart β ₁The AR activity is at β ₁Bigger variation is possible among the-Arg389 genotype patient, and causes more favourable clinical response potentially.In transgenic mice research, the contriver has checked that beta blocker is to key signal transmission and Ca in the heart during 6 months ²⁺Operation albumen (Ca ²⁺Handling protein) influence of Biao Daing.In the Gly389 mouse, handle the not influence of these protein expressions.On the other hand, the overall therapeutic action of β-blocking-up is significant in the Arg389 mouse, and has the reverse variation of rebuilding unanimity on molecular level.Next, be used to the preservation DNA from BEST, BEST provides the research of extensive phenotype analytical and coupling placebo; Because the result of transfectional cell and transgenic mice, the contriver has supposed β in advance ₁-Arg389 is to the best genotype of surviving.At this, supposed explicit mode (dominant model).Therefore, two genotype groups be the Arg389 homozygote and have the allelic patient of one or two Gly389 (that is, to Gly be isozygoty or heterozygote; This group is known as " Gly389 carrier ").Point out from the clinical endpoint result of BEST, compare with placebo, at β ₁Mortality ratio, hospital care in heart failure or mortality ratio+hospital care in heart failure does not obtain benefit among-the Gly389 carrier patient from the bucindolol treatment, but at β ₁All three kinds of results with the bucindolol treatment among the-Arg389 patient have obtained clinical relevant improvement.The baseline clinical parameter or the cause of disease in heart failure that comprise heart rate, blood pressure and LVEF are comprising all β ₁Can not indicate end reaction in whole group of the AR genetic mutation.In addition, β ₁-Gly49 polymorphism is to the not significantly influence of these relations.Combine and see, the result of these researchs points out strongly, β ₁389 variants of AR can be predicted the reaction to bucindolol in heart failure.

In addition, the role of hereditary variant in α 2cAR gene also is used to suppose the effect of bucindolol.The present inventor's hypothesis, the difference to the bucindolol reaction is because α between individuality in heart failure _2cThe inheritable variation of AR gene.Therefore the present invention addresses α with BEST _2cWhether Del322-325 AR allelotrope can represent the problem of prediction heart failure to the pharmacogenetics locus of bucindolol reaction, and described BEST is extensive multicenter placebo-controlled trial (seeing the embodiment of following detailed description).Be used to preservation DNA from BEST (research of extensive phenotype analytical and coupling placebo is provided).At this, supposed explicit mode.Therefore, two genotype groups are 1) α _2cWild-type homozygote (patient who does not have 322-325 position disappearance on the equipotential gene in office) and α _2cDel322-325 heterozygote or homozygote (have the patient of this disappearance, are called " α in one or two allelotrope _2cThe Del322-325 carrier ").Point out from the clinical endpoint result of BEST, compare with placebo, at α _2cMortality ratio, hospital care in heart failure or mortality ratio+hospital care in heart failure does not obtain benefit among the Del322-325 carrier patient from the bucindolol treatment, but at α _2cWild-type is isozygotied, and all the three kinds of results with the bucindolol treatment have obtained clinical relevant improvement among the patient.The baseline clinical parameter or the cause of disease in heart failure that comprise heart rate, blood pressure and LVEF are comprising all α _2cCan not indicate end reaction in whole group of the AR genetic mutation.Combine and see, the result of these researchs points out strongly, α _2cThe Del322-325 polymorphism can be predicted the reaction to bucindolol in heart failure.

Therefore, the present invention relates to utilize at Arg389 β ₁Between AR polymorphism and the bucindolol therapy and at Del322-325 α _2cThe method of the genetic affinity between AR polymorphism and the bucindolol therapy.

1. adrenergic receptor and beta blocker

Treatment in heart failure has related to target adrenergic receptor (AR).At least 9 adrenergic receptor hypotypes (Dohlman et al., 1991 are arranged; With Liggett et al., 1993), wherein at least 3 hypotypes are B-adrenergic receptors.

Reduce and process is closed the remarkable interindividual variation of treatment result by the myocardiac penetrance of family's cluster analysis, the family of phenotype, point out common hereditary variant to close latent effect in the therapeutic response at susceptibility, disease process.Although the polymorphism in the adrenergic receptor identified, also there is not research to relate to such patient data, wherein identified any polymorphism and to the dependency between the clinical response of therapeutical agent.The present invention relates to two kinds of polymorphisms: 1) coding β ₁The polymorphism of 389 amino acids and 2 among-the AR) coding for alpha _2CThe polymorphism of 322-325 amino acids among the-AR.Yet,, do not prove any dependency with bucindolol in the relation of still determining before the present invention between these concrete heritable variations and any treatment result without any clinical evidence yet.

A. β ₁Adrenergic receptor

β ₁Adrenergic receptor (β ₁-AR) be the main hypotype that the myocardial cell go up to express.Human heart had both been expressed β ₁AR expresses β again ₂AR hypotype (Bristow et al, 1986; Bristow et al., 1988).Every kind of receptor-mediated variable force and chronotropic response (Bristow et al., 1986 that endogenous catecholamine and external source are used agonist; Brodde et al., 1986; Brodde et al., 1992).

When being activated, when for example being activated by norepinephrine, β ₁The reaction of AR cardiac trigger shrinkability.In addition, β ₁Acceptor has central role in the process in myocardosis and other disease path.The activation of this acceptor strengthens and relevant myopathy and arrhythmia approach plays an important role in the natural medical history of heart failure.In case the myocardosis process begins, chronic β ₁Disease process is quickened in the activation of-adrenergic, because depleted heart is attempted by discharging more norepinephrines and increasing β ₁-receptor signal compensates its impaired function.The theory part of beta-receptor blocking-up is based on passing through blocking-up β ₁-acceptor and minimizing norepinephrine signal offset this myocardosis approach.

β ₁-adrenergic receptor cloned and check order (Frielle et al., 1987).This gene has been located in No. 10 chromosomal q24-q26 and has gone up (Yang-Feng et al., 1990).People β ₁AR has 477 amino acid whose aminoacid sequences of derivation.

At β ₁On 1165 coding nucleotides of AR gene, can discovery or cytosine(Cyt) or guanine in the crowd, this causes on 389 coded amino acids or Arg or Gly (Mason et al., 1999).This position is in the cell intracellular domain of acceptor, and described acceptor relates to and pungency G-albumen (G _S) coupling.In the inoblast of expressing two kinds of acceptors that equate level through transfection, β ₁-Arg389 acceptor and β ₁-Gly389 acceptor is compared, and demonstrates the hormesis bigger to adenylyl cyclase (Mason et al., 1999).More rare β ₁AR polymorphism Gly49 is also identified, but some the relevant inconsistent report of its functional effect (Rathz et al., 2002 is arranged; With Levin et al., 2002).

β ₁-AR 389Arg/Arg polymorphism is actually β ₁The form that adrenergic receptor is the most general and in American population, account for 50% (few slightly in the African American) greatly.The another kind of variant of this acceptor has glycine (Gly) and is considered to wild-type on 389, this for no other reason than that it cloned at first.In other known non-human animal's species, having arginine (Arg) on codon 389 is the structure of this acceptor preferred (and being unique), and 389 zones are arranged in the important function structural domain at all.389Arg/Arg also is the high functionality variant (Masonet al., 1999) of this acceptor; Its signal transduction effect is than the Gly heterozygote on 389 or the big 3-4 of Gly/Gly times (seeing embodiment and Mason et al., 1999).β ₁The increase of-AR 389Arg/Arg signal transduction ability can be applicable to cAMP and generates (Mason et al., 1999), the contraction of separation of human cardiac muscle (Mason et al., 1999) and produce myocardosis (Mialet Perez et al., 2003) in the transgenic mice.

Under specific inheritable variation background, assessed some β ^-Blocker also has Different Results.Sofowora et al. report, based on hemodynamic reaction, the patient that Gly389 isozygotys is lower to the susceptibility of atenolol USP 23 effect, and this is a kind of selectivity B-adrenergic receptor, and the described variation of prompting author may be especially relevant with static blood pressure response.Johnson et al. (1993) report, by measuring blood pressure, the Arg389 homozygote more may produce reaction to selectivity beta blocker metoprolol.Perez et al. (2003) utilizes the target transgenic mice to assess 389 varients of β 1AR under complete heart function background.At these on 389 or cross to express the Arg or cross in the transgenic mice of expressing the Gly that isozygotys of isozygotying, the Arg/Arg mouse has been lost the isoproterenol reactivity that is used to increase myocardial function to a greater degree, and rebuild degree and histology measurement according to myocardial dysfunction, chamber, have the more myocardosis of severity.They have also reported with non-selective beta blocker carvelilol treatment and have caused the bigger improvement of patient's left ventricular function, it with isozygoty or heterozygous state in the Arg389 polymorphism relevant.

Liu et al. (2003) has reported their and has found, compares with Gly389, and Arg389 is relevant with bigger reaction (according to changes in heart rate) to metoprolol.The author has also warned the expansion result to arrive above beyond their the patient storehouse, and their patient storehouse is healthy, young, male sex Chinese volunteer.They study in particular and do not find any long-term effect of metoprolol about described polymorphism.

2004 disclosed comment (Lohse, 2004) point out, although perhaps the benefit with beta blocker treatment is relevant for the Arg389 polymorphism, still less than about β ₁-adrenergic receptor polymorphism is for the research of heart failure to the influence of beta blocker reaction.Another comment is pointed out, although some studies show that the polymorphism of adrenergic receptor perhaps changes the reaction to the beta blocker treatment, because very low patient's quantity in difference research, so can not draw definite conclusion or the recommendation of disposing for the patient.

In addition, several reports are not at β ₁Detect any dependency between 389 polymorphisms of-AR and the reaction to the beta blocker treatment.Therefore, the disclosure of invention has remarkable meaning, although specification sheets has been mentioned reference White et al. (the discussion #1 of face as follows), the document is declared by metoprolol-treatment heart failure patient has been checked in the assessment of mortality ratio or mortality ratio+hospital care associating terminal point in heart failure, and is not found dependency.O ' Shaughnessy et al. (2000) report is not found blood pressure or the heart rate reaction difference relevant with 389 polymorphisms to β blocking-up (-two kinds of antagonists of atenolol USP 23 or bisoprolol).It is variant with 389 polymorphisms to the affinity of beta blocker that acceptor do not observed in another piece article (Joseph et al. (2004)) yet.In addition, nearest research report fails to find that any evidence shows " the pharmacogenetics effect " of Arg389 polymorphism for the metoprolol treatment.White et al.，Eur.J.Heart Fail 5：463-8，2003.

Therefore, beta blocker is as the validity and the β of a class therapeutical agent ₁Dependency among the-AR between 389 polymorphisms is not still established.In addition, especially do not have the dependency of acquisition about bucindolol, bucindolol is different from other beta blocker in several importances.Because the difference between these beta blockers is significantly, β ₁The AR-389 variant may depend on specific medicament to the influence of beta blocker reaction.

Present disclosure provides this data, and when in idiotype, especially under the background of Arg389 polymorphism during assessment BEST data, bucindolol has clear and definite therapeutic efficiency.This data are wonderful because in BEST research in full-fledged research colony observed mortality ratio effect be lower than with other beta blocker such as carvedilol, metoprolol CR/XL and the viewed result of bisoprolol.In addition, the science data that this paper provided have proved the dependency between bucindolol therapeutic efficiency and the two kinds of genetic variants for the first time.

At length, the invention provides and determine whether and bucindolol is write out a prescription to patient's method, wherein β ₁AR and α _2CThe polymorphic nucleotide of-AR or the identity of amino acid position are determined, and open or do not open the prescription of bucindolol based on the result of this diagnostic check.Similarly, based on genotype, can give to have unfavorable β ₁The genotypic patient of AR opens another kind of medicine, thereby attempts obtaining the clinical response of raising.In two kinds of situations, the decision of pharmacological agent is based on patient's β ₁The AR genotype.

Therefore, the present invention relates to, based on individuality whether at β ₁Be the Arg389 that isozygotys in the AR gene, whether be the α of wild-type form on amino acid position 322-325 _2CAR the or whether both is comes the especially method of the bucindolol treatment of heart failure patient of assess patient.As an alternative, the present invention relates to relevant β ₁AR389Gly carrier and α _2cThe method of the dual-gene type of ARDel322-325 carrier.

B. α _2CAdrenergic receptor

α _2C-AR is positioned at the cardiac sympathetic nerve tip, and regulates presynaptic (prejunctional) neurone release norepinephrine to the synaptic cleft zone.Norepinephrine and α _2CThe combination of-AR causes the reaction of reverse feedback sympatholytic, and this weakens further neuronal norepinephrine and discharges.Musculus cdna partly excises model hint α _2c-AR mainly is responsible for the speed steady in a long-term that the control norepinephrine discharges.(Hein et al.，1999)。α in this way _2c-AR has " protectiveness " effect in heart, the lasting rising of the noradrenaline levels that runs in the depleted human heart of buffering opposing.

Reported people's genetic polymorphism (Small et al., 2000) of α 2c-AR gene A DRA2C.The ADRA2C that loses 12 Nucleotide translates into and lack 4 continuous amino acid (α in the ring in acceptor control three cell _2cDel322-325).(Small et al.，2000)。This deletion polymorphism is more general in the African American, and African American's gene frequency is 0.4, but not the African American has 0.04 gene frequency.Generally, this polymorphism is present in about 15% the U.S. population.

Compare α with the Arg389 polymorphism _2CThe 322-325 Del polymorphism of adrenergic receptor is uncommon, and is the low-function variant (Small et al., 2000) of this acceptor.Lose this 4 reductions that residue is indicating function of receptors, this can test by cell transfecting and support that wherein function of receptors has been lowered 50-85%.(Small et al.，2000)。A _2CAcceptor suppresses norepinephrine usually consumingly in the adrenergic nerve tip presynaptic discharges (Hein et al., 1999).

The 322-325 disappearance has been destroyed function of receptors (Small et al., 2000) basically, causes higher levels of norepinephrine and suprarenin motility (Neumister et al., 2005).The result that inhibitory control goes down is that the basis release of norepinephrine is increased by essence, causes the more sympathetic activity of high state.(Hein et al.，1999)。This is meaningful especially in heart failure, because α _2c" protectiveness " braking action that Del322-325 receptor deficiency opposing sympathetic nerve power increases.

In a research, α _2cThe Del322-325 receptor polymorphisms only exists among 7.4% the chronic heart failure Caucasian as homozygous genotype; but in 52.6% chronic heart failure Black people, exist; cause by stimulating the afunction of assessing (Small et al., 2002) suppressing adenylyl cyclase.This defective is to α _2c-AR function has function effect, and has been called " polymorphism " and " sudden change " interchangeably, respectively the feature of the relative ubiquity among the reflection crowd and far-reaching structure/function thereof.In this application, described variant mainly is called as " polymorphism ", is the general variant that is present in many objects in heart failure to reflect this.Yet its importance is derived from its function effect, and it greatly reduces receptor active.Based on the effect in the heredity excision mouse, wild-type, Full Featured α _2cAcceptor discharges norepinephrine and has the presynaptic restraining effect; Lost this inhibition because described knock out mice shows, this causes noradrenaline levels to raise and the pathologic hypertrophy.(Hein et al.，1999)。Be tested and appraised α _2cThe Del322-325 genotype is for example understood the clinical importance of this polymorphism for the people as the research of the risks and assumptions of heart failure; Compare with non-carrier with heterozygote, it has 5.54 not adjustment for heart failure in having this genotypic homozygote Black people (95%CI 2.68,11.45 than (odds ratio) than number; P＜0.001).(Small et al.，2002)。With β ₁Arg389 variant associating, this effect is stronger, have 12.67 not adjustment than number than (95%CI 2.70,59.42; P=0.001).Yet, compare with present disclosure, it is relevant with the treatment distant view still not indicate these polymorphisms.

C. beta blocker

Although β ₁Agonist is used to treat the depleted patient's of ventricular function acute exacerbation such as dobutamine, and heart contacts with the prolongation of using agonist or the endogenous catecholamine agonist that improves the health generation causes the heart failure that worsens more.In fact, β ₁AR and β ₂It is insensitive that AR becomes in heart failure, and it is considered to resist a kind of self-protective mechanism of the high-level catecholamine that exists in the heart failure.Use beta antagonists and can improve ventricular function and clinical effectiveness, this supposition is because these harmful effects of blocking-up catecholamine.And in fact, during with the heart failure of β blocking treatment, expression and the function of heart β AR improve.The success of most of deleterious effect of catecholamine and Beta receptor blockers treatment is because β ₁The variant of AR hypotype.(Zhu et al., 2001; With Bristow et al., 2003)

B-adrenergic receptor antagonist (being also referred to as beta blocker) has shown the main treatment pattern as a kind of chronic heart failure.Think that at first these medicaments can not be used for heart failure, because think that it is very crucial that the suprarenin motility is increased in the support failure heart.In fact, in early days in the experience about first-generation compound Proprasylyte, use standard dose often be associated (Stephen, 1968) with the deterioration of heart failure.Yet, use low initial dose and slowly upwards titration, the s-generation (selectivity β ₁-blocker) or the third generation (non-selective beta blocker-vasodilator) compound demonstrated and can reverse contractile dysfunction and structure and molecule is rebuild, and can improve M ﹠ M in heart failure (Bristow, 2000); The CIBIS-II investigator and the council (CIBIS-II Investigators and Committees).Cardiac Insufficiency bisoprolol research II:(CIBIS-II, 1999); MERIT-HF study group.The effect of metoprolol CR/XL in chronic heart failure: the at random intervention test (MERIT-HF, 1999) of metoprolol CR/XL in congestive heart failure.Packer et al. (2001); BEST experimental study person, (2001); Loweset al., 2002).Think that partly these beneficial effects are because the unfavorable biological action of persistence by noradrenaline levels rising mediation that the protection failure heart avoids finding in syndrome, this provide protection has limited metabolism and physiological reverse effect (Bristow, 2000; Cohn et al., 1984; And Liggett, 2001).In addition, beta blocker has demonstrated and can partly reverse molecular phenotype (Lowes et al. in heart failure, 2002), thus these medicaments can prevent and can reverse carrying out property myocardial failure and reconstruction (Eichhorn and Bristow, Circulation 1996).

Interesting is, nearest research shows, compares with the Gly389 individuality, and heart rate and/or blood pressure are to stronger (Liu et al., 2003 in the normotensive Arg389 individuality of being reflected at of beta blocker metoprolol and atenolol USP 23; With Sofowora et al., 2003).And in the hyperpietic, in Arg389 patient blood pressure to the reaction of metoprolol greater than the reaction in Gly389 patient (Johnson et al., 2003).A disclosed research in heart failure has been found that at β ₁The AR polymorphism does not have tangible related or trend (White et al., 2003) with hospital care and death between the association response to the metoprolol treatment.In this research, although directly do not carry out genotypic comparison with patient with placebo with the patient of metoprolol treatment, the patient near 45% has slight heart failure (NYHA Class II), and on average follow-up period has only 12 months.These differences can illustrate this potential contradiction.Yet bucindolol and metoprolol have some significant differences (Bristow, 2000 aspect their pharmacological property; With Bristow et al., 1997).Especially, bucindolol reduces norepinephrine, expansion peripheral vasculature and more effectively blocks people β ₁-adrenergic receptor.

Although all common pharmacological properties that have been used to treat the beta blocker of HF (heart failure) are their blocking-up β ₁AR, transduction stimulates (Zhu et al., 2001 up to about 90% pathologic adrenergic in the human heart of depletion according to estimates; With Bristow et al., 2003), obtainable beta blocker has a large amount of different characteristics, comprises β AR subtype-selective, to α ₁The affinity of AR, partial agonist activity, sympatholytic (Bristow et al., 2004) and vasorelaxation (Bristow, 2000; With Bristow et al., 1997).

The chemical structure of some beta blockers is provided in Fig. 1, and it shows that these medicaments have tangible textural difference.In addition, they have different pharmacological characteristics.As shown in Fig. 2, in the research and development or the different antiadrenergic agents in II or the III clinical trial phase relatively described these difference.For example carvedilol is a kind of β efficiently ₁-AR and β ₂-AR blocker and α ₁-AR blocker.Compare, bucindolol is a kind of weak α ₁-AR blocker, and metoprolol and bisoprolol can not be blocked α at all ₁-AR.Noticeable, because the orthosympathetic characteristic bucindolol of retardance is unique in beta blocker, correlated with its formation is that carvedilol, metoprolol and bisoprolol do not have this specific character.Compare with other Beta receptor blockers, bucindolol reduces noradrenaline levels in the whole body (Lowes et al., 2000 uniquely; Bristow et al., 1997; BEST NEJM, Bristow, 2004), and be β ₃The full agonist of-adrenoceptor (Strosberg, 1997).

Bucindolol is third generation beta blocker-vasodilator, its chemical name and structure be (2-{2-hydroxyl-3{{2-(3-indyl)-1,1-dimethyl ethyl } amino } propoxy--the benzonitrile hydrochloride).It at first is used to hypertension, is used for heart failure subsequently.Because its low contrary exciting and vasodilation character, non-selective β-the blocking-up of bucindolol can be tolerated by heart failure patient well, and part is because this reason, selects bucindolol to check beta blocker can reduce the hypothesis of mortality ratio in heart failure in late period at NIH in 1994 and VA cooperation clinical trial group (VA Cooperative ClinicalTrials Group).The check of this hypothesis is BEST test, its May 31 nineteen ninety-five to implementing between 29 days July in 1999.

Beta blocker viability assessment test (" BEST ") is because the suggestion of the data and the safety supervision council (theData and Safety Monitoring Committee) is stopped in advance, the risk ratio is 0.90 (C.I.s 0.78-1.02) (BEST Investigators, 2001 for main (primary) terminal point of full reason mortality ratio at this moment; Domanski et al., 2003).Yet the result of whole BEST group is positive for the high-order secondary terminal point of mortality ratio or hospital care in heart failure, and it has been reduced by 19% (p value＜0.0001) (Domanski et al., 2003) by bucindolol.In fact this terminal point is counted as the preferred main terminal point of HF crucial test.

The reason that stops BEST is 1) the III level, the BEST testing data that produces among the non-Black people patient has been confirmed recently disclosed from CIBIS-II (CIBIS Investigators, 1999) and MERIT-HF (MERIT-HF Study Group, 1999) Shi Yan information, the essence survival rate of the heart failure patient of these types all is benefited from β-blocking treatment, 2) forfeiture of researchist's intermediate equilibria constantly increases, they believe that the effect of β-blocking-up in heart failure has been proved to be and 3) in the subgroup (IV level and Black people) formerly in beta blocker test in heart failure, do not studied for the ineffectivity and the trend of adverse events.Subsequently the further research and development of bucindolol are abandoned,, but do not known whether bucindolol can successful being pushed to the market even because bucindolol goes through.

Therefore, in this is tested as the large-scale survival rate of assessment terminal point with overall survival rate, the BEST clinical trial is ended prematurely, because confirmed the interests that in other test, shown recently, and the effect of bucindolol can not expand to previous patient's subgroup (BEST Investigators, 2001) of not assessing in large-scale clinical trial.At that time, do not observe between those patients and have tangible mortality difference with bucindolol or placebo treatment.Apparent in view different with the result of BEST, reported between with the similar research of beta-adrenergic antagonist bisoprolol (be called " CIBIS-II " test), metoprolol (being called " MERIT-HF " test) and carvedilol (being called " COPERNICUS " test) and to have had very favorable difference (mortality ratio reduction 34-35%) with antagonist and those patients with placebo treatment.BEST researchist infers that a possible explanation for difference among the result is " unique pharmacological property that may derive from bucindolol ".

In the CIBIS-II test, research is also stopped too early, but is because mortality ratio is significantly lower in those patients of bisoprolol treatment.CIBIS-II Investigators，1999。Similarly, in the MERIT-HF research with metoprolol, early stopping is crossed in this research, because preassigned is satisfied and is exceeded.MERIT-HF Study Group，1999。The COPERNICUS research that relates to carvedilol is also stopped too early, because observe obvious benefit in treatment.Packer et al.，2001。BEST researchist points out that their result has proposed the problem of relevant beta blocker equivalence.

In addition, formerly relate in the non-mortality ratio research of carvedilol (Yancy et al., 2001), do not observe response difference between Black people and non-Black people object, this is another the concrete and relevant difference about bucindolol.In the BEST test, the Black people patient who suffers from the heart failure in late period demonstrates the worse result than non-Black people patient.Bristow，1997。

A comment of different tests is set forth and " can not be proposed clear and definite explanation to using the benefit minimizing that bucindolol obtained in the BEST research." Bouzamondo et al., 2001 (if find the BEST experimental is removed, evidence shows with the beta blocker treatment has realized the risk reduction).Although the author says their research and point out decentraction force failure crowd subgroup that beta blocker is treated and have different reactions that they do not get rid of the possibility that different beta blockers have different qualities, they do not say that polymorphism is its reason yet.Also see Sallach et al., 2003. (" although some authors propose [BEST test difference] is because the patient colony that is checked, other people thinks to lack that mortality ratio reduces is because bucindolol self.”)

Therefore, between bucindolol and other beta blocker, have treatment difference, and have obvious problem about the overall therapeutic efficiency of bucindolol.Therefore, any relation between bucindolol and the specific hereditary variant is not conspicuous.

Benefit based on the retrospective analysis of genetic data disclosed herein is to emphasize that bucindolol helps unique pharmacological property of its validity in the treatment heart failure patient.Two in these features also is helpful to the interaction between medicine and the adrenergic receptor genetic mutation.

In these features first is the ability (reducing noradrenaline levels in blood and the tissue) that sympatholytic effect or medicine directly reduce the suprarenin motility.As what point out above, in being used to treat beta blocker in heart failure, bucindolol is unique (BEST Trial Investigators, 2001 in this; Lowes et al., 2000; Bristow et al., 2004).The sympatholytic effect of bucindolol may be because β ₂-receptor blocking adds inadequate α ₁-blocking-up makes the activation of suprarenin motility, and to β ₁-and β ₂The low reverse agonist activity of-acceptor makes the adrenergic activation that suppresses based on cardiac muscle drop to minimum.It is to generate nitrogen protoxide and β that bucindolol can help other characteristic of sympatholytic effect ₃Receptor agonism (Strosberg, 1997).These back two kinds of character add or deduct weak α ₁The slight vasodilation character of-receptor blocking effect possible explanation bucindolol (Gilbert et al., 1990), different with carvedilol, this is not enough to cause the activation of reflectivity adrenergic.

When existing in right amount, the effect of (norepinephrine still less reduces) sympatholytic is a kind of favourable character, helps the therapeutic anti adrenergic effect of bucindolol.This is the latent effect mechanism that is better than simple β-blocking-up, because removed excessive norepinephrine from system.Norepinephrine can cause various heart trouble approach to the toxic and excessive words of cardiac muscle.Yet it does the time spent when undue expansion, and the sympatholytic interaction energy causes damage, and can increase mortality ratio (Bristow et al.2004).As discussed below, the genetic targets of bucindolol is to allowing this specific character only to bring into play its function in an advantageous manner.

With second pharmacological characteristics of the interactional bucindolol of pharmacogenetics target be the β of high-affinity ₁The effect of-receptor blocking (Hershberger et al., 1990; Asano et al., 2001).Bucindolol is to people β ₁-acceptor and β ₂-acceptor has high-affinity (Hershberger et al., 1990).In addition, by the non-agonism to translation or protein conversion, bucindolol reduces β ₁The density of-acceptor (Asano et al., 2001).Because its good tolerability like this, bucindolol can be used with very high beta blocker amount, and each all helps it to high functionality people β in these character ₁The beneficial effect of-acceptor 389Arg/Arg genetic mutation (embodiment, Mason et al., 1999).Although bucindolol has intrinsic class sympathetic activity (ISA) in rat heart muscle, bucindolol lacks ISA (Bristow et al., 1994 in the functional human heart tissue; Sederberg et al., 2000; Bristow et al., 1998, embodiment 7).This can clearly in the plate A of Figure 13 and B see, wherein at β ₁In AR Arg/Arg or the Gly carrier genotype,, in people's right ventricle girder of isolating depletion, on generation power, obviously do not increase even exist under the condition of transduceing with diterpene di compound Forskolin (forskolin) enhancing signal.By contrast, as shown in Figure 13 plate C, xamoterol as positive control ISA compound at β ₁All demonstrating in low and high signal transduction activation in the AR Arg/Arg genotype has increased power, just shows but have only in the Gly carrier under the overactivity state that the Forskolin pre-treatment provides.At last, as seen in fig. 13, in the isolating human heart of preparation, bucindolol is to β ₁AR Arg/Arg contrast Gly carrier acceptor has unique effect.Under low level signal transduction (low receptor activation) condition in failure heart (plate A), bucindolol is as people's cardiac muscle β ₁The neutral antagonist of Arg/Arg acceptor (no agonist or reverse agonist activity) works; but when signal transduction just as adenylyl cyclase during by Forskolin direct activation high (plate B); bucindolol plays a role as reverse agonist and makes the acceptor inactivation; as pointed by the significant slope factor of statistics, maximum concentration 10 in passing through the accessible blood plasma of therapeutic dose ^-6M.This effect do not occur in Gly carrier acceptor, wherein bucindolol plays a role as reverse agonist in low active state, and when Forskolin exists as neutral antagonist.These Notes of Key Data bucindolols are at the β of antagonism overactivity state ₁Have unique effect in the 389Arg/Arg acceptor, wherein the form of acceptor is presumably the most myocardiac form.

These character may be exactly at β under bucindolol treatment background ₁The Arg389 of AR heredity variant and at α _2cViewed wondrous and unexpected result's reason in the Del322-325 of the AR heredity variant.

II. polymorphism analysis

Because described hereditary variant is at β ₁The coding region of-AR and α-AR gene and influence coded protein is so can determine the existence of Arg389 or Del322-325 polymorphism from nucleotide sequence or protein sequence.Therefore, can use various method for this purpose.

A. nucleic acid

Certain embodiments of the present invention relate to multiple nucleic acid, comprise other nucleic acid elements that amplimer, oligonucleotide probe and genomic dna analysis relate to.In some aspects, nucleic acid comprises wild-type, mutant or polymorphism nucleic acid.

Therefore, term " β ₁-adrenergic receptor " polymorphism or " β ₁AR " polymorphism is the term of this area, expression β ₁The nucleic acid of-adrenergic receptor gene or gene product or the polymorphism in the aminoacid sequence.Only be the purpose of reference, the sequence of GenBank registration number J03019 (Gly389) and AF169007 (Arg389) (these two sequences are merged in herein with reference) is respectively β ₁The example of the Gly-of-adrenergic receptor gene order and Arg-389 form.

Equally, term " α _2c-adrenergic receptor " polymorphism or " α _2cAR " therefore polymorphism is the term of this area, expression α _2cPolymorphism in-adrenergic receptor gene or the gene product.Only be the purpose of reference, the corresponding wild-type (non-disappearance) of GenBank registration number NM00683 sequence and the corresponding absence type of AF280400 sequence (these two sequences are merged in herein with reference).

In order to identify pleomorphism site, β ₁AR or α _2cFirst Nucleotide of-AR gene coding region initiator codon (in dna molecular the VITAMIN B4 of ATG and in the RNA molecule VITAMIN B4 of AUG) is set to Nucleotide " 1 " and increases number along encoding sequence.Similarly, first amino acid (methionine(Met)) of institute's translated protein product is set to amino acid " 1 ".

Term " nucleic acid " is well-known in the art." nucleic acid " ordinary representation of Ying Yonging comprises the DNA that examines base or the molecule (that is chain) of RNA in this article.For example, the nuclear base comprise DNA (for example, VITAMIN B4 " A ", guanine " G ", thymus pyrimidine " T " or cytosine(Cyt) " C ") or RNA (for example, A, G, uridylic " U " or C) in natural purine or the pyrimidine bases found.Term " nucleic acid " comprises term " oligonucleotide " and " polynucleotide ", separately all as the subclass of term " nucleic acid ".The molecule of about 100 the nuclear bases of the about 3-of term " oligonucleotide " expression length.Term " polynucleotide " expression length is greater than at least one molecule of about 100 nuclear bases.The encoding sequence of " gene " expression gene product and the intron and the promotor of gene product.

In some embodiments, nucleic acid of the present invention is included in 1165 people β with cytosine(Cyt) or guanine in the cDNA sequence ₁The α of AR cDNA sequence or existence or disappearance 964-975 position Nucleotide _2CWhole in the AR cDNA sequence or 5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,450,460,470,480,490,500,510,520,530,540,550,560,570,580,590,600,610,620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790,800,810,820,830,840,850,860,870,880,890,900,910,920,930,940,950,960,970,980,990,1000,1100,1165,1200,1300,1400,1500 or more a plurality of continuous nucleotide, or any scope that can derive therebetween, or with above nucleotide sequence complementation.Skilled in the art will recognize that how to design and use to be used to the primer and the probe of hybridizing and increasing, comprise and use primer and the required restriction of probe homology.

These definition are usually directed to single chain molecule, but also will comprise part, basic or complete and the extra chain of described single chain molecule complementary in specific embodiments.Therefore, nucleic acid can comprise the one or more complementary strands of the particular sequence that comprises molecule or the duplex molecule or the three chain molecules of " complement ".As what use in this article, single-chain nucleic acid can be with prefix " ss " expression, and duplex molecule can be with prefix " ds " expression, and three chain nucleic acid can be represented with prefix " ts ".

Aspect concrete, nucleic acid encoding protein matter, polypeptide or peptide.In certain embodiments, the present invention relates to comprise the novel composition of at least one protein molecule.As what use in this article, " protein molecule ", " protein composition ", " protein compound ", " protein chain " or " protein material " ordinary representation, but be not limited to, greater than about 200 amino acid or from the protein of the endogenous full length sequence of gene translation; Greater than about 100 amino acid whose polypeptide; And/or about 100 the amino acid whose peptides of about 3-.The term of relevant " protein " that all are above-mentioned mutual alternative in this article uses.

1. the preparation of nucleic acid

Can prepare nucleic acid by the known any technology of those of ordinary skills, such as, chemosynthesis, enzymatic production or biological production.Nucleic acid (for example, synthetic oligonucleotide) limiting examples comprises the nucleic acid through external chemosynthesis preparation, this is by utilizing phosphotriester, phosphorous acid ester or phosphoramidate chemistry and solid phase technique, this technology for example is described in European patent 266, in 032, it incorporates this paper into by reference; Perhaps pass through deoxynucleoside H-phosphonic acid ester intermediate, this is described in Froehler et al., 1986 and United States Patent (USP) 5,705,629 in, each piece document is through with reference to being incorporated herein.In the method for the invention, can use one or more oligonucleotide.The multiple different mechanisms of oligonucleotide synthetic is disclosed, and for example sees United States Patent (USP) 4,659,774,4,816,571,5,141,813,5,264,566,4,959,463,5,428,148,5,554,744,5,574,146,5,602,244, every piece all is incorporated herein through reference.

The limiting examples of enzymatic production nucleic acid comprises by enzyme at amplified reaction such as PCR ^TMThe nucleic acid of producing in (for example see, United States Patent (USP) 4,683,202 and United States Patent (USP) 4,682,195, each piece of writing is through with reference to being incorporated herein), or by being described in United States Patent (USP) 5,645, the nucleic acid that 897 oligonucleotide synthesis method generates, it is incorporated herein by reference.The limiting examples of biological production nucleic acid is included in the viable cell recombinant nucleic acid of producing (promptly duplicating), such as the recombinant DNA carrier that duplicates in bacterium (for example see, Sambrook et al.2001, it is by with reference to being incorporated herein).

2. nucleic acid purification

Can be on polyacrylamide gel, cesium chloride centrifugation gradient, chromatographic column or by the known any alternate manner of those of ordinary skills (for example see, Sambrook et al.2001, it is by with reference to being incorporated herein) purification of nucleic acid.In some aspects, nucleic acid is that pharmacy can be accepted nucleic acid.The pharmacy acceptable composition is known for those skilled in the art, and is described in herein.

In some aspects, the nucleic acid that the present invention relates to is isolating nucleic acid.As what use in this article, term " isolating nucleic acid " expression is separated and be free on or otherwise be free on total genome of one or more cells and transcribe the nucleic acid molecule (for example, RNA and dna molecular) of nucleic acid system.In certain embodiments, " isolating nucleic acid " expression separated and be free on or otherwise be free on cellular constituent or vitro reactions composition for example macromole such as the nucleic acid of systems such as lipid or protein, atom molecule.

3. nucleic acid fragment

In certain embodiments, described nucleic acid is nucleic acid fragment.As what use in this article, term " nucleic acid fragment " is the fragment of nucleic acid, such as, as limiting examples, those encoding parts β ₁AR locus or β ₁AR gene order or part α _2cThe nucleic acid fragment of AR locus or gene order.Therefore, " nucleic acid fragment " can comprise any part of gene order, comprises from about 2 Nucleotide to comprising that promoter region is to the full-length gene of polyadenylic acid signal and the random length that comprises whole coding regions.

Can design multiple nucleic acid fragment based on specific nucleic acid sequence, and can be random length.By giving sequence assignment, for example, first residue is that 1, the second residue is 2 etc., just can set up the algorithm that defines all nucleic acid fragments:

N to n+y

Wherein n be from 1 to the integer of the last numbering of sequence and y to be nucleic acid fragment length deduct 1, wherein n+y is no more than the last numbering of sequence.Therefore, for 10 aggressiveness, the corresponding base 1 to 10,2 to 11,3 of nucleic acid fragment is to 12... or the like.For 15 aggressiveness, the corresponding base 1 to 15,2 to 16,3 of nucleic acid fragment is to 17... or the like.For 20 aggressiveness, the corresponding base 1 to 20,2 to 21,3 of nucleic acid fragment is to 22... or the like.In certain embodiments, nucleic acid fragment can be probe or primer.As what use in this article, " probe " ordinary representation is used for the nucleic acid of detection method or composition.As what use in this article, " primer " ordinary representation is used for extending or the nucleic acid of amplification method or composition.

4. nucleic acid complement

The present invention also comprises and nucleic acid complementary nucleic acid.When nucleic acid can carry out base pairing according to standard Watson-Crick, Hoogsteen or anti-Hoogsteen complementary pairing principle and another nucleic acid, described nucleic acid was exactly " complement " or " being complementary to " another nucleic acid.As used herein, " another nucleic acid " can represent nucleic acid separately or the sequence of spatially separating in a part.In preferred embodiments, complement is hybridization probe or the amplimer that is used to detect polymorphic nucleic acid.

Just as used herein, term " complementation " or " complement " also represent to comprise can with another nucleic acid chains or the continuous kernel base of double-stranded hybridization or the nucleic acid of semicontinuous nuclear base (being that one or more nuclear base structures are not present in this molecule) sequence, even be less than all nucleotide bases with corresponding nuclear base formation base pairing.Yet in some diagnosis or detection embodiment, complementary nucleic acid is preferred fully.

5. detection of nucleic acids and assessment

According to before being described in Small et al., the method for (2002) is implemented genotype detection exactly, and the document is incorporated this paper into through reference.It should be appreciated by those skilled in the art that available other standard technique is carried out genotype detection and any technology can be used for the present invention.The ordinary method of nucleic acid detection method is provided in down, and the back is to identify that polymorphism comprises the specific embodiment of single nucleotide polymorphism (SNP).

Determine that needing the Id concrete genotype detection method of beta blocker treatment is not a part of the present invention, but relate to isolating nucleic acid mixture from individuality in brief, wherein comprise the β of two copies that exist in the individuality ₁AR gene or its fragment, and definite β ₁Among the AR on 1165 positions the identity of nucleotide pair or determine at α _2cOn Nucleotide 964-975 position, whether disappearance is arranged in the AR gene.At β ₁AR and α _2cPreferred polymorphism and polymorphic site comprise the content in the following table 1 in the AR gene:

Table 1

β 1-adrenergic receptor polymorphism
					Nucleotide position 1165	Nucleotide G or C	Amino acid position 389	Amino acid Gly or Arg	Code name Gly389, Arg389
α 2c-adrenergic receptor polymorphism
					Nucleotide position 964-975	Nucleotide deletion	Amino acid position 322-325	Aminoacid deletion	Code name α 2cDel322-325

These polymorphisms were formerly all reported.Wild-type beta ₁The AR nucleotide sequence comprises the guanine of 1165 in Nucleotide usually.Wild-type beta ₁The AR protein sequence comprises the glycine of 389 in amino acid usually.It has been generally acknowledged that wild-type α _2cThe disappearance that the AR nucleotide sequence is represented there is not the disappearance of Nucleotide 964-975 position and therefore do not had amino acid 322-325.

Those skilled in the art will readily appreciate that nucleic acid molecule can be that the particular location of duplex molecule and reference on a chain also relates to the corresponding position on the complementary strand.Therefore, when determining pleomorphism site, when mentioning nucleic acid molecule just when the VITAMIN B4 on (justice or coding) concrete site of chain, thymus pyrimidine (uridylic), cytosine(Cyt) or guanine, the meaning also comprises thymus pyrimidine (uridylic), VITAMIN B4, guanine or the cytosine(Cyt) in corresponding site on negative (antisense or non-coding) chain of nucleic acid molecule complementation chain respectively.Therefore, can and still comprise same pleomorphism site with reference to arbitrary chain, and can design oligonucleotides and the hybridization of arbitrary chain.What run through this paper is, when differentiating pleomorphism site, with reference to positive-sense strand, this is just for purpose easily.

Be typically, use such as being disclosed in the standard technique of Jones (1963) in waiting, the document is incorporated this paper into through quoting, from picking up from the biological specimen of individuality, such as isolating nucleic acid mixture in blood sample or the tissue samples.Suitable tissue samples comprises whole blood, seminal fluid, saliva, tear, urine, fecal matter, sweat, oral cavity thing (buccal), skin and hair.Described nucleic acid mixture can comprise genomic dna, mRNA or cDNA, and in back two kinds of situations, biological specimen must be from expressing β ₁Obtain in the organ of AR gene.What those skilled in the art should understand that in addition, is that mRNA or cDNA prepared product are not used for detecting position in the polymorphism of intron or 5 ' and 3 ' nontranscribed domain.If separate β ₁The AR gene fragment, it must contain genotypic pleomorphism site to be determined.

The prediction patient determines that for the doctor it is useful how treating heart failure patient to the ability of beta-2-agonists reaction.If patient's genotype indicates this patient to have sound response to agonist, so with may show medium reaction or responseless patient compares, this patient is better candidate for beta blocker treatment, and the doctor can and wrongly determine which individuality should accept the treatment of alternative form with still less test.

In the employed in the present invention genotype detection method, can be by one from be present in individuality or whole β of two copies ₁AR gene and/or α _2cIn the AR gene directly amplification comprise the target region of pleomorphism site and determine that by ordinary method the sequence of institute's amplification region determines Nucleotide (nucleotide pair) identity at the pleomorphism site place.Those skilled in the art will be readily appreciated that in the individuality that isozygotys in this site will only detect a kind of Nucleotide on described pleomorphism site, yet if individuality is a heterozygosis for this site, will detect two kinds of different IPs thuja acids so.Polymorphism can directly be identified, is known as positive type and identifies, or get by deriving, and is called as negative type identification.For example, known SNP is under the situation of guanine and cytosine(Cyt) in the reference crowd, is that the individuality that isozygotys can positivity determine that this site is guanine or cytosine(Cyt) for this site; If or individual be heterozygosis in this site, this site is guanine and cytosine(Cyt) so.As an alternative, can negativity determine that this site is not guanine (and being cytosine(Cyt)/cytosine(Cyt) therefore) or is not cytosine(Cyt) (and being guanine/guanine therefore).

Target region can use any oligonucleotide directed amplification method to increase, and includes but not limited to polymerase chain reaction (PCR) (U.S. Patent No. 4,965,188), ligase chain reaction (LCR) (LCR) (Barany et al., 1991; WO90/01069) be connected analysis (OLA) (Landegren et al., 1988) with oligonucleotide.The oligonucleotide that can be used as primer or probe in these methods will hybridize to the nucleic acid region that contains or be close to pleomorphism site specifically.Be typically, oligonucleotide is that long and preferred 15-30 the Nucleotide of 10-35 Nucleotide is long.Most preferably, oligonucleotide is that 20-25 Nucleotide is long.The exact length of oligonucleotide will depend on many factors that the conventional institute of technician considers and puts into practice.

Can comprise based on the amplification system of transcribing (U.S. Patent No. 5,130,238 with other known nucleic acid amplification method amplified target zone; EP 329,822; U.S. Patent No. 5,169,766, WO89/06700) and isothermal method (Walker et al., 1992).

Also can before or after amplification, use as known in the art several based on the polymorphism in one of method of hybridizing analysis target region.Usually when implementing such method, use allele specific oligonucleotide.Allele specific oligonucleotide can be right as the probe of isolabeling not, and a right member of its middle probe demonstrates that a kind of variant to target sequence mates fully and another member demonstrates different variants are mated fully.In some embodiments, can use one group of allele specific oligonucleotide or oligonucleotide that one-time detection is surpassed a pleomorphism site.

Can two kinds entity all in solution, implement the hybridization of allele specific oligonucleotide and target polynucleotide, perhaps when the such hybridization of enforcement so that covalently or non-covalently mode is attached on the solid support of oligonucleotide or target polynucleotide.For example, can adhere to by mediations such as antibody-AI, poly-L-Lysine, streptavidin or affinity element-vitamin H, salt bridge, hydrophobic interaction, chemistry connection, UV-crosslinked bakings.Can directly be attached on the solid support more later at synthetic allele specific oligonucleotide on the solid support or synthetic.Be applicable to that the solid support in the detection method of the present invention comprises the substrate that is prepared by silicon, glass, plastic cement, paper etc., for example, it can be made into hole (as 96 orifice plates), bar (slide), thin slice (sheet), film, fiber, substrate (chip), dish and pearl.Thereby can handle, wrap by or auxiliary allele specific oligonucleotide of derivatize solid support or target nucleic acid fixing.

Also can be by with one or all gene of two copies or its fragment and the β that determines individuality such as the nucleic acid array that is described in WO95/11995 and inferior hybridization array ₁The genotype of one or more pleomorphism site in the AR gene.Described array comprises one group of allele specific oligonucleotide, and its representative is contained in the various pleomorphism sites in genotype or the haplotype.

Also can use the mispairing detection technique to determine the identity of polymorphism, the mispairing detection technique includes but not limited to use riboprobe (riboprobes) (Winter et al., 1985; Meyers et al., 1985) and discern the RNA enzyme protection method of the protein of Nucleotide mispairing such as intestinal bacteria mutS protein (Modrich, 1991).As an alternative, single strand conformation polymorphism (SSCP) be can pass through and (Orita et al., 1989 analyzed; Humphries, et al., 1996) or denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990; Sheffield et al., 1989) evaluation variant allelotrope.

Also can use polymerase-mediated primer extension method to identify polymorphism.Several such methods were described in patent and scientific literature.Can be by being described in United States Patent (USP) 5,605,798 mass spectrometry method detects the extension primer that contains polymorphism.Another kind of primer extension method is allele-specific PCR (Ruano et al., 1989; Ruano et al., 1991; WO 93/22456; Turki et al., 1995).

Also can use the DNA difference digestion method (Small et al., 2002) of some restriction enzyme or be tested and appraised β ₁Any other method of 65 Nucleotide of AR gene 11 detects people β ₁The polymorphic variation that the AR gene nucleotide is 1165.

A. hybridization

Use length between 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,50,60,70,80,90 or 100 Nucleotide, preferred 17-100 Nucleotide or reach 1-2 kilobase or longer probe and primer in aspect some, allow to form and not only stablize but also have an optionally duplex molecule of the present invention.General preferred surpassing the molecule that has complementary sequence on the long continuous fragment of 20 bases, to increase the stability and/or the selectivity of the hybrid molecule that obtained.The nucleic acid molecule that the common preferred design of technician has one or more 20-30 Nucleotide or when expectation even longer complementary sequence is used for hybridization.Such fragment can be produced easily, and is for example, directly synthetic or carry out recombinant production by will selected sequence introducing in the recombinant vectors by chemical process.

Therefore,, can utilize the complementary segment selectivity of they and DNA and/or RNA to form the ability of duplex molecule, perhaps utilize them that the ability of primer DNA amplification or RNA from sample is provided for nucleotide sequence of the present invention.Use according to expection, expectation utilizes different hybridization conditions to realize probe or the primer selectivity in various degree to target sequence.

For the application that needs highly selective, expectation uses the condition of high relatively rigorous degree to form hybrid usually.For example, relatively less salt and/or hot conditions are such as about 50 ℃ of conditions that about 0.02M is provided to about 0.10M NaCl under about 70 ℃ of temperature.The rigorous degree condition of this height seldom allows between probe or primer and template or target chain any possible mispairing and is particularly suitable for separating specific gene or the detection specificity polymorphism.Usually know that the methane amide of increasing amount can provide more rigorous condition by adding gradually.For example, under the rigorous degree condition of height, can carry out the hybridization of filter membrane under the following conditions: at 0.5M NaHPO in conjunction with DNA ₄, 7% sodium lauryl sulphate (SDS), 1mMEDTA, 65 ℃, and in 68 ℃, 0.1 * SSC/0.1%SDS, clean (Ausubel et al., 1989).

Can be by increasing salt concn and/or reducing the condition that temperature provides low rigorous degree.For example, can by about 37 ℃ to about 55 ℃ down about 0.1 to 0.25M NaCl medium rigorous degree condition is provided, can provide the rigorous degree condition of hanging down to about 0.9M salt by about 0.15M under about 20 ℃ to about 55 ℃.Under low rigorous degree condition, under suitable rigorous degree condition, cleaning can for example carried out (Ausubel et al., 1989) among 42 ℃ of following 0.2 * SSC/0.1%SDS.Can control hybridization conditions at an easy rate according to expected result.

In other embodiments, hybridization can for example realize under the following condition: 50mMTris-HCl (pH8.3), 75mM KCl, 3mM MgCl ₂, 1.0mM dithiothreitol (DTT), about 20 ℃ to about 37 ℃ of temperature.The hybridization conditions that other utilized can comprise about 10mMTris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl ₂, about 40 ℃ to about 72 ℃ of temperature.

In certain embodiments, the nucleic acid parallel connection of application defined nucleotide sequence of the present invention is appropriate to determine that the hybridization means will be favourable such as mark.Be known in the art suitable widely indicatory device, comprise fluorescence, radioactivity, enzyme or other part, such as affinity element/vitamin H, they can be detected.In preferred embodiments, the technician can expect to use fluorescent mark or enzyme label and substitutes radioactivity or other environmentally harmful reagent such as urase, alkaline phosphatase or peroxidase.In the situation of enzyme label, the colorimetric indication substrate is known and can be used to provide visible or the detectable detection means of spectrophotometer, thereby identifies and the sample specific hybrid that comprises complementary nucleic acid.Aspect other, may there be specific nuclease cleavage site, and can be by there being or lacking the detection that specific nucleotide sequence is determined in the nucleic acid cutting.

In general, can predict probe described herein or primer can be used to detect the expression or the genotype of corresponding gene, and be used in the embodiment of using solid phase as solution hybridization such as the reagent among the PCR.In relating to the embodiment of solid phase, test dna (or RNA) is adsorbed or is attached to selected surface or matrix in other mode.Subsequently this fixed, single-chain nucleic acid and selected probe are hybridized under desired conditions.Selected condition will depend on specific environment (size etc. that for example, depends on G+C content, target nucleic acid type, nucleic acid source, hybridization probe).The method that those skilled in the art optimize hybridization conditions according to the application-specific purpose is well-known.Cleaning after thereby hybrid molecule removes the probe molecule of non-specific binding, hybridization is being detected and/or quantitatively by the amount of determining binding label.Typical solid-phase hybridization method is disclosed in United States Patent (USP) 5,843,663,5,900,481 and 5,919,626.Can be used to implement other hybridizing methods of the present invention and be disclosed in United States Patent (USP) 5,849,481,5,849,486 and 5,851,772.The relevant portion of these and other reference of pointing out in this specification sheets this part is incorporated herein by reference.

B. nucleic acid amplification

Nucleic acid as amplification template can separate from cell, tissue or other sample according to standard method (Sambrook et al., 2001).In certain embodiments, template nucleic acid being carried out or do not carry out substantive purifying implements to analyze to full cell or tissue homogenate or biological fluid sample.Nucleic acid can be genomic dna or separated or whole-cell rna.When using RNA, can expect at first RNA to be changed into complementary DNA.

The meaning of the term of Shi Yonging " primer " comprises can cause any nucleic acid of newborn nucleic acid synthetic in the process that template relies in this article.Usually, primer is 10-20 and/or 30 oligonucleotide that base pair is long, but also can use longer sequence.Can provide primer with two strands and/or single stranded form, although single stranded form is preferred.

Design is used for selective cross and β ₁AR locus or its variant and fragment thereof the primer pair of corresponding nucleic acid contact under the condition that allows selective cross with template nucleic acid.According to the application of expectation, can select only to allow the high rigorous degree hybridization conditions of complete complementary sequence and primer hybridization.In other embodiments, can under low rigorous degree, hybridize, thereby allow amplification and primer sequence to comprise the nucleic acid of one or more mispairing.In case form hybridization, the templa-primer mixture promotes template dependency nucleic acid synthetic enzyme to contact with one or more.Carry out many wheel amplifications, also claim " circulation ", up to the amplified production that produces capacity.

Can detect, analysis or quantitative amplification product.In some applications, can implement to detect by visual means.In some applications, detection can relate to via mixing radio-labeling or fluorescently-labeled chemoluminescence, radioactivity scintillation analysis or even via (the Affymax technology of system that uses electronics and/or thermal pulse signal; Bellus, 1994) carry out the indirect evaluation of product.

A lot of template dependency methods can be used for increasing and have an oligonucleotide sequence that exists in the given template samples.Understanding one of maximum amplification method is that the polymerase chain reaction (is known as PCR ^TM), its detailed content is disclosed in United States Patent (USP) 4,683,195,4,683,202 and 4,800,159 and Innis etal., 1988, each piece of writing wherein with integral way by with reference to being incorporated herein.

Another kind of amplification method is ligase chain reaction (LCR) (" LCR "), and it is disclosed in european patent application No.320 308, and it is incorporated herein by reference with integral way.United States Patent (USP) 4,883,750 have described the similar methods with LCR, are used to make probe pair to combine with target sequence.A kind of United States Patent (USP) 5,912 that is disclosed in, 148 PCR-based ^TMAnd the method for oligonucleotide ligation assay (OLA) (below further detailed description will be arranged) also can be used.

The alternative method that can be used to implement target nucleic acid sequence of the present invention that is used to increase is disclosed in United States Patent (USP) 5,843,650,5,846,709,5,846,783,5,849,546,5,849,497,5,849,547,5,858,652,5,866,366,5,916,776,5,922,574,5,928,905,5,928,906,5,932,451,5,935,825,5,939,291 and 5,942,391, Britain applies for 2 202 328 and PCT application PCT/US89/01025, each piece of writing wherein with integral way by with reference to being incorporated herein.The Qbeta replicative enzyme of describing in PCT application PCT/US87/00880 also can be used in the amplification method of the present invention.

A kind of isothermal amplification method, wherein use restriction enzyme and ligase enzyme realize amplification in a chain of restriction site, contain Nucleotide 5 '-target molecule of [α-sulfo-]-triphosphoric acid, this method also can be used for amplification of nucleic acid (Walker et al., 1992) in the present invention.Be disclosed in United States Patent (USP) 5,916,779 strand displacement amplification (SDA) is the another kind of method of implementing the nucleic acid isothermal amplification, and this method relates to the strand displacements of taking turns and synthetic more, that is, and and nick translation.

Other nucleic acid amplification method comprises based on the amplification system of transcribing (TAS), comprises amplification (NASBA) and 3SR (Kwoh et al., 1989 based on nucleotide sequence; PCT applies for WO 88/10315, and it is incorporated herein by reference with integral way).European application 329 822 discloses a kind of nucleic acid amplification method, relates to circulation synthesizing single-stranded RNA (" ssRNA "), ssDNA and double-stranded DNA (dsDNA), and it can use according to the present invention.

PCT application WO 89/06700 (it with integral way by with reference to being incorporated herein) discloses a kind of amplification of nucleic acid sequences scheme, and it copies based on promoter region/primer sequence and the hybridization of target single stranded DNA and many RNA sequences of transcribing described sequence subsequently.This scheme is not a round-robin, that is, do not produce from gained rna transcription thing from new template.Other amplification method comprises " RACE " and " monolateral PCR " (Frohman, 1990; Ohara et al., 1989).

C. detection of nucleic acids

After any amplification, can expect from template and/or excessive primer, to separate amplified production.In one embodiment, use standard method to separate amplified production (Sambrook et al., 2001) by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis.Isolating amplified production can from gel, cut out and from gel wash-out be used for further processing.Use the low melting-point agarose gel, can extract nucleic acid subsequently by the heating gel and reclaim isolating band.

Also can use rotating centrifugal post known in the art and/or chromatographic technique to realize separate nucleic acid.There are many kinds to can be used for chromatography of the invention process, comprise absorption, distribution, ion-exchange, hydroxyapatite, molecular sieve, anti-phase, post, paper, thin layer and vapor-phase chromatography and HPLC.

In certain embodiments, to separating or not having isolating amplified production to show.Typical display packing relates to the ethidium bromide staining gel and under UV light makes band visual.As an alternative, if with radioactivity-or fluorescently-labeled Nucleotide to integrate mode mark amplified production, isolating amplified production can or make it visual under suitable excitation spectrum to the x-ray film exposure.

In one embodiment, after separating amplified production, the nucleic acid probe of mark is contacted with the flag sequence of amplification.Probe is preferably puted together with chromophoric group, but also can radio-labeling.In another embodiment, but probe and binding partners are puted together such as antibody or vitamin H or the another kind of binding partners that carries the test section.

In specific embodiments, implement described detection by the Southern trace with the hybridization of label probe.The technology of Southern trace is well-known (seeing Sambrook et al., 2001) to those skilled in the art.An aforesaid example is at United States Patent (USP) 5,279, description arranged in 721, and it is by with reference to being incorporated herein, and it discloses the autophoresis that is used for nucleic acid and the apparatus and method of transfer.Described device allows need not the peripheral operation gel and carries out electrophoresis and trace and be very suitable for implementing the method according to this invention.

Can be used for implementing other nucleic acid detection methods of the present invention and be disclosed in United States Patent (USP) 5,840,873,5,843,640,5,843,651,5,846,708,5,846,717,5,846,726,5,846,729,5,849,487,5,853,990,5,853,992,5,853,993,5,856,092,5,861,244,5,863,732,5,863,753,5,866,331,5,905,024,5,910,407,5,912,124,5,912,145,5,919,630,5,925,51 7,5,928,862,5,928,869,5,929,227,5,932,413 and 5,935,791, wherein each is incorporated herein by reference.

D. other detections

Can use other genetic screening method within the scope of the invention, for example, be used for detecting the sudden change in genomic dna, cDNA and/or the RNA sample.Be used for that the method for check point sudden change comprises denaturing gradient gel electrophoresis (" DGGE "), restriction fragment length polymorphism analysis (" RFLP "), chemistry or enzymatic cutting method, directly order-checking is through PCR ^TMTarget region, single-strand conformation polymorphism analysis (" SSCP ") and the additive method known in the art of (seeing above-mentioned) amplification.

A kind of method of screening point mutation is based on the right RNase cutting of base mismatch in RNA/DNA or the RNA/RNA heteroduplex.As what use in this article, term " mispairing " is defined as in double-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule one or more not pairings or the mispairing zone to Nucleotide.Therefore this definition comprises because the mispairing that insertion/deletion mutantion and list or the sudden change of polybase basic point cause.

United States Patent (USP) 4,946,773 have described a kind of RNaseA mispairing cutting detection method, and it relates to anneals single stranded DNA or RNA detection sample with rna probe, and handles nucleic acid duplex with RNase A subsequently.Be to detect mispairing, compare to the contrast duplex of similar processing with single stranded product RNase A processing, carry out electrophoretic separation according to size.Comprising the sample than small segment (cleaved products) that can not see in the contrast duplex is recorded as the positive.

Other investigators have described the application of RNase I in mispairing detects.In the document of PromegaBiotech, describe RNase I and be used for the application that mispairing detects.Promega sells the test kit that contains RNase I, it is reported that RNase I cuts 3 in 4 known mispairing.Also having in addition to describe uses MutS protein or other DNA-repair enzyme to detect single base mismatch.

The alternative method that can be used for implementing detection disappearance of the present invention, insertion or replacing sudden change is disclosed in United States Patent (USP) 5,849,483,5,851,770,5,866,337,5,925,525 and 5,928,870, wherein each is merged in herein by reference with its integral body.

E. the specific embodiment of polymorphism nucleic acid screening method

All members of these species are not often spread all in the spontaneous mutation that takes place in the genome evolution process of biology immediately, thereby produce the polymorphism allelotrope that coexists as in this species colony.Polymorphism often is the cause of disease of heredopathia.A few class polymorphisms are identified.For example, variable nucleotide type polymorphism (VNTR) is to be produced by the spontaneous inline copy that two or trinucleotide of Nucleotide repeat motif.If such variation changes the length of the dna fragmentation that is produced by the restriction enzyme cutting, described variation is called as restriction fragment length polymorphism (RFLP).RFLP has been widely used in the genetic analysis of humans and animals.

Another kind of polymorphism is to be produced by the replacement of mononucleotide.This single nucleotide polymorphism (SNP) seldom causes the variation of restriction endonuclease sites.Therefore, SNP almost can not detect with the analysis of restriction fragment length.SNP is that modal inheritable variation and every 100-300 base occur once, and has had been found that several SNP sudden changes, and they influence mononucleotide in the protein coding gene in the mode that enough causes genetic diseases actually.The example of SNP disease has hemophilia, sicklemia, hereditary hemochromatosis thesaurismosis, delayed alzheimer's disease etc.

Develop the method for several screening polymorphisms and enumerated some examples below.Reference Kwok and Chen (2003) and Kwok (2001) provide the summary of some these methods; These two reference are incorporated herein by reference especially.

The feature that relates to the SNP of ABCC2 can be determined by any method or its suitable modifications used in these methods.These methods comprise described site direct or indirect order-checking, use restriction site to be produced or the destructive restriction enzyme, use the allele-specific hybridization probe, use at the coded proteinic specific antibody of the not isoallele of polymorphism or any other biological chemistry interpretation procedure by each allelotrope of described site.

The i.DNA order-checking

The method that the sign polymorphism is the most generally used is that the locus that comprises polymorphism and side thereof is directly carried out dna sequencing.Can use dideoxy chain termination (being also referred to as " Sanger method " (Sangeret al., 1975)) or " chemical degradation method " (being also referred to as " Maxam-Gilbert method " (Maxamet al., 1977)) to finish such analysis.Can use sequence measurement associating genome sequence specific amplification technology to promote recovery (Mullis et al., 1986 of desired gene such as the polymerase chain reaction; European patent application 50,424; European patent application 84,796, european patent application 258,017, european patent application 237,362; European patent application 201,184; United States Patent (USP) 4,683,202; 4,582,788; With 4,683,194), all reference by with reference to being incorporated herein.

Ii. exonuclease resistance

Can be used for determining that there is the special exonuclease resistance nucleotide derivative (United States Patent (USP) 4,656,127) of other method utilizations of Nucleotide identity in the pleomorphism site place.With pleomorphism site immediately 3 ' district's allelotrope sequence complementary primer under study condition with described DNA hybridization.If pleomorphism site comprises with existing and has the circumscribed resistance derivative of specific nucleotide complementary Nucleotide on this DNA, this derivative will mix the end of hybridized primer by polysaccharase so.Mixing like this makes primer have resistance and allow its detection thus exonuclease cutting.Because the identity of the circumscribed resistance derivative of Nucleotide is known, can determine the specific nucleotide that in the dna polymorphism site, exists.

Iii. microsequencing method

The Nucleotide that several in addition primers that are used for detecting the DNA pleomorphism site instruct mixes existing (Komher et al., 1989 described of method; Sokolov, 1990; Syvanen 1990; Kuppuswamy et al., 1991; Prezant et al., 1992; Ugozzoll et al., 1992; Nyrenet al., 1993).These methods depend on mixes labeled dideoxynucleotide Nucleotide to distinguish the base at pleomorphism site place.Because signal is proportional with the amount of the deoxynucleotide that mixes, so the polymorphism that occurs in the circulation of identical Nucleotide produces and the proportional signal of this round-robin length (Syvanen etal., 1990).

Iv. in solution, extend

French Patent 2,650,840 and PCT application WO91/02087 the method based on solution of the Nucleotide identity of definite pleomorphism site has been discussed.According to these methods, use and pleomorphism site be 3 ' allelotrope sequence complementary primer immediately.If with the Nucleotide complementation of pleomorphism site place, the dideoxyribonucleoside acid derivative of mark will be mixed the end of primer, use described mark dideoxyribonucleoside acid derivative to determine the identity of this site Nucleotide.

V. hereditary bit (Bit) is analyzed or solid phase is extended

PCT application WO92/15712 described the applying marking terminator and with the method for the mixture of pleomorphism site 3 ' sequence complementary primer.Therefore existing Nucleotide is complementary in mark terminator that is mixed and the target molecule pleomorphism site to be evaluated is also identified.Be fixed on the solid phase at this primer or target molecule.

Vi. oligonucleotide connects analysis (OLA)

The another kind of solid phase method (Landegren et al., 1988) that this is to use different methods to learn.Use can hybridize to two kinds of oligonucleotide of the contiguous sequence of target DNA strand.A kind of in these oligonucleotide is biotinylated, and another kind ofly can be detected ground mark.If found accurate complementary sequence in target molecule, oligonucleotide will be hybridized, then with their end adjacency, and produce and connect substrate.Described connection allows to use the plain oligonucleotide that reclaims mark of affinity.Based on this method and the other nucleic acid detection method of uniting PCR (Nickerson et al., 1990) are described also.Use PCR to realize the index amplification of target DNA at this, use OLA subsequently and detect target DNA.

Vii. ligase enzyme/polymerase-mediated hereditary bit analysis

United States Patent (USP) 5,952,174 described a kind of also relate to can with the method for two kinds of primers of the contiguous sequence of target molecule hybridization.Be fixed with formation hybridization product on the solid support of target.Hybridization takes place at this, so separated the opening of primer and apart from one another by single Nucleotide.At polysaccharase, ligase enzyme with contain under the condition that the ribonucleoside triphosphote mixture of at least a deoxynucleoside triphosphate exists, hatch this hybridization product, be connected with the hybridization oligonucleotide that allows any paired adjacency.Adding ligase enzyme causes that signal, extension take place and is connected two kinds of required incidents.This compares with only using the method for extending or being connected separately, higher specificity and lower " noise " are provided, and it is different with detection based on polysaccharase, this method is attached to solid phase by making the hybridization of polymerase step and secondary and Connection Step linked together being used for signal, thereby has strengthened the specificity of polymerase step.

Viii. invasive cleavage reaction

The invasive cleavage reaction can be used for assessing the specific polymorphism of cell DNA.The technology that is known as INVADER  has been utilized this reaction (for example, de Arruda et al., 2002; Stevens etal., 2003, they are incorporated herein by reference).In general, three kinds of nucleic acid molecule are arranged: the 1) oligonucleotide of target site upstream (" upstream oligonucleotide "), 2) cover the probe oligonucleotides (" probe ") and 3 of target site) have a single stranded DNA (" target ") of target site.But upstream oligonucleotide and probe not overlapping they contain contiguous sequence.Probe contain the donor fluorophore such as fluorescein and acceptor dye such as Dabcyl.First base pair of Nucleotide overlapping (" intrusion ") the probe one target two strands of upstream oligonucleotide 3 ' end.The probe of structure specificity 5 ' nuclease cutting subsequently causes fluorophore/quencher to separating, and this has increased the amount that can detect fluorescence.See Lu et al., 2004.

In some cases, on solid surface or with array format, implement to detect.

Ix. detect the additive method of SNP

Enumerated several other specificity methods that are used for that polymorphism detects and identify below, and can former state or in conjunction with in conjunction with identifying β among the present invention ₁The AR gene pleiomorphism is suitably revised and is used.The SNP network address of NCBI on the World Wide Web Www.ncbi.nlm.nih.gov/SNPIn on several additive methods have also been described, they are by with reference to being incorporated herein.

In specific embodiment, the haplotype of expansion can be in colony determines on any given locus, and this allows people to identify that clearly which SNP will be redundant and which is essential in association study.The latter is called as ' haplotype label SNP (htSNP) ', it is to catch the gene of linkage disequilibrium or the marker of haplotype in zone.See Johnson et al. (2001) and Ke and Cardon (2003), wherein each piece of writing is used as illustrative methods by with reference to being incorporated herein.

By using the long range PCR method of TaKaRa LA Taq reagent and other standard reaction condition, the pcr amplification of VDA analysis and utilization genomic fragment.Long can increase apart from amplification method about 2,000-12, the DNA of 000bp size.Can pass through Affymetrix high flux screening center (HighThroughput Screening Center) and computer software analysis and implement the hybridization of product and variation detection arrays (VDA).

A kind of method that is called chip analysis (Chip Assay) is used the pcr amplification of genomic fragment by standard or long range PCR scheme.Analyze hybridization product (Halushka et al. (1999), it incorporates this paper into through reference) by VDA.SNPs is classified as " determining " or " possibility " based on the Computer Analysis of crossing pattern usually.By comparing such as nucleotide sequencing with alternative detection method, verified by this method " determining " SNP of 100%; And verified " possibility " SNP of 73%.

Other methods only relate to pcr amplification and use the respective limits enzymic digestion subsequently.Also having other to relate to checks order to the purified pcr product from known group district.

Also in other methods, the overlapping fragments of indivedual exons of pcr amplification or big exon.Design primer and use following condition to implement the pcr amplification of genomic dna from open or database sequence: 200ng dna profiling, every kind of primer 0.5 μ M, each 80 μ M of dCTP, dATP, dTTP and dGTP, 5% methane amide, 1.5mM MgCl ₂, 0.5U Taq polysaccharase and 0.1 volume Taq damping fluid.Implement thermal cycling and analyze gained PCR product under various conditions by PCR single strand conformation polymorphism (PCR-SSCP) analysis, described condition for example contains or does not contain 5% glycerine, has 5 or 10% polyacrylamide gel of 15% urea.The enforcement electrophoresis that spends the night.Thereby amplification demonstrates the PCR product of mobility shifting and it is checked order and identifies nucleotide diversity again.

In the method that is called CGAP-GAI (DEMIGLACE), with sequence and comparison data (from the PHRAP.ace file), be used for the sequence base and judge that quality score (from the PHRED quality document), range information (from PHYLIP dnadist and neighbour program) and the base decision data (from PHRED '-d ' swith) of (call) are loaded into storer.Aligned sequences is also checked the discordance of each vertical block of gained chunk (' bar slice ').Any such bar is considered to candidate SNP (EDMIGLACE).Use multiple filter to get rid of the bar that to represent real polymorphism by DEMIGLACE.These filters comprise: if (i) the flanking sequence quality score descends 40% or more, and the sequence in from SNP considers, getting rid of any given; (ii) get rid of base judge in peak amplitude all bases of being lower than the sort of Nucleotide type judge 1 15 judgement; (iii) get rid of the zone that has the sequence of a large amount discordance with the consensus sequence that participates in SNP calculating; (iv) from consider, remove and have certain and substitute any base of judging and judge, in described alternative judgement peak value account for judge peak area 25% or more; (v) get rid of the variation that only on a read direction, occurs.The PHRED quality score is converted into the word error probability value at each Nucleotide in described.Use standard Baysian method is calculated the posterior probability that has the heterogeneous evidence of Nucleotide on given position.

In the method that is called CU-RDF (RESEQ), use is used from the DNA of blood separation at the Auele Specific Primer of every kind of SNP and is implemented pcr amplification, and typically clean (cleanup) scheme with remove do not use primer and free nucleotide after, use same or nested primer directly checks order.

In the method that is called DEBNICK (METHOD-B), carry out the comparative analysis of cluster est sequence and confirm by dna sequencing based on fluorescence.In a kind of methods involving that is called DEBNICK (METHOD-C), the comparative analysis condition of cluster est sequence is as follows: in mispairing site phred quality＞20, do not have mispairing in average phred quality＞=20 on 5 bases of 5 ' of SNP-FLANK and 3 ', 5 ' and 3 ' 5 bases at SNP, each allelotrope implements at least twice and confirm by the inspection track.

In the method that is called ERO (RESEQ), design new primer sets and the DNA of being used for increasing from 10 different mouse species according to the disclosed STS of electronics.Amplified production from each strain carries out gel-purified subsequently and uses application ³³The two deoxidations of the standard of p-mark terminator, cycle sequencing technology check order to it.All the reaction of ddATP terminated is loaded in the neighbouring lane of sequencing gel subsequently, is whole ddGTP reactions etc. subsequently.Identify SNP by visual inspection radioactivity photo.

Be called in the authentication method of ERO (RESEQ-HT) at another, design new primer sets and the DNA of being used to increase from 10 kinds of different mouse species according to the disclosed mouse dna sequence dna of electronics.By using exonuclease I and shrimp alkaline phosphotase, preparation is used for order-checking from the amplified production of each strain.Use ABI Prism Big Dye Terminator Ready Reaction test kit (Perkin-Elmer) enforcement order-checking and go up processing sequence sample at 3700 DNA analysis instrument (96 kapillary sequenator).

FGU-CBT (SCA2-SNP) is a kind of method, wherein uses primer SCA2-FP3 and SCA2-RP3 pcr amplification to contain the zone of SNP.The about 100ng genomic dna of amplification in the 50ml reaction volume, reaction system contains final concentration 5mM Tris, 25mM KC1,0.75mMMgCl ₂, 0.05% gelatin, every kind of each 20pmol of primer and 0.5U Taq archaeal dna polymerase.Sample is by sex change, annealing and extension, and use, for example, the PCR product of QIAquick gel extraction kit (Qiagen) purifying from sepharose cutting-out band, and on ABI Prism377 automated DNA sequenator, use dyestuff to stop chemical method with the PCR primer it is checked order.

In the method that is called JBLACK (SEQ/RESTRICT), genomic dna is implemented two independent PCR reactions.By the product of sequencing analysis from first reaction, the FspI restriction site that indication is unique.By determine the sudden change in the 2nd PCR reaction product with Fsp I digestion.

In the method that is described as KWOK (1), by relatively selecting individual application dyestuff-termination chemical method that the high quality genomic sequence data that the direct dna sequencing of PCR product obtains is identified SNP (seeing Kwok et al., 1996) at random from 4.In a methods involving that is called KWOK (2), identify SNP such as bacterial artificial chromosome (BAC) or based on the high quality genomic sequence data of the artificial chromosome (PAC) of P1 by relatively inserting the clone from overlapping big fragment.Development subsequently contains the STS of this SNP, and checks order and determine exist (the seeing Taillon-Miller et al., 1998) of in each kind of groups SNP by merging DNA (pooled DNA).Be called in the similarity method of KWOK (3) at another, identify SNP by the high quality genomic sequence data that relatively inserts clone BAC or PAC from overlapping big fragment.The SNP of Fa Xianing represents two kinds of mutant dna sequences between the donor karyomit(e) by this method, but the gene frequency in general groups is not still determined.In KWOK (5) method, by relatively the high quality genomic sequence data that the direct dna sequencing of PCR product obtains being identified SNP from homozygote DNA sample and one or more merging DNA sample application dyestuff termination chemical method.The sequence data of finding from public database develops used STS.Specifically, to complete hydatidiform mole (CHM) by these STS of pcr amplification, described complete hydatidiform mole be presented on all locus all be isozygoty and the DNA sample from 80 CEPH father and mother (seeing Kwok et al., 1994).

In another such method KWOK (OverlapSnpDetectionWithPolyBayes), insert the overlap of people's gene group cloned sequence by the big fragment of Computer Analysis and find SNP.In order to obtain data, directly obtain cloned sequence from the large scale sequencing center.This is necessary, can not obtain by GenBank because base quality sequence is not present in GenBank/.Original data processing relates to analyzes cloned sequence and about conforming additional base quality information.Specify a unified base mass value 40 (specific inaccuracy is 10, among the 000bp 1) for and not have the finishing of related base matrix amount sequence (' perfect base ', specific inaccuracy are per 10, are less than 1 among the 000bp) sequence.Abandon and do not have the sketch of base mass value sequence.To handle the back sequence and be input to local data base.Also preserve the version of sheltering the known person tumor-necrosis factor glycoproteins of each sequence.Implementing tumor-necrosis factor glycoproteins with program " MASKERAID " shelters.Overlapping detection: it is overlapping to detect imagination with program " WUBLAST ".Next be several filtration steps, they are in order to get rid of false overlapping detected result, promptly because the similarity between the paired cloned sequence of sequence replicating but not real overlapping generation.Total overlapping length, whole percentage similarity, has the sequence difference number between the Nucleotide of high-alkali matrix value " high quality mispairing ".The result also compares with result, overlapping result's report of the person of finishing and the result of NCBI kind sequence linkage group construction project at the restriction fragment collection of illustrative plates of the genomic clone at University of Washington gene order-checking center.SNP detects: detect software with ' POLYBAYES ' SNP and analyze the overlapping right of cloned sequence at candidate SNP locus.At the real sequence variations of representative but not the wrong probability of order-checking the sequence difference of paired sequence is marked.This processing needs the base mass value of two sequences all to exist.Extract the candidate of high score out.Search is limited to the variation of replacement type list base pair.Degree of confidence scoring by POLYBAYES computed in software candidate SNP.

In the method that is called KWOK (TaqMan analysis), use TaqMan to analyze to determine the genotype of 90 random individuals.In the method that is called KYUGEN (Q1), merging is specified the dna sample of colony and is analyzed by PLACE-SSCP.Proofread and correct each allelic peak heights in the combined analysis by in the heterozygote those, be used to calculate gene frequency subsequently.Quantitatively be higher than 10% gene frequency by this method reliably.Allelotrope=0 (zero) is illustrated in and has found allelotrope in the individuality, but does not see corresponding peak in checking the merging storehouse.Gene frequency=0-0.1 indication detects a small amount of allelotrope in merging the storehouse, but can not be reliably quantitative because the peak is too low.

Be called in the method for KYUGEN (Method1) at another, the PCR product is carried out the back mark and under SSCP condition (PLACE-SSCP), it analyzed by automatic capillary electrophoresis system with fluorescence dye.In a series of tests, use or do not use two kinds and merge storehouse DNA (Japanese storehouse and CEPH father and mother storehouse) four or more a plurality of body DNA are analyzed.Identify allelotrope by visual inspection.Individual DNA with different genotype is checked order and identifies its SNP.After with the peak height correction signal deviation in the heterozygote, estimate gene frequency from the peak height that merges the storehouse sample.For PCR, primer tagged and have 5 at its end '-ATT or 5 '-GTT is used for the back mark of two chains.In reaction mixture dna sample (10ng/ul) is increased, described reaction mixture contains damping fluid (10mM Tris-HCl, pH8.3 or 9.3,50mM KCl, 2.0mM MgCl ₂), each 0.25 μ M of every kind of primer, every kind of each 200 μ M of dNTP and 0.025U/ μ l be mixed with the Taq archaeal dna polymerase of anti--Taq antibody in advance.By two chains of the segmental permutoid reaction of the Klenow of dna polymerase i with nucleotide difference mark PCR product with R110 and R6G modification.By adding the EDTA stopped reaction, and make uncorporated Nucleotide dephosphorylation by adding calf intestinal alkaline phosphatase.For SSCP, the internal markers of aliquot fluorescent mark PCR product and TAMRA mark is added in the deionized formamide, and sex change.Use ABI Prism 310 genetic analysis instrument in kapillary, to carry out electrophoresis.Use Genescan software (P-E Biosystems) and collect data and processing data.Using big-dye to stop chemistry directly checks order to the DNA (being included in the DNA of individual that demonstrates different genotype on the SSCP) of individual (2 to 11) on ABI Prism 310 sequenators.By Phred/Phrap the multiple sequence track trail file that obtains from ABI Prism 310 is handled and compared, and use the Consed reader to observe.

Be called in the method for KYUGEN (Method2) at another, the sequence that has the individual of different genotype and determine them by sex change HPLC (DHPLC) or PLACE-SSCP (Inazuka et al., 1997) search is to identify SNP.For the back mark of two chains, with endways with 5 '-primer of ATT or 5 '-GTT label implements PCR.Use WAVE dna fragmentation analytical system (Transgenomic) and implement the DHPLC analysis.The PCR product is injected in the DNASep post, and under with the definite condition of WAVEMaker program (Transgenomic) it is separated.Use two chains of nucleotide difference mark PCR product with R110 and R6G modification by the Klenow fragment permutoid reaction of dna polymerase i.Stop described reaction by adding EDTA, and make uncorporated Nucleotide dephosphorylation by adding calf intestinal alkaline phosphatase.In kapillary, implement SSCP with rear electrophoresis with ABI Prism 310 genetic analysis instrument.Genescan software (P-E Biosystems).On ABI Prism 310 sequenators, use big-dye termination chemistry individual DNA (being included in those that demonstrate different genotype on DHPLC or the SSCP) is checked order.By Phred/Phrap the multiple sequence track trail file that obtains from ABI Prism 310 is handled and compared, and use the Consed reader to observe.Identify SNP by PolyPhred software and visual inspection.Use the track chromatographic data that PHRED handles est sequence among the Unigene.In order to identify possible SNP, from produce by program PHRAP, BRO and POA at report base mispairing each Unigene bunch the multiple sequence comparison result.BRO proofreaies and correct the EST orientation that may report by mistake, yet POA identifies and analyze the gene mixing/chimeric non-linear comparison structure that indication may produce false SNP.Weigh about following evidence with the Bayesian inference: truly polymorphism ratio order-checking mistake, mistake comparison or indeterminate property, mistake cluster or chimeric est sequence, assessment data are such as original chromatogram height, acuteness, overlapping and interval; The sequencing error rate; Background susceptibility; Source, cDNA library etc.

In the method that is called MARSHFIELD (Method-B), identify the overlapping human DNA sequence who comprises imagination insertion/deletion polymorphism by the retrieval public database.From consensus sequence, select the PCR primer of each pleomorphism site both sides.With human gene group DNA described primer amplification individuality or that merge.Gained PCR product separates in denaturing polyacrylamide gel and assesses gene frequency with PhosphorImager from DNA merging storehouse.

F. linkage disequilibrium

With the polymorphism of another kind of polymorphism linkage disequilibrium, wherein identify a kind of polymorphism indicating the identity of chain polymorphism." linkage disequilibrium " is (in this article as " LD ", be also referred to as " LED " in the art) represent that allelotrope (that is, specifying the variant form of gene) or the particular combinations of polymorphism on two locus demonstrate the situation of beguine according to the higher frequency of randomness expection.Determined as those skilled in the art, as to use in relevant linkage disequilibrium " significantly ", expression p value or the α value on the statistics can be 0.25 or 0.1 and can be 0.1,0.05,0.001,0.00001 or littler.Can determine at β by the nucleotide sequence that assessment and 389 polymorphisms are in the polymorphism in the linkage disequilibrium ₁389 polymorphisms in the AR albumen.Can implement the present invention by this way to one or more polymorphism, to allow haplotype analysis.According to being that the usual and common meaning is used " haplotype " to those skilled in the art.Its expression is along the two or more allelotrope of one of homologous chromosomes or the integrator gene type of polymorphism.

B. evaluating protein matter

As an alternative, can be by detecting β ₁Any method of AR protein 389 amino acids variation is determined the polymorphic variation.The invention is not restricted to any concrete grammar and realize this purpose.For example, can obtain sample and definite β of fluid or tissue from individuality ₁AR protein 389 amino acids.Such detection method can be a whole bag of tricks, comprises can using based on detection of antibodies, (Western trace, ELISA) or amino acid analysis (high pressure lipuid chromatography (HPLC) or mass spectrometry), detects protein and whether has Arg or Gly.

Therefore, in certain embodiments, the present invention relates to comprise the composition of at least a protein molecule, such as β ₁AR protein or in conjunction with β ₁The proteinic protein of AR is such as antibody.As used in this article, " protein molecule ", " protein composition ", " protein compound ", " protein chain " or " proteinaceous substances " ordinary representation but be not limited to greater than the protein of about 200 amino acid whose or translations from the total length endogenous sequence of gene; Greater than about 100 amino acid whose polypeptide; And/or from about 3 to about 100 amino acid whose peptides.Use can be replaced in this article in the term of all above-mentioned relevant " protein ".

Can be according to any technology well known by persons skilled in the art, comprise by standard biological learn a skill marking protein, polypeptide or peptide, prepare protein composition from natural resource isolated protein compound or chemosynthesis proteinaceous substances.Disclose Nucleotide and protein, polypeptide and the peptide sequence of several genes before this, and can find in the known Computer Database for those of ordinary skills.Genbank that such database is a NCBI and GenPept database (the National Center for Biotechnology Information ' sGenbank and GenPept databases) ( Http:// www.ncbi.nlm.nih.gov/).Can use disclosed technology or the known technology of persons skilled in the art to increase herein and/or express the coding region of these knowns.As an alternative, the multiple commercial production thing of those skilled in the art's known protein matter, polypeptide and peptide.

1. protein purification

Can expect purifying β from sample ₁AR or purifying are in conjunction with β ₁The protein of AR is such as antibody.Such technology is used and the present invention's intention of protein involved purifying without limits widely.Purified technology of protein is well-known to those skilled in the art.On certain level, these technology relate to the cellular material rough classification are become polypeptide and non-polypeptide fraction.If from other protein, isolated described polypeptide, thereby can use chromatogram and electrophoretic technique to carry out that further purifying is realized part or purifying (or being purified to homogeneity) completely to polypeptide of interest.The analytical procedure that is particularly useful for making pure peptide is ion exchange chromatography, exclusion chromatography; Polyacrylamide gel electrophoresis; Isoelectric focusing method.A kind of special effective means of purified peptide is fast protein liquid chromatogram or or even HPLC.

Some aspect of the present invention can relate to the purifying of coded protein or peptide and the substantive purifying in specific embodiments.The declaration of will of the term of Shi Yonging " protein of purifying or peptide " can be from other component composition isolated in this article, and wherein protein or peptide can be purified to respect to its natural any degree that obtains state.So the protein of purifying or protein or the peptide that peptide also represents to be free on its natural surroundings.

Usually, " purifying " thus will represent to carry out protein or the peptide combinations that multiple other component has been removed in classification, and this protein or peptide combinations have kept its expressed biological activity basically.When using term " basic purifying ", this statement will represent that protein or peptide constitute the composition of composition main ingredient, account for about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more such as protein in composition.

Based on present disclosure, the whole bag of tricks of quantitative protein or peptide purification degree is known to those skilled in the art.These comprise, for example, determine the specific activity of active ingredient, or by the polypeptide amount in the SDS/PAGE analysis and evaluation component.A kind of preferred method that is used to assess component purity is the specific activity that calculates component, compares with the specific activity of original extract, and calculates purity thus, uses " purifying multiple " to assess in this article.Certainly, but the effective unit that is used to represent live vol will depend on whether selected particular detection technology and expressed protein or peptide show detection of active behind the purifying.

The multiple technology that is suitable for protein purification is well-known to those skilled in the art.These comprise, for example, utilize ammonium sulfate, PEG, antibody etc. or precipitate by heat denatured, and are then centrifugal; Chromatographic step is such as ion-exchange, gel-filtration, anti-phase, hydroxylapatite and affinity chromatography; Isoelectrofocusing; Gel electrophoresis; Associating with these technology and other technology.As well known in the art, believe that the order of implementing various purification steps can change, perhaps some step can be omitted, and still causes preparing the appropriate method of the protein or the peptide of basic purifying.

Usually always needn't require provides protein or peptide with their purified state.In fact, can consider that the product of less basic purifying will have effectiveness in certain embodiments.By using less united purification step, perhaps, can realize partial purification by utilizing the multi-form of identical general purification scheme.For example, it should be understood that the cation exchange column chromatography that utilizes HPLC equipment to implement upward causes Billy the purifying of more " doubly " usually with the same technology of low-pressure chromatography system.The method that shows low relative degree of purification can have superiority aspect the expressed protein active at the protein overall recovery or keeping.

The migration of known peptide under the SDS/PAGE different condition can change, also very remarkable sometimes (Capaldi et al., 1977).Therefore it should be understood that under different deposition conditions apparent molecular weight purifying or partially purified expression product can change.

High performance liquid chromatography (HPLC) is characterised in that the sharp separation with high peak resolving power.This is by utilizing very thin particle and high pressure to realize to keep enough flow velocitys.Separation can be finished in the time of several minutes or as many as hour.In addition, only need the very sample of small volume, because particle is very little and closely fills so that outer volume only accounts for the very little part of bed volume.Equally, do not need very large concentration of specimens, sample not dilution basically because band is so narrow.

Gel chromatography, or molecular sieve chromatography is the separation chromatography of specific type, and it is based on bulk of molecule.Gel chromatography theory behind is when walking around or when passing aperture than macromole or than small molecules, and the post for preparing with the molecule of the inert substance that contains aperture will separate them according to molecular size.As long as preparation particulate material does not adsorb described molecule, unique factor of determining flow velocity is a size.Therefore, as long as shape of molecule is constant relatively, molecule will by reduce gradually size sequence from post by wash-out.For the molecule that separates different sizes, gel chromatography is incomparable, does not rely on all other factorses such as pH, ionic strength, temperature etc. because separate.Also almost not absorption, than sub-district band broadening, and elution volume is only simple relevant with molecular weight.

Affinity chromatography be a kind of depend on material to be separated and and its specificity bonded molecule between the chromatographic process of pathoklisis.This is a kind of interaction of receptor-ligand type.By making a kind of binding partners and insoluble matrix covalent coupling synthesize column material.Column material subsequently can be from solution the described material of specific adsorption.By condition changing is realized wash-out (for example, changing pH, ionic strength and temperature) for the bonded condition does not take place.

It is a kind of that to can be used for the particular type affinity chromatography that purifying contains the compound of carbohydrate be the lectin affinity chromatography.Lectin is the class material in conjunction with multiple polysaccharide and glycoprotein.Lectin passes through cyanogen bromide and agarose coupling usually.The Conconavalin A that is coupled to Sepharose is stand-by this class material of first-selection, and it has been widely used in separating polyose and glycoprotein, and other lectin comprises French beans (lentil) lectin, is used for the wheat germ agglutinin and snail (Helix pomatia) lectin of purifying N-acetyl-glucosamine residue.Lectin self carries out purifying with the affinity chromatography of using carbohydrate ligand.With lactose purifying lectin from Semen Ricini and peanut; From French beans (lentil) and sword bean, extract lectin with maltose; With N-ethanoyl-D-galactosamine from soybean purifying lectin; The N-acetyl glucosamine is in conjunction with the lectin from wheat germ; D-galactosamine has been used for obtaining lectin and the L-Fucose will combine with the lectin from lotus from clam.

Matrix should be on any obvious degree self the material of adsorbed molecules and its do not have chemistry, physics and thermostability widely.Part should be in the mode that do not influence its binding characteristic by coupling.Part also should provide combination relatively closely.And it should be able to be under the situation of not destroying sample or part the described material of wash-out.A kind of affinity chromatography of most common form is an immune affinity chromatographic.Be applicable to that production of antibodies of the present invention is in following discussion.

2. antibody

Another embodiment of the invention is an antibody, is in some cases and people β ₁Immunoreactive human monoclonal antibodies takes place in the AR peptide sequence.It should be understood that antibody capable is used to detect β ₁AR, the β that especially specific polymorphism causes ₁AR.The intent of the present invention is that the antibody that is particularly useful under the background of the present invention is and the β with Arg389 ₁AR protein is compared, diacritically in conjunction with the β with Gly389 ₁The proteinic antibody of AR, thus between two kind of groups, distinguish.

As used in this article, term " antibody " is intended to represent widely any immunoconjugator, such as IgG, IgM, IgA, IgD and IgE.Usually, preferred IgG and/or IgM are because they are modal antibody and because they are the antibody of easy preparation in testing apparatus under the physiology situation.

Term " antibody " is used to represent any antibody molecule with antigen binding domain, and comprises that antibody fragment is such as Fab ', Fab, F (ab ') ₂, single domain antibody (DABs), Fv, scFv (strand Fv) etc.Being used to prepare and using various construction and segmental technology based on antibody is well-known in the art.The means that are used to prepare and characterize antibody also be in the art well-known (see, for example, Harlow et al., 1988; Incorporate this paper into by reference).

A. production of antibodies

In certain embodiments, the present invention relates to antibody.For example, the monoclonal antibody of all or part can be used to determine 389 amino acid.As mentioned above, except the antibody that produces anti-full length protein, also can produce the antibody that the littler construction that comprises the epi-position core area is reacted, wherein the epi-position core area comprises wild-type and mutant epi-position.Being used to prepare and using various construction and segmental technology based on antibody is well-known in the art.Preparation and the means that characterize antibody also are well-knownly (for example to see Harlow and Lane, 1988 in the art; Incorporate this paper into by reference).

Monoclonal antibody (mAbs) is realized has some advantage, for example, repeatability and can be mass-produced, usually advantageous applications they.Therefore the invention provides people, mouse, monkey, rat, hamster, rabbit and even the monoclonal antibody in chicken source.

The method that produces monoclonal antibody (mAbs) begins along the same routes of preparation polyclonal antibody usually.Briefly, can prepare polyclonal antibody from the antiserum(antisera) of immunized animal with immunogen peptide composition immune animal according to the present invention and by collecting.Alternately, in some embodiments of the present invention, collect serum from the philtrum that may be exposed to specific antigen.Being exposed to specific antigen can occur in the Working environment, is exposed to the concurrent exhibition of specific antigen at peptide, polypeptide or proteinic polyclonal antibody so those people are professional.In some embodiments of the present invention, by using immunologic detection method, use the people's who exposes from occupational polyclonal serum to identify the antigenic region of spending more in the white tree toxalbumin toxin.

Animal species can be used in the production antiserum(antisera) on a large scale.Typically being used for producing sero-fast animal is rabbit, mouse, rat, hamster, cavy or goat.Because the blood volume of rabbit is big relatively, preferentially select rabbit to be used to produce polyclonal antibody.

As well-known in the art, the immunogenicity of given composition can change.Therefore often strengthen host's immunity system inevitably, such as realizing by peptide or polypeptide immunogen are coupled on the carrier.Typical or preferred carrier is keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA).Other albumin also can be used as carrier such as Protalbinic acid, mice serum albumin or albumin rabbit serum.To make polypeptide and carrier proteins bonded means be well-known in the art and comprise glutaraldehyde, m-maleimide benzoyl-N-hydroxysuccinimide eater, carbodiimide and two-two nitrogenize benzidines (bis-biazotized benzidine).

Also well-knownly in the art be that can be construed to by application is the immunogenicity that the nonspecific immune reaction stimulant of adjuvant strengthens specific immunogenic composition.Suitable molecule adjuvant comprises all acceptable immune-stimulating compounds, such as cytokine, toxin or synthetic composition.

Applicable adjuvant comprises IL-1, IL-2, IL-4, IL-7, IL-12, gamma-interferon, GMCSP, BCG, aluminium hydroxide, MDP compound, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A and monophosphoryl lipid A (MPL).RIBI is also included within the present invention, and it comprises the three kind components (MPL in 2% shark alkene/Tween 80 emulsions, trehalose two mycophenolic acids (TDM) and cell wall skeleton (CWS)) of extraction from bacterium.Even can use MHC antigen.Typically, often preferred adjuvants comprises Freund's complete adjuvant (a kind of nonspecific immune response stimulant, it comprises the Mycobacterium tuberculosis of deactivation), the non-Freund's complete adjuvant of Fu Shi and aluminum hydroxide adjuvant.

Except adjuvant, can expect to use jointly biological response modifier (BRM), it has shown immunity or the downward modulation that can raise the T cell and has suppressed cell activity.Such BRMs includes, but not limited to CIMETIDINE (CIM; 1200mg/d) (Smith/Kline, PA); Low-dose cyclophosphamide (CYP; 300mg/m ²) (Johnson/Mead, NJ), cytokine is such as gamma-interferon, IL-2 or IL-12 or relate to the protein of the genes encoding of immune subsidiary function, such as B-7.

The amount that is used to produce the immunogenic composition of polyclonal antibody changes according to immunogenic characteristic and institute immune animal.Various approach can be used for using immunogen (subcutaneous, intramuscular, intracutaneous, intravenously and intraperitoneal).Can monitor the generation of polyclonal antibody by the blood sample of each time point collection immune animal after immunity.

Also can carry out the second time, strengthen injection.Repeat to strengthen and titrating process, till reaching suitable tiring.When obtaining the immunogenicity of desirable level, can implement bloodletting and separation and preservation serum to immune animal, and/or animal capable is used to produce mAbs.

Can such as by with reference to the technology of incorporating United States Patent (USP) 4,196,265 illustrated of this paper into, prepare mAbs easily by using well-known technology.Typically, no matter this technology is the wild-type or the composition of mutant if relating to selected immunogenic composition, and for example, purifying or partially purified polypeptide, peptide or structural domain come the suitable animal of immunity.Mode with the effective stimulus antibody produced cell is used immune composition.

If desired, can use filtration, centrifugal and various chromatography is further purified mAbs such as HPLC or affinity chromatography.Can be by comprising enzyme, such as the digestion of stomach en-or papoid and/or obtain the fragment of monoclonal antibody of the present invention from monoclonal antibody by the method for chemical reduction cutting disulfide linkage.As an alternative, the monoclonal antibody fragment that the present invention includes can be used the self acting peptide synthesizer and synthesize.

The present invention also intended use molecular cloning method produces mAbs.For this reason, from separating from immunoglobulin (Ig) phagemid (phagemid) library of the RNA of immunized animal spleen preparation combination, and the phagemid of selecting to express suitable antibody by the cell and the control cells of elutriation antigen expressed.This method is better than conventional hybridization oncocyte technology part and is once can produce and screen about 10 ⁴Antibody doubly, and by H and the new specificity of L chain combination results, described specificity has further increased the chance of finding suitable antibody.

B. immunologic detection method

As what discussed in some embodiment, the immunologic detection method that the present invention relates to is used for combination, purifying, removal, determine and/or the antigenic region of detection of biological ratio of component such as polypeptide and peptide additionally.Immunologic detection method of the present invention can be used for identifying peptide, polypeptide or the proteinic antigenicity district that therapeutic is used, especially reduce peptide in the target object, polypeptide or proteinic immunogenicity or antigenicity.

Immunologic detection method comprises that enzyme linked immunological absorption detects (ELISA), and radioimmunoassay detects (RIA), immunoradiometric assay(IRMA), fluoroimmunoassay, chemiluminescence analysis, bioluminescent assay and Western trace, also has the known method of other multiple those of ordinary skills.The step of various applicable immunologic detection methods has had description in scientific literature, such as, for example, Doolittleet al., 1999; Gulbis et al., 1993; De Jager et al., 1993; With Nakamura et al., 1987, each piece of writing is incorporated this paper into by reference.

Usually, immune combining method comprises obtaining infers the sample that comprises protein, polypeptide and/or peptide, comprises, according to circumstances, is effectively allowing to make sample have according to first antibody of the present invention mono-clonal or polyclonal antibody under the condition of formation immunocomplex.

These methods comprise the method that is used for from protein, polypeptide and/or the peptide of organoid, cell, tissue or body sample purifying.In these examples, antibody is removed antigen protein, polypeptide and/or peptide composition from sample.Antibody will preferably be connected to the solid support such as base for post matter form, and infer that the sample that comprises protein, polypeptide and/or peptide antigenicity component will being used for fixing antibody.From post, wash not desired components off, stay the antigen for the treatment of wash-out that forms immunocomplex with immobilized antibody.

The immunity combining method also comprises and is used for detecting and the quantitative method of the amount of sample antigen component, and any immunocomplex that detects and quantitatively form in the cohesive process.Therefore, people will obtain to infer the sample that comprises antigen or antigenicity structural domain, with the antibody contact sample of antigen or antigenicity structural domain, the amount of the immunocomplex that detects subsequently and quantitatively form under given conditions.

According to Detection of antigen, the biological specimen of being analyzed can be the sample that any supposition comprises antigen or antigenicity structural domain, such as, for example, the tissue extract of tissue slice or sample, homogenization, cell, organoid, separation and/or purified form any above-mentioned comprise antigenic composition or even any biofluid that contacts with cell or tissue, comprise blood and/or serum.

In the condition for validity that is enough to allow immunocomplex to form and for some time, contact selected biological specimen with antibody, usually just simply be added to antibody compositions in the sample and mixtures incubated for some time, antibody is enough to and the antigen that exists, i.e. combination forms immunocomplex.After this, usually clean sample-antibody compositions, such as tissue slice, elisa plate, dot blotting (dot blot) or western trace, thereby remove the antibody type of any non-specific binding, only allow to detect and elementary immunocomplex internal specific bonded antibody.

Usually, the formation of detection immunocomplex is well-known in the art and can realizes by using several different methods.These methods are usually based on detection label or marker, such as any radioactivity, fluorescence, biological and label enzyme.Relate to the United States Patent (USP) of using such marker and comprise 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241, wherein each piece of writing is incorporated herein by reference.Certainly, just as known in the art, can by use second binding partner such as two anti-the and/or plain part of vitamin H/affinity in conjunction with arranging to find other advantages.

Self can be connected to detectable label the antibody that is used to detect, and wherein people only detect this label subsequently simply and get final product, and allows to determine the amount of elementary immunocomplex in the composition thus.As an alternative, the first antibody that is bonded in the elementary immunocomplex can detect by second binding partner that the antibodies affinity is arranged.In these situations, but second binding partner can be connected with detection label.Second binding partner self is antibody normally, therefore is named as " second " antibody.At the condition for validity that is enough to allow second immunocomplex to form with in the time, with mark, second binding partner or antibody contacts elementary immunocomplex.Usually clean the two anti-or parts of second immunocomplex subsequently, and detect the label that keeps in second immunocomplex subsequently with the mark of removing any non-specific binding.

Additive method comprises by two-step approach detection of primary immunocomplex.As mentioned above, second binding partner of antibodies affinity is arranged,, be used to form second immunocomplex such as antibody.After the cleaning, be enough to allow the condition for validity of immunocomplex (the 3rd immunocomplex) formation and in the time, making the contact of second immunocomplex once more there being two resistive connections to close the 3rd binding partner or the antibody of affinity.The 3rd part or antibody are connected to detectable label, allow to detect the 3rd immunocomplex that forms thus.If desired, such system can provide signal to amplify.

The immunologic detection method of Charles Cantor design uses two kinds of different antibody.The first step is biotinylated, mono-clonal or polyclonal antibody are used to detect target antigen, and the second step antibody is used for detecting the vitamin H that is attached to the compound bio element subsequently.In the method, sample to be detected is at first hatched in the solution that contains the first step antibody.If target antigen exists, thereby some antibody combine with antigen and form biotinylated antibody/antigen mixture.Subsequently by in successive Streptavidin (or affinity element), biotinylation DNA and/or complementary biotinylation dna solution, hatching the antibody/antigen mixture, add extra vitamin H site to the antibody/antigen mixture in each step, thereby amplified the antibody/antigen mixture.Repeat amplification procedure until suitable amplification level, under this level sample is hatched in the solution of the second step antibody that contains antibiotin.This second step antibody is labeled, and for example uses enzyme labelling, and described enzyme can be used for utilizing chromophoric substrate to detect the antigen/antibody mixture by histoenzymology.By suitable amplification, can produce macroscopic view and go up the visible combination.

Another known immunologic detection method utilizes immunity-PCR (polymerase chain reaction) method.Till the incubation step of biotinylation DNA, PCR method is all similar to the Cantor method, yet, replace many wheel Streptavidins and biotinylation DNA to hatch, with low pH that discharges antibody or the slow DNA/ vitamin H/Streptavidin/antibody complex that cleans of high salt.The cleaning solution of gained is used for implementing having the PCR reaction of the suitable control of suitable primer subsequently.At least in theory, can utilize huge amplifying power of PCR and specificity to detect the monoclonal antibody original molecule.

i.ELISA

As detailed above, according to the simplest and/or direct meaning, immunoassay is a binding analysis.Some preferred immunoassay is that various types of enzyme linked immunological absorption well known in the art detects (ELISAs) and/or radioimmunoassay detects (RIA).It also is very useful that the immunohistochemistry of using-system section detects.Yet, be understood that easily described detection is not limited to these technology, and/or western trace, dot blotting (dot blot), facs analysis and/or similar method can be used also.

In an ELISA example, antibody is fixed on the selected surface that shows the protein affinity, such as the hole on the polystyrene microtiter plates.Subsequently, supposition is contained the antigenic composition that tried,, be added in the hole such as clinical sample.After combination and/or cleaning, can detect bonded antigen with the immunocomplex of removing non-specific binding.Usually but another antibody that is connected to detection label by adding is realized detecting.Such ELISA is simple " sandwich ELISA ".Also can realize detecting but then add the 3rd antibody that is connected with detection label that the second antibody affinity is arranged by adding second antibody.ELISA can have the combination of proteins difference of Gly389 relatively based on antibody to the protein with Arg389.

In another ELISA example, infer that contain antigenic sample is fixed on the hole surface and/or contacts with antibody subsequently.In conjunction with and/or clean to remove after the non-specific binding immunocomplex, detect the bonded anti-antibody.When initial antibodies is connected to detectable label, can directly detect immunocomplex.In addition, but can use the second antibody that is connected with detection label of first antibody affinity to detect immunocomplex.

The ELISA that another kind of antigen is fixed relates to use antibody competition in detection.In such ELISA, the anti-antigenic antibody of mark is added in the hole, allow its combination, and/or detect by their label.Subsequently and the bag between incubation period, mixed the antigenic amount in unknown sample of determining with the anti-antigen-antibody of mark by the hole by sample.But in sample antigenic existence cause combined hole anti-antigen-antibody amount reduction and therefore reduced final signal.This also is applicable in unknown sample and detects anti-antigen-antibody, wherein unlabelled antibody be coated with antigenic hole and combine and also reduced the antigenic amount of the antibody that can be used for bonding mark.

Do not consider used form, ELISAs has some common feature, such as bag by, hatch and in conjunction with, clean with the kind of removing non-specific binding and detect the bonded immunocomplex.These are described below.

During with antigen or antibody sandwich plate, usually with antigen or antibody-solutions with the orifice plate overnight incubation or specify several hrs.Subsequently with cleaning orifice to remove not the material of absorption fully.For detecting antiserum(antisera), use any remaining available hole surface of antigenicity neutral nonspecific proteins matter " bag quilt " then.These comprise bSA (BSA), casein or milk power solution.Bag is allowed to seal the lip-deep non-specific adsorption of immobilization site and therefore reduces the background that is caused by antiserum(antisera) non-specific binding on the surface.

In ELISAs, may more be accustomed to using the second or the 3rd detection means, rather than direct method.Therefore, combine with the hole at protein or antibody, with the non-reactive material bag by to reduce background, to clean with after removing unconjugated material, under the condition that effectively allows immunocomplex (antigen/antibody) to form, fixed surface is contacted with biological specimen to be detected.The detection of immunocomplex subsequently needs second binding partner or the antibody of mark, and second binding partner or antibody connect markd the 3rd antibody or the 3rd binding partner.

" under the condition that effectively allows immunocomplex (antigen/antibody) to form " expression preferably includes with solution such as BSA, calf gamma Globulin (BGG) or phosphate-buffered salt (PBS)/Tween solution dilution antigen and/or antibody.The purpose that adds these reagent also is to help to reduce non-specific background.

" suitable " condition is also illustrated in and allows to hatch under effective bonded temperature or the time enough.Incubation step about about 1 to 2 to 4 hours usually, temperature preferably according to 25 ℃ to 27 ℃ order, perhaps can be spent the night about 4 ℃.

After all incubation step of ELISA, thereby non-compound material is removed on the surface of cleaning contact.The example of cleaning step comprises with solution such as PBS/Tween or borate buffer solution cleaning.Detect form specific complex between sample and the initial binding substance and carry out subsequently cleaning after, also can determine even produced the immunocomplex of small quantity.

For the means of detection are provided, the second or the 3rd antibody will have the respective labels of the detection of allowing.This can hatch with suitable chromophoric substrate just can colorific enzyme.Therefore, for example, people will be desirably under the condition that helps further forming immunocomplex with urase, glucose oxidase, alkaline phosphatase or the contact of catalase link coupled antibody and hatch for first and second immunocomplex for some time (for example, at room temperature contain PBS solution hatched 2 hours in such as PBS-Tween).

Hatching and cleaning subsequently with the antibody of mark with after removing unconjugated material, the amount of quantitative label, for example, peroxidase as the situation of enzyme label by with chromophoric substrate such as urea or purpurum bromocresolis or 2,2 '-azino-two-(3-ethyl-phenyl thiazole quinoline-6-sulfonic acid (ABTS) or H ₂O ₂Hatch together and realize.Quantitatively by measuring the degree that color takes place, for example, use visible spectrophotometer to realize subsequently.

Ii. immunohistochemistry

Antibody of the present invention also can with the FF and/or formaldehyde solution fixed that is used to study, the paraffin-embedded tissue block combined utilization by immunohistochemical method (IHC) preparation.For example, immunohistochemistry can be used for distinguishing Fortilin or assess the amount of Fortilin at cell.The method for preparing tissue block from these specific sample has been successfully applied in the IHC research of previous various predictive factorses, and/or is well-known (Brown et al., 1990 to those skilled in the art; Abbondanzo et al., 1990; Allred et al., 1990).

Briefly, can be by at room temperature making 50mg freezing " powdered " tissue rehydration in the phosphate buffer soln in little plastic capsule (PBS); Become sphere by the centrifugal particle that makes; They are resuspended in the viscosity embedding medium (OCT); Put upside down plastic capsule and/or once more by centrifugal its precipitation that makes; In-70 ℃ of iso-pentane, carry out quick-frozen; Cutting plastic capsule and/or remove and organize the refrigerated cylinder; Fixing organization cylinder on the cold micro-microtome frame of perseverance; And/or 25-50 serial section, prepare freezing microtome section.

Can in the plastics micro-centrifuge tube, make 50mg sample rehydration by relating to; Precipitation; Resuspension was fixed in 4 hours in 10% formaldehyde solution; Cleaning/precipitation; Be resuspended in 2.5% agar of temperature; Precipitation; In frozen water, make its cooling so that the agar hardening; From pipe, remove tissue/agar block; Filter and/or embedded block in paraffin; And/or the similarity method of cutting permanent section up to 50 continuously prepares permanent section.

III. treatment

In case determine individual β ₁The genotype of-AR or protein sequence can make the personalization course of treatment of treatment.In the preferred embodiment of described method, interested feature is that the patient handles the clinical response that is shown to some treatment, for example medicine is such as but not limited to beta blocker, such as bucindolol, target β ₁The medicine of AR or the reaction that medical treatment of conditions is handled.As used herein, " medical condition " includes but not limited to that any being proved to be has one or more healths of expecting treatment and/or the patient's condition or the disease of mental symptoms, and has the disease of similar pathological and physiological condition with the disease of identifying recently with other before comprising, be such as but not limited to heart failure, pheochromocytoma, migraine, arrhythmia, hypertension, DCM (dilated cardiomyopathy), ischemic heart disease (myocardosis, ischemic heart disease (myocardosis, stenocardia, impatient infraction) and various anxiety disorder.As what use in this article, the quantitative measurment of any or all that term " clinical response " expression is following: to the quantitative measurment of therapeutic efficiency and usefulness and adverse events (that is side effect).

Therefore can be to having the β that isozygotys of medical condition ₁The individual enforcement of Arg389 comprises that beta blocker is such as but not limited to the treatment of bucindolol.Beta blocker can use separately or with at least a other reagent, co-administered such as stable compound.Beta blocker also can be with previous to demonstrate mishandling medical device means by the morbid state of the described device of needs co-administered.For example, have bradycardic heart failure patient usually and can not accept beta blocker.If but individual genotype is Arg389 (a favourable genotype), can implantable pacemaker, and open the bucindolol prescription.

A. route of administration

Can use beta blocker by many approach, that described approach includes, but are not limited to is oral, in the intravenously, intramuscular intra-arterial, marrow. in the sheath, in the ventricle, in the intradermal, tracheae, in intravesical, intraocular, transdermal, subcutaneous, intraperitoneal, the nose, interior, partial, the my humble abode of intestines or rectal administration.About the further detailed explanation of preparation and the technology used can (Maack Publishing Co., Easton find in Pa.) at the Remington ' of latest edition sPharmaceutical Sciences.In certain embodiments, the preparation bucindolol is used for Orally administered.

B. formulation

When considering clinical application, will be to be suitable for the form pharmaceutical compositions that purpose is used.Usually, this must make the composition of preparation not have pyrogen and other may be to the deleterious impurity of human or animal in fact.

People expect to use suitable salt usually and buffer reagent provides stable delivery vector and permission to be absorbed by target cell.When reconstitution cell is introduced the patient, also will use buffer reagent.Waterborne compositions of the present invention comprises the carrier or the aqueous medium of significant quantity.Phrase " medicine or pharmacology on acceptable " expression does not have side effects when being administered to the animal or human, the molecular entity and the composition of anaphylaxis or other adverse effects.As what use in this article, " medicinal acceptable carrier " comprise solvent, buffer reagent, solution, dispersion medium, Drug coating, antibacterium and anti-mycotic agent, etc. blend the acceptable compounding pharmaceutical that is used for such as absorption delay agent, such as the medicine that is fit to use to the people.The application of these media and reagent is well-known in the art for pharmaceutically active substance.Except any discomfort in this scope is closed the conventional media or reagent of activeconstituents of the present invention, their being applied in the intent of the present invention in therapeutic composition.Supplementary active ingredients also can be integrated in the composition, as long as they do not make the carrier or the cell inactivation of composition.

Active composition of the present invention can comprise typical pharmaceutical preparations.These can be via any common approach, as long as target tissue can be used described composition by the sort of approach according to using of composition of the present invention.This comprises using of per os, nose or cheek.As an alternative, use and by intracutaneous, subcutaneous, intramuscular, intraperitoneal or intravenous injection or to be injected directly into heart tissue.As mentioned above, such composition is normally used as the medicine acceptable composition usually.

Active compound also can be used through peritonaeum or intraperitoneal.Explanation as an example can be prepared in the water such as hydroxypropylcellulose at the suitable tensio-active agent that is mixed with as the solution of the active compound of acceptable salt on free alkali or the pharmacology.Dispersed system also can and prepare in oil in glycerine, liquid macrogol and its mixture.Under usual preservation and application conditions, these prepared products comprise sanitas usually to prevent microbial growth.

The medicament forms that is suitable for injecting use comprises, for example, and sterilization aqueous solution or dispersed system and be used for preparing immediately the sterile powder of sterile solution for injection or dispersed system.Usually, these prepared products be the sterilization and be to exist to a certain extent with the fluid form that is easy to inject.Prepared product should be stable under the condition of making and preserving and should be with the opposing microorganism, is saved such as the mode of bacterium and fungal contamination effect.The suitable solvent or dispersion medium can comprise, for example, and water, ethanol, polyol (for example, glycerine, polyoxyethylene glycol and liquid macrogol etc.), their suitable mixture and vegetables oil.Can, for example, by using dressing, such as Yelkin TTS, in the dispersed system situation by keeping essential granular size and keeping suitable flowability by the application surface promoting agent.Can pass through various antibacteriums and anti-mycotic agent, for example, P-hydroxybenzoic acid lipid, butylene-chlorohydrin, phenol, Sorbic Acid, thimerosal wait and prevent action of microorganisms.In many situations, will preferably include isotonic agent, for example, sugar or sodium-chlor.Can be by application delay absorption agent in composition, for example, aluminum monostearate and gelatin prolong the absorption of Injectable composition.

For Orally administered, polypeptide of the present invention can be mixed with vehicle usually and use with non-absorbable mouth wash shua and dentifrice form.Can be by activeconstituents being mixed The suitable solvent, such as preparing mouth wash shua in the dobell's solution (Dobell ' s Solution) with essential amount.Selectable, activeconstituents can be mixed in the anticorrosion washing lotion that comprises Sodium Tetraborate, glycerine and saleratus.Activeconstituents also can be scattered in dentifrice, comprising: gel, paste, powder and slurry.Activeconstituents can be added in the paste dentifrice with the treatment significant quantity, and described dentifrice can comprise water, tackiness agent, abrasive, seasonings, foaming agent and wetting agent.

Usually can prepare composition of the present invention with neutrality or salt form.Drug acceptable salt comprises, for example, and derived from the acid salt (forming) of mineral acid (for example, hydrochloric acid or phosphoric acid) or organic acid (for example, acetate, oxalic acid, tartrate, amygdalic acid etc.) with proteinic free amine group.The also salt that can derive and form with proteinic free carboxy from mineral alkali (for example, sodium hydroxide, potassium, ammonium, calcium or iron) or organic bases (for example, Isopropylamine, Trimethylamine 99, Histidine, PROCAINE HCL, PHARMA GRADE etc.).

Depend on preparation, preferably use solution with form that can be compatible and with the treatment significant quantity with formulation.Preparation can be used such as injection solution, drug release capsules etc. with various formulations easily.For gi tract are used aqueous solution outward, for example, solution is cushioned usually suitably to be handled and liquid diluent such as at first provides for example to use enough salt or glucose at the condition of oozing.Such aqueous solution can be used for, for example, intravenously, intramuscular, subcutaneous and intraperitoneal are used.Preferably, use well known by persons skilled in the art, especially according to the aqueous medium of sterilization disclosed by the invention.Explanation as an example, the grade that single dose can be dissolved in 1ml ooze in the NaCl solution and or be added to inject in the hypodermoclysis fluid of 1000ml or in the infusion site of being recommended and (for example see, " Remington ' s Pharmaceutical Sciences " the 15th edition, 1035-1038 and 1570-1580 page or leaf).Some variation is taken place in condition necessarily that depend on treatment target on dosage.In any case the people who is responsible for using will determine the optimal dose of individual subject.In addition, for using of people, prepared product should satisfy the standard of the desired sterilization of FDA biological product standards office (Office of Biologics standards), pyrogen, Generally Recognized as safe and purity.

1. what control/expansion/continue/prolongation discharged uses

Another aspect of the present invention provides by sending the method for the treatment of heart failure as the beta blocker of sustained release preparation to having the genotypic patient of β 1Arg389 of isozygotying.As used herein, term " control ground ", " expansion ground ", " constantly " or " prolonging ground " discharge composition of the present invention and common are represented as in this article " control ground discharges ", and comprise lasting or discontinuous and linear or nonlinear release composition of the present invention.Control ground discharges the beta blocker preparation and has many advantages.

A. tablet

Control ground discharges the tablet that is fit to the object of the invention and is disclosed United States Patent (USP) 5,126,145, and it incorporates this paper into through reference.Such tablet is included in the hydrophobic components of the water dissolvable medicinal adhesive of the high viscosity Vltra tears of the about 5-30% in the mixture (admixture), about 2-15%, about 2-20% such as the wax material, for example, lipid acid and the approximately activeconstituents of 30-90%.

B. film

The present invention prevents or treats to have the β 1Arg389 genotype patient's of isozygotying method after also being provided at invasive cardiac procedures, comprises and uses polymeric membrane biodegradable, biocompatibility, wherein comprises beta blocker and gives the patient such as bucindolol.Compare with width with their length, polymeric membrane is extremely thin.Described film has identical selected thickness usually, and about 60 microns are arrived about 5mm.Thickness be about 600 microns to 1mm with approximately 1mm is to the film of about 5mm, and thickness is about 60 microns to about 1000 microns and about 60 all can be used on preparation to about 300 microns film and be used for inserting the intravital treatment of patient graft.Described film can be administered to the patient with the similar manner that adheres to the surgery method therefor.For example, beta blocker, such as bucindolol, film preparation can be sprayed at during the surgical management or drop on heart tissue position or the artery, or formed film can be placed on the selected tissue location.In alternate embodiment, the dressing that described film can be used as sustained release overlays on the medical treatment device, and such as support, it will further go through below.

Can construct implant with biodegradable or abiotic degradable polymer, wherein beta blocker is distributed in whole polymeric matrix uniformly.Many suitable biodegradable polymers that are used to prepare biological degradable membrane of the present invention are well known in the art, comprise poly-acid anhydrides and aliphatic polyester, preferred poly(lactic acid) (PLA), polyglycolic acid (PGA) and their mixture and multipolymer, more preferably 50: 50 PLA: PGA multipolymer and 75: 25 PLA most preferably: PGA multipolymer.Also can use single enantiomer of PLA, preferred L-PLA, it can use or unite PGA separately and use.Also can applying polycarbonate, poly-fumaric acid esters and caprolactone prepare implant of the present invention.

Mix the beta blocker of polymeric film of the present invention, such as bucindolol, amount be the significant quantity that demonstrates the effect of measurable treatment disease, described disease has similar pathological and physiological condition, be such as but not limited to heart failure, pheochromocytoma, migraine, arrhythmia, hypertension, ischemic (aschemia), myocardosis and various anxiety disorder.Can be by various technology such as composition of the present invention being incorporated in the film by solution method, suspension method or fusion pressing.

C. transdermal label apparatus

Transdermal delivery relates to through the dermal delivery therapeutical agent so that it distributes in vivo by blood circulation.Transdermal delivery is equivalent to replace intravenous syringe needle to come constantly, control the ground delivering drugs as admission port with skin.Therapeutical agent passes the skin of skin, diffuses into capillary vessel or small blood vessel and be transported to the main recycle system subsequently in skin.

It is well-known in the art that transdermal patch device by skin control ground, administering therapeutic agent constantly is provided.Such device for example, is disclosed United States Patent (USP) 4,627,429; 4,784,857; 5,662,925; 5,788,983; With 6,113,940, they are merged in herein by reference.Distinctive is that these devices comprise the impermeable back sheet of the medicine of device for limiting outside surface and permeable skin cling film, such as the air retaining wall of viscous layer, sealing, produce the storage storehouse by this way and be used for placing therapeutical agent between them.In one embodiment of the invention, the preparation of beta blocker is introduced into the storage storehouse of transdermal patch and is used by the Arg389 patient of β 1AR gene pure.

D. medical treatment device

The intention of another embodiment is beta blocker is mixed medical treatment device such as bucindolol, and this device is placed in the target position of expectation in the body subsequently, so beta blocker flows out medical treatment device at this.As what use in this article, " medical treatment device " represented in order to prevent or to treat medical conditions and temporarily or for good and all introduce mammiferous device.Thereby these devices comprise with subcutaneous, transdermal or operating mode and are introduced into any device that places in organ, tissue or the chamber.Medical treatment device comprises, but be not limited to, support, artificial graft, artificial heart valve, artificial heart and be connected and repair organ and vascular round-robin device (fixture), vein valve, abdominal aortic aneurysm (AAA) graft, IVCF (inferior venal caval filters), conduit comprise permanent drug infusion catheter, spring ring embolism (embolic coils), are used for embolism materials (for example, PVA foam material), reticulation repair materials, Dracon blood vessel particle shaping metal sheet, bar and the screw and the vascular suture material of blood vessel embolism.

In one embodiment, medical treatment device wraps quilt such as support or graft with matrix.Can be used to wrap matrix from various material preparations by support according to the present invention or graft.Basic demand for matrix is that it has enough elasticity and flexibility, does not break thereby keep the surface that support or synthetic graft expose.

C. dosage

Amount that use to the patient or the bucindolol of prescribing can be about, at least about, or about at the most 0.1,0.5,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500mg perhaps can be from any scope of wherein drawing.Alternately, use or recipe quantity can be about weight in patients approximately, at least about, or about at the most 0.001,0.002,0.003,0.004,0.005,0.006,0.007,0.008,0.009,0.01,0.02,0.03,0.04,0.05,0.06,0.07,0.08,0.09,0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8,0.9,1.0,1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8,1.9,2.0,2.1,2.2,2.3,2.4,2.5,2.6,2.7,2.8,2.9,3.0,3.1,3.2,3.3,3.4,3.5,3.6,3.7.3.8,3.9,4.0,4.1,4.2,4.3,4.4,4.5,4.6,4.7,4.8,4.9,5.0mg/kg, perhaps can be from any scope of wherein drawing.

When providing with discontinuous amount, the bucindolol of each picked-up can be considered to one " dosage ".The medical worker can prescribe or use the multiple doses bucindolol for specific or uncertain time process.

Can use bucindolol 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,30,40,50,60,70,80 or more times or can be from any time of wherein drawing.Purpose of the present invention also plan can be in the uncertain time cycle to the patient take described medicine or as long as the patient show prescription or use bucindolol at the symptom of medical conditions just take medicine to the patient. are same; 2、3、4、5、6、7、8、9、10、15、20、25、30、35、40、45、50、55、1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、1、2、3、4、5、6、7、1、2、3、4、5、1、2、3、4、5、6、7、8、9、10、11、12、，。 As an alternative, medicine can be used in the general mode in these any time cycles and the time can prolong above 1 year.

D. other treatment is selected

In certain embodiments of the invention, can to relate to the beta blocker of using be not that bucindolol or it are muscular strength agent (ionotrope), diuretic(s), ACE-I, AII antagonist, BNP, Ca to described method ⁺⁺-blocker or hdac inhibitor.At assessment β ₁-AR and/or α _2cAfter-the AR, these medicaments can replace bucindolol or prescribe except bucindolol or be administered to the patient.

As second treatment plan, described medicament can or be used before or after bucindolol or take with the bucindolol while.Described treatment can improve one or more symptoms of pathologic cardiac hypertrophy or heart failure such as the motor capacity that increase is provided, the cardiac ejection volume that increases, the left ventricle diastole that reduces end is eventually pressed, the pulmonary capillary wedge pressure that reduces, the cardiac output or the cardiac index that increase, the Ppa pulmonary artery pressure that reduces, the left ventricle that reduces end eventually shrinks and the diastole degree, the left side and the right ventricle wall stress that reduce, the wall tension force and the wall thickness that reduce, the quality of life that increases, with the disease-related M ﹠ M that reduces.

In another embodiment, expectation bucindolol and other treatment pattern combined utilization.Therefore, except above-mentioned therapy, also can provide more " standard " medicine heart therapy to the patient.Other therapy example comprises, but be not limited to other beta blocker, anti--the hypertension agent, cardiotonic drug, anti--the thrombus agent, vasodilator, hormone antagonist, inotropic agent (iontropes), diuretic(s), endothelin antagonist, calcium channel blocker, phosphodiesterase inhibitor, ACE inhibitor, hypertensin 2 type antagonist and cytokine blocker/inhibitor and hdac inhibitor.

Can be by making heart cell and single composition or comprising the pharmaceutical preparation contact of two kinds of medicaments or by making cell contact the described combination of realization simultaneously with two kinds of different compositions or preparation, wherein a kind of in two kinds of compositions comprises expression constructs and another kind comprises described medicament.As an alternative, use the therapy of bucindolol can be before using other medicament or subsequently at interval several minutes in the time in several weeks, use.Be applied to respectively in the embodiment of cell at different medicaments and expression constructs, usually to guarantee that the important time cycle does not have still can bring into play the useful compound action of pair cell above the time between sending at every turn such as medicament and expression constructs.In such example, the intent of the present invention is to make usually cell and two kinds of modalities (modalities) to contact in their each other about 12-24 hours, more preferably in each other about 6-12 hour, and to have only about 12 hours time-delay be most preferred.In some cases, can expect the extended treatment time cycle significantly, such as several days (2,3,4,5,6 or 7) the time that is spaced apart between using separately to several weeks (1,2,3,4,5,6,7 or 8).

Can also conceivablely be, perhaps bucindolol or in addition medicament surpass using and will expect once.In this, can use various combinations.Explanation is that " A " and other medicament are the situations of " B " at bucindolol as an example, and based on 3 and 4 times use altogether, following permutation and combination is enumerated as an example:

A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B

A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A

A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations are included in the present invention too.

1. rem

Rem and application process, dosage etc. are well-knownly (for example to see to those skilled in the art, " Physicians Desk Reference ", Klaassen ' s " The Pharmacological Basisof Therapeutics ", " Remington ' s Pharmaceutical Sciences ", " The MerckIndex; Eleventh Edition ", relevant portion in them is incorporated herein by reference), and can make up according to content disclosed herein and the present invention.The condition of treatment target is made the variation of some necessity to dosage with depending on.In any case the people who is responsible for using will determine the optimal dose at individual subject, and in this individuation scope that fixes on those of ordinary skills really and grasped.

The limiting examples that can be used for rem of the present invention comprises anti-high lipoprotein agent, arteriosclerosis agent, anticoagulation/thrombolytics, coagulant, anti-heart disorder agent, hypotensive agent, vasoconstrictor, congestive heart failure therapeutical agent, antianginal agent, antiseptic-germicide or their combination.

In addition, it should be noted and to develop the disease treatment target gene that overlaps that makes new advances with following any described content that it is the same with the beta blocker that uses in present embodiment (as follows).Have and overlap although expect some such genes, may develop the target gene that makes new advances.

A. hyperlipoproteinemia agent

In certain embodiments, use the medicament of reduction than hyperlipidemia and/or lipoprotein concentration, be called as " hyperlipoproteinemia agent " in this article, can with cardiovascular therapy combination according to the present invention, especially in treatment atherosclerosis and vascular tissue thicken or block.In some aspects, the hyperlipoproteinemia agent can comprise aryloxy alkanoic acid/fibric acid derivative, resin/bile acid chelating agent, HMGCoA reductase inhibitor, nicotinic acid derivates, Triiodothyronine or thyroid hormone analogs, other class medicaments (miscellaneous agent) or its combination.

I. aryloxy alkanoic acid/fibric acid derivative

The limiting examples of aryloxy alkanoic acid/fibric acid derivative comprises Sgd-24774, enzafibrate, binifibrate, Win-35833, S-8527, chlorine Bei Te (clofibrate-S), clofibric acid, etofibrate, fenofibrate, gemfibrozil (lobid), nicofibrate, pirifibrate, Ronifibrate, simfibrate and Phyllocormin N chlorine Bei Te.

Ii. resin/bile acid chelating agent

The limiting examples of resin/bile acid chelating agent comprises Colestyramine (cholybar, questran), colestipol (DEAE-sephadex) and poly osamine.

Iii.HMG CoA reductase inhibitor

The limiting examples of HMG CoA reductase inhibitor comprises lovastatin (mevacor), Pravastatin (pravochol) or Simvastatin (zocor).

Iv. nicotinic acid derivates

Limiting examples nicotinate, Olbetam, pentaerythritol tetranicotinate, S-486, nicomol and the oxiniacic acid of nicotinic acid derivates.

V. Triiodothyronine and analogue

The limiting examples of Triiodothyronine and its analogue comprises etoroxate, Thyropropic Acid and thyroxine.

Vi. other hyperlipoproteinemia agent

The limiting examples of other hyperlipoproteinemia agent comprises Acifran, diazasterol, Benfluorex, β-Ben Yajiaji butyramide, vitamin BT, chondroitin sulfate, oxygen steroid ether, detaxtran, dextran sulfate sodium, 5,8,11,14, the 17-timnodonic acid, eritadenine, rofurazanol, meglutol, AC-233, mytatrienediol, ornithine, gamma oryzanol, Pantethine, pentaerythrite tetra-acetate, α-fenbutyramidum, pirozadil, probucol (lorelco), β-Gu Zaichun, sultosilic acid-piperazine salt, tiadenol, Verdiana and xenbucin.

B. arteriosclerosis agent

The limiting examples of arteriosclerosis agent comprises Pyridocarbates (pyridinol carbamate).

C. anticoagulation/fibrinolytic agent

In certain embodiments, use assist in removing or the medicament of pre-preventing thrombosis can with use the conditioning agent combination, especially in treatment arteriosclerosis and vascular system (for example, artery) are blocked.The limiting examples of anticoagulation and/or fibrinolytic agent comprises anti-coagulant, anti-coagulant antagonist, anti-platelet agents, thrombolytic agent antagonist or their combination.

In some aspects, antithrombotic agent that can be Orally administered, such as, for example, acetylsalicylic acid and warfarin (but arteries and veins fourth) they are preferred.

I. anti-coagulant

The limiting examples of anti-coagulant comprises Acenocoumarol, ancrod, bacterium indenes diketone, bromindione, clorindjone, cumetharol, Methopyranorin, dextran sulfate sodium, temparin, Oragulant, tromexan, ethylenebis tonka bean camphor, Fluindione, heparin, r-hirudin, Lyapolate Sodium, oxazidione, many sulfuric acid poly-pentose, phenindione, phenprocoumon, phosvitin, Pirodomast, tioclomarol and warfarin.

Ii. anti-platelet agents

The limiting examples of anti-platelet agents comprises acetylsalicylic acid, dextran, Dipyridamole (dipyridamole), heparin, sulfinpyrazone (sulfinpyrazone) and ticlopidine (Ticlid).

Iii. thrombolytic agent

The limiting examples of thrombolytic agent comprises tissue plasminogen activator (activase), plasmin, uPA, urokinase (abbokinase), streptokinase (streptase), Eminase/APSAC (Eminase).

D. blood coagulant

In certain embodiments, wherein the patient has hemorrhage or increases hemorrhage possibility, can use the preparation that can strengthen coagulation of blood.The limiting examples of coagulation of blood promotor comprises thrombolytic agent antagonist and anticoagulant antagonist.

I. anti-coagulant antagonist

The limiting examples of anti-coagulant antagonist comprises protamine and vitamin K1.

Ii. thrombolytic agent antagonist and antithrombotics

The limiting examples of thrombolytic agent antagonist comprises hexosamine (amicar) and tranexamic acid (amstat).The limiting examples of antithrombotics comprises anagrelide, Argatroban, Cilostazole (cilstazol), Daltroban, defibrotide, enoxaparin, not uncommon heparin, indobufen, lamoparan, Ao Zegerui, Pirodomast, sprinkles appropriate bright, Fragmin, ticlopidine and trifluoro to loose.

E. anti-arrhythmic agents

The limiting examples of anti-arrhythmic agents comprises I class anti-arrhythmic agents (sodium channel blockers), II class anti-arrhythmic agents (beta-adrenergic blocking agent), II class anti-arrhythmic agents (prolong drug again polarizes), IV class anti-arrhythmic agents (calcium channel blocker) and other class anti-arrhythmic agents.

I. sodium channel blockers

The limiting examples of sodium channel blockers comprises IA class, IB class and IC class anti-arrhythmic agents.The limiting examples of IA class anti-arrhythmic agents comprises disopyramide (norpace (disopyramide)), procainamide (pronestyl) and Quinidine (quinidex).The limiting examples of IB class anti-arrhythmic agents comprises lignocaine (xylocaine), tocainide (tonocard) and mexiletine (mexitil).The limiting examples of IC class anti-arrhythmic agents comprises encainide (enkaid) and flecainide (tambocor).

Ii. beta blocker

Beta blocker, be also referred to as beta-adrenergic blocking agent, beta-adrenergic antagonist or II class anti-arrhythmic agents, limiting examples comprise acebutolol (sectral), alprenolol, amosulalol, Arottnolol, atenolol USP 23, befunolol, betaxolol, bevantolol, bisoprolol, Bopindolol, bucumolol, bufetolol, bufuralol, bunitrolol, bupranolol, betabloc, butofilolol, Carazolol, carteolol, carvedilol, Celiprolol, Cetamolol, Cloranolol, Sch-19927, ICI-141292, esmolol (brevibloc), indenes dawn alcohol, Trate, levobunolol, Corindolan, metipranolol, metoprolol, moprolol, nadolol, nadoxolol, nifenalol, nipradolol, oxprenolol, penbutolol, pindolol, practolol, Pronethalol, Proprasylyte (inderal), sotalol (betapace), Sulfinalol, talinolol, Tertatolol, timolol, toliprolol and xibinolol.In some aspects, beta blocker comprises the aryloxy propanol amine derivative.The limiting examples of aryloxy propanol amine derivative comprises acebutolol, alprenolol, Arottnolol, atenolol USP 23, betaxolol, bevantolol, bisoprolol, Bopindolol, bunitrolol, butofilolol, Carazolol, carteolol, carvedilol, Celiprolol, Cetamolol, ICI-141292, indenes dawn alcohol, Corindolan, metipranolol, metoprolol, moprolol, nadolol, nipradolol, oxprenolol, penbutolol, pindolol, Proprasylyte, talinolol, Tertatolol, timolol and toliprolol.

Iii. polarization prolongs agent again

Prolong polar medicament again, be also referred to as III class anti-arrhythmic agents, limiting examples comprise amiodarone (cordarone) and sotalol (betapace).

Iv. calcium channel blocker/antagonist

Calcium channel blocker, be also referred to as IV class anti-arrhythmic agents in addition, limiting examples comprise that aralkylamine (for example, Bepridil, Odizem, Fendiline, methoxyverapamil, prenylamine, terodiline, verapamil), dihydrogen pyridine derivative (felodipine, Isrodipine, nicardipine, nifedipine, nimodipine, nisoldipine, nitrendipine), bridged piperazine derivatives (for example, CN, flunarizine, lidoflazine) or blending calcium channel blocker be such as bencyclane, Pagano-Cor, magnesium, the non-ground of rice you or Parker former times woods.In certain embodiments, calcium channel blocker comprises long-acting dihydropyridine (nifedipine type) calcium antagonist.

V. other class anti-arrhythmic agents

The limiting examples of other class anti-arrhythmic agents comprises vidarabine (adenocard), ground Gao Ping (lanoxin), acecainide, ajmaline, amoproxan, amidonal, Bretylium Tosylate, Meregon, gram hat isobutylamine, Acid Capobenique, cibenzoline, disopyramide, dihydrochinidin, Ricainide, SCH 1000, lignocaine, fragrant Lamine, Lorcainide, U.S. Australia ice is fixed, Moracizine, pirmenol, Prajmaline, Propafenone, Bi Linuoning, galactoquin, quinidine sulfate and quinotoxol.

F. hypotensive agent

The limiting examples of hypotensive agent comprises sympatholytic, α/Beta receptor blockers, alpha blocker, the agent of anti-II type Angiotensin, Beta receptor blockers, calcium channel blocker, the mixed hypotensive agent of vasodilator.

I. alpha block agent

The alpha block agent, be also referred to as alpha antiadrenergic agent or alpha-adrenergic antagonist, limiting examples comprise Amosulal YM-09538, Arottnolol, Glamidole, Doxazosin, Dihydroergotoxine, fenspiride, Indoramine, Trate, Varson, Prazosin, terazosin, phenylmethylimidazoline, trimazosin and Yohimbine.In certain embodiments, the alpha block agent can comprise quinazoline derivant.The limiting examples of quinazoline derivant comprises alfuzosin, bunazosin, Doxazosin, Prazosin, terazosin and trimazosin.

Ii. α/Beta receptor blockers

In certain embodiments, hypotensive agent is α-be again the beta-adrenergic antagonist.The limiting examples of α/Beta receptor blockers comprise Trate (normodyne, trandate).

Iii. the agent of anti-II type Angiotensin

The limiting examples of anti-II type Angiotensin agent comprises angiotensin converting enzyme inhibitor and angiotensin II receptor antagonists.The limiting examples of angiotensin converting enzyme inhibitor (ACE inhibitor) comprises alacepril, enalapril (vasotec), captopril, Yipingshu, delapril, enalaprilat, fosinopril, lisinopril, Moveltipril, perindopril, quinapril and Ramipril.Angiotensin-ii receptor blockers, be also referred to as angiotensin II receptor antagonists, ANG receptor blocking agent or ANG-I type-1 receptor blocking agent (ARBS), limiting examples comprise that blood vessel Kant sand is smooth, eprosartan, irbesartan, losartan and valsartan.

Iv. sympatholytic

The limiting examples of sympatholytic comprises central action sympatholytic or peripheral action sympatholytic.The central action sympatholytic is also referred to as central nervous system (CNS) sympatholytic, limiting examples comprise clonidine (catapres), guanabenz (wytensin), guanfacine (tenex) and methyldopa (aldomet).The limiting examples of peripheral action sympatholytic comprises ganglionic blockader, adrenergic neuron blocker, beta-adrenergic blocking agent or alpha 1 adrenergic blocker.The limiting examples of ganglionic blockader comprises mecamylamine (inversine) and trimetaphan (arfonad).The limiting examples of adrenergic neuron blocker comprises guanethidine (ismelin) and serpentine (serpasil).The limiting examples of beta-adrenergic blocking agent comprises that the heart executes moral (sectral), atenolol USP 23 (tenormin), betaxolol (kerlone), carteolol (cartrol), Trate (normodyne, trandate), metoprolol (lopressor), nadolol (corgard), penbutolol (levatol), pindolol (visken), Proprasylyte (inderal) and timolol (blocadren).The limiting examples of alpha 1 adrenergic blocker comprises Prazosin (minipress), Doxazosin (cardura) and terazosin (hytrin).

V. vasodilator

In certain embodiments, the cardiovascular treatment agent can comprise vasodilator (for example, cerebral vasodilator, coronary vasodilator or peripheral vasodilator agent).In some preferred embodiment, vasodilator comprises coronary vasodilator.The limiting examples of coronary vasodilator comprises that A Meng chases after sweet smell, dibazol, benfurodil hemisuccinate, benziodarone, chloracizin, ethoxylcumarin O deethylase, cloridarol, clonitrate, dilazep, Dipyridamole, droprenilamine, efloxate, Erythrityl Tetranitrate, Pagano-Cor, Fendiline, floredil, ganglefene, herestrol two (β-diethyl amino benzyl ethyl ether), hexobendine, nitrolamine tosylate, Khellinum's rope, lidoflazine (lidoflanine), Mannityl Nitrate, medibazine, nicorglycerin, trinitrol, Pentaerythritol Trinitrate, Parker former times woods, pyrrole amine second theophylline, trapidil, methylchromone, trimetazidine, triethanolamine trinitrate biphosphate and visnadine.

In some aspects, vasodilator can comprise chronic treatment vasodilator or hypertensive emergency vasodilator.The limiting examples of chronic treatment vasodilator comprises hydralazine (apresoline) and minoxidil (loniten).The limiting examples of hypertensive emergency vasodilator comprises Sodium Nitroprusside (nipride), diazoxide (hyperstat IV), hydralazine (apresoline), minoxidil (loniten) and verapamil.

Vi. other class hypotensive agents

The limiting examples of other class hypotensive agents comprises ajmaline, γ-ammonia butyric acid; diiodobuphenine; cicletanine; ciclosidomine; cryptenamine tannates; fragrant Lip river dopan; Manoplas; Sufrexal; mebutamate; mecamylamine; methyldopa; methyl-4-pyridyl ketone amido thiocarbamide (methyl4-pyridyl ketone thiosemicarbazone) that contracts; muzolimine; Pargyline; pempidine; Pinacidil; benodaine; Flufenone; Protoveratrine; tetrahydroserpentine; Rescimetol; rilmenidine; Saralasin; sodium nitrorusside; ticrynafen; Trimetaphani Camsylas; tyrosine oxidase and urapidil.

In some aspects, hypotensive agent can comprise aryl ethanol amine derivative, benzothiadiazine derivatives, N-carboxyalkyl (peptide/lactan) derivative, dihydrogen pyridine derivative, guanidine derivative, hydrazine/phthalazines, imdazole derivatives, quaternary ammonium compound, serpentine derivative or sulfone amide derivative.

Aryl ethanol amine derivative. the limiting examples of aryl ethanol amine derivative comprises Amosulal YM-09538, bufuralol, Sch-19927, Trate, Pronethalol, sotalol and Sulfinalol.

Benzothiadiazine derivatives. the non-specific example of benzothiadiazine derivatives comprises Altizide (althizide), Hydrex, benzthiazide, hydrochlorothiazide benzyl ester, fourth hydrochlorothiazide, chlorothiazide, chlorthalidone, cyclopenthiazide, cyclothiazide, diazoxide, epithiazide, P-2105, Fenquizone, hydrochlorothiazide, Hydroflumethiazide, Methyclothiazide, meticrane, metolazone, paraflutizide, polythiazide (polythizide), Tetrachloromethiazide and trichlormethiazide.

The limiting examples of N-carboxyalkyl (peptide/lactan) derivative .N-carboxyalkyl (peptide/lactan) derivative comprises alacepril, captopril, Yipingshu, delapril, enalapril, enalapril spy, fosinopril, lisinopril, Moveltipril, perindopril, quinapril and Ramipril.

Dihydrogen pyridine derivative. the limiting examples of dihydrogen pyridine derivative comprises amlodipine, felodipine, Isrodipine, nicardipine, nifedipine, nilvadipine, nisoldipine and nitrendipine.

Guanidine derivative. the limiting examples of guanidine derivative comprises betanidine, Debrisoquine, guanabenz, guanacline, Quanadrel, guanazodine, guanethidine, guanfacine, Vatensol, guanoxabenz and envacar.

Hydrazine/phthalazines. the limiting examples of hydrazine/phthalazines comprises budralazine, cadralazine, nepresol, endralazine, hydracarbazine, Hydralazine, Pheniprazine, propyldazine and todralazine.

Imdazole derivatives. the limiting examples of imdazole derivatives comprises clonidine, lofexidine, phentolamine, thiamenidine and tolonidine.

Quaternary ammonium compound. the limiting examples of quaternary ammonium compound comprises Prparat 9295, chlorisondamine chloride, hexamethonium, Pentacynium Bis, Penthonium, pentolinium tartrate, phenactropinium chloride and trimethidinium methosulfate.

The serpentine derivative. the limiting examples of serpentine derivative comprises bietaserpine, deserpidine, Rescinnamine, serpentine and syrosingopine.

Sulfone amide derivative. the limiting examples of sulfone amide derivative comprises ambuside, clopamide, FRUSEMIDE, indapamide, quinethazone, tripamide and xipamide.

Vii. vasopressor

Usually the use vasopressor promotes the blood pressure between shock stage, and shock can appear at during the surgical operation.Vasopressor, be also referred to as the hypotension agent, limiting examples comprise Knoll), angiotensin aminocompound, dimetrophine, Dopamine HCL, etifelmine, S 40032-7, Gepefrine, metaraminol, midodrine, norepinephrine, pholedrine and synephrine.

G. congestive heart failure therapeutical agent

Anti--Angiotensin II agent that the limiting examples of congestive heart failure therapeutical agent comprises, afterload-preload reduce therapeutical agent, diuretic(s) and muscular strength agent (inotropic agnet).

I. afterload-preload reduces

In certain embodiments, the animal patient that can not accept angiotensin antagonist can be treated with combination treatment.Such therapy can combined administration hydralazine (apresoline) and sorbide nitrate (isordil, sorbitrate).

Ii. diuretic(s)

The limiting examples of diuretic(s) (for example comprises thiazine or benzothiadiazine derivatives, Altizide, Hydrex, benzthiazide, hydrochlorothiazide benzyl ester, the fourth hydrochlorothiazide, chlorothiazide, gram urine plug, chlorthalidone, cyclopenthiazide, epithiazide, P-2105, P-2105, Fenquizone, hydrochlorothiazide, Hydroflumethiazide, Methyclothiazide, meticrane, metolazone, paraflutizide, polythiazide, Tetrachloromethiazide, trichlormethiazide), organomercurial (for example, Percapyl, meralluride, mercamphamide, mercaptomerin sodium, mercumallylic acid, mercumatilin sodium, subchloride of mercury, mersalyl), pteridine (for example, furterene, triamterene), purine (for example, vinegar bitter edible plant alkali, the 7-xanturil, pamobrom, protheobromine, Thesal), the steroid that contains aldosterone antagonists (for example, canrenone, folinerin, spironolactone), sulfone amide derivative (for example, acetazolamide, ambuside, azosemide, bumetanide, Butazolamide, Chloraminophenamide, clofenamide, clopamide, the clorexolone, ditane-4,4 '-benzene disulfonic acid amide, Toluidrin, Ethoxzolamide, Furosemide, indapamide, mefruside, methazolamide, piretanide, quinethazone, torasemide, tripamide, xipamide), uridylic (for example, aminometradine, amisometradine), protect potassium antagonist (for example, guanamprazine, triamterene) or mix diuretic(s) such as aminozine, arbutin, chlorazanil, Ethacrynic Acid, etozolin, hydracarbazine, Isosorbide, N.F,USP MANNITOL, metochalcone, muzolimine, Parker former times woods, Tienilic Acid (ticrnafen) and urea.

Iii. muscular strength agent

The muscular strength agent, be also referred to as cardiotonic drug, limiting examples comprise acefylline, acedoxin, 2-amino-4-picoline, amrinone, benfurodil hemisuccinate, Bucladesine, cerberosine, camphor pyrrole acid amides, Convallatoxin, apocynei, ibopamine, Deslanoside, Cardigin, purple foxglove, digoxigenin, digoxin, dobutamine, Dopamine HCL, the DOPA donaxine, methimazole, norcassamidine, Oxileina, gitalin, gitoxin, guanidoacetic acid, heptyl amice alcohol, hydrastinine, Denopamine, lanatoside, methoxy phenol amine, milrinone, cerberoside, oleandrine, Strophanthin G, oxyfedrine, prenalterol, caradrin, bufogenin, scillarenin, scillaridin, Strophanthin K, the pyridine of sulphur benzene miaow, Theobromine and xamoterol.

Aspect concrete, the muscular strength agent is cardiac glycoside, beta-adrenergic agonist or phosphodiesterase inhibitor.The limiting examples of cardiac glycoside comprises digoxin (lanoxin) and digoxigenin (crystodigin).The limiting examples of beta-adrenergic agonist comprises salbutamol, bambuterol, bitolterol, Carbuterol, clenbuterol, clorprenaline, ibopamine, Dioxethedrine, dobutamine (dobutrex), Dopamine HCL (intropin), the DOPA donaxine, ephedrine, Novedrin, Ethylnoradrenaline, Partusisten, formoterol, Hexoprenaline, Denopamine, neoisuprel, Racemic isoproterenol, Mabuterol, orciprenaline sulfate, Methoxyphenamine, oxyfedrine, pirbuterol, procaterol, protokylol, reproterol, rimiterol, ritodrine, soterenol, terbutaline, the fen of bent holder quinoline, tulobuterol and xamoterol.The limiting examples of phosphodiesterase inhibitor comprises amrinone (inocor).

Iv. antianginal agent

The antianginal agent can comprise organic nitrates, calcium channel blocker, beta blocker and their combination.

Organic nitrates is also referred to as nitrovasodilators, limiting examples comprise pannonit (nitro-bid, nitrostat), sorbide nitrate (isordil, sorbitrate) and Isopentyl nitrite (aspirol, vaporole).

2. surgical intervention means

In some aspects, second therapeutical agent can comprise the surgical operation of some type, and it comprises, for example, and preventative, diagnostic or interim, the curative and surgical management of appeasing.Surgery, and especially treat surgery and can unite with other therapy, such as of the present invention and one or more other medicaments.

Such surgical intervention agent for blood vessel and the cardiovascular disorder and the patient's condition is well-known to those skilled in the art, and can comprise, but be not limited to, organ is implemented surgical operation, cardiovascular mechanical prosthetic is provided, reconstructive vascular operation, coronary artery reperfusion, conduit surgical blanking (catheterablation), provides implantable cardioverter to recover defibrillator, mechanical cycles support or their combination to object.The limiting examples that can be used for mechanical cycles support of the present invention comprises intra-aortic balloon pump (intra-aortic balloon counterpulsation), left ventricular assist device or their combination.

IV. embodiment

Comprise that following examples are used for proving the preferred embodiments of the invention.It should be appreciated by those skilled in the art that the disclosed technology representative function good technical that the inventor finds in the present invention's practice in following examples, and therefore can be considered to constitute the preference pattern of the present invention's practice.Yet, according to content of the present invention, it should be appreciated by those skilled in the art and can in specific embodiments, make many variations, and still obtain same or similar result that they do not break away from the spirit and scope of the present invention.

Embodiment 1

Cells transfected

A. method

According to described (Mason et al., 1999) in the past personnel selection Arg389 and Gly389 cDNA stable transfection Chinese hamster inoblast (CHW cell).Determine to have the clone of equal expression level according to the radioactivity binding partner, the antagonistic action that the cAMP that studying these clones stimulates norepinephrine with definite bucindolol gathers.Lacking and existing the bucindolol of various concentration and down handling monolayer cell 20 minutes with 10 μ M norepinephrines at 37 ℃, and by the column chromatography separation [ ³H] cAMP (Salomon, 1991).

B. result: in the transfectional cell to the reaction of bucindolol

For functional antagonistic action research, the expression level of acceptor in the CHW cell is respectively 123 ± 19 and 137 ± 16fmol/mg for Arg389 and Gly389 clone.Lacking or existing under the condition of bucindolol of various concentration, cellular exposure is in 10 μ M agonist norepinephrines, and definite cAMP level.As shown in Figure 3, lacking under the condition of bucindolol, compare with Gly389, Arg389 shows that the bigger cAMP to norepinephrine stimulates, and they represent the main phenotype (Mason et al., 1999) of two kinds of acceptors.Although the Arg389 acceptor has the stimulation of norepinephrine mediation in fact greatly, bucindolol is this reaction of antagonism effectively still.For the cell of expressing β 1-Arg389, the difference of the absolute reduction aspect that the cAMP that is caused by bucindolol generates is bigger: cause that by bucindolol the maximum of cAMP reduces by 435 ± 80fmol/ml in the Arg389 cell, by contrast, 115 ± 23fmol/ml cAMP in the Gly389 cell (P＜0.008, N=4).For reaction, do not find on the bucindolol usefulness difference (be respectively ED50=46 ± 4.5 and 35 ± 11nM, P=0.94, N=4).In other test, in expressing the cell of arbitrary receptor variant, concentration does not cause stimulation (data not shown) to cAMP up to the independent bucindolol of 10 μ M.Therefore these results show that the Arg389 acceptor can provide the clinical response bigger to bucindolol in heart failure therapy.

Embodiment 2

In the transgenic mice to the reaction of β-blocking-up

Use α-myoglobulin heavy chain promotor, utilize transgenic mice to determine the allele-specific of long-term application beta blocker Proprasylyte is reacted with directed ventricle expressing human β 1AR (Arg389 or Gly389 form).Two kinds of receptor expression levels are equal to.The part that will produce this mouse and they recently is characterized in other places play-by-play (Perez et al., 2003).For current research, handled two kinds of mouse genotypes at 3 monthly ages and non-transgenic mouse continuously 6 months with the water (contrast) that is present in the Proprasylyte (0.5mg/ml) in the tap water or does not contain Proprasylyte.Excise heart subsequently and prepare the ventricle protein extract.These extracts are carried out the Western trace determine following protein expression to use previously described method (Perez et al., 2003): G α s, G α i2, G-protein linked receptor kinases-2 (GRK2), 5 type adenylyl cyclases (AC5), always phospholamban (phospholamban) (T-PLN), phosphorylation phospholamban (P-PLN) and sarcoplasmic reticulum calcium ATPase-2A (SERCA).By relatively untreated mice and Proprasylyte are handled in the mouse protein expression and assessed therapeutic action in genotype with ANOVA.This research is ratified by experimentation on animals management committee of University of Cincinnati (the University of Cincinnati Animal Use and CareCommittee).

According to nearest report (Perez et al., 2003), the transgenic mice with heart orientation expression β 1-Gly389 or β 1-Arg389 demonstrates expressing aspect some heart signal and the Ca++ operation albumen (handling protein) in time (as far back as 6 monthly ages time observe) multiple change.Be the potential of assessment to the genotype specific reaction of long-term β-blocking-up, with placebo or beta blocker Proprasylyte handle every kind of β 1AR of expression genotypic 3 the monthly age mouse six months, remove ventricle and prepare protein extract.Utilize the Western trace quantitatively to specify protein expression, relatively the expression that is caused by processing in the β 1AR genotype group changes.Show that as Fig. 4 Proprasylyte is handled not influencing (P=0.67) from appointment protein expression in the heart of Gly389 mouse.On the contrary, in the heart of the Arg389 mouse of handling, observe overall therapeutic response (on expressing or increase or reduce) (P＜0.002) from Proprasylyte.The direction of these trend that caused by β-blocking-up comprises increasing G α s, P-PLN and T-PLN and reduction G α i and GRK2, all is considered to be in the restorative biochemical reaction (Liggett, 2001) under cardiac hypertrophy/background in heart failure.Subsequently, combine and see, protein expression profile relevant with long-term β-blocking-up in this transgene mouse model is pointed out, compare with having the genotypic heart failure patient of β 1-Gly389, can be expected at reaction more favourable in the β 1-Arg389 heart failure patient the beta blocker bucindolol.

Embodiment 3

Bucindolol is to the clinical study of placebo

A. material and method

1. patient colony

Agree also that to participating in beta blocker existence test assessment (BEST) (Lowes et al., 2002) patient who carries out the research of DNA subgroup carries out gene type assay at coding β 1AR pleomorphism site.BEST goes into group from December 31 31 days to 1998 May of nineteen ninety-five; Research and design has been described (BEST Trial Investigators, 2001 elsewhere in detail; With Lowes et al., 2002).Briefly, this research is to the third generation, non-selective beta blocker-vasodilator bucindolol (Bristow in 2708 III/IV level heart failure patients, 2000) carry out at random, multicenter, placebo-controlled trial (BEST Trial Investigators, 2001).In first week, if patient's administered twice every day 3mg bucindolol of butt joint receptor 1 activity medicine is and tolerance then be that the basis upwards is titrated to twice of 50mg every day (if or weight in patients＞75kg be twice of 100mg every day) with a week.In 2708 patients, 1040 patients agree to carry out subgroup research and have enough DNA for preparing from blood sample.This research is through BEST DNA Watch-dog committee and University of Cincinnati's institutional review board (the University of Cincinnati Institutional Review Board) approval.

2. gene type assay

Use standard technique (Jones, 1963) from whole blood, to extract DNA.Use according to the method for describing in detail in the past (Small et al., 2002) exactly and implement gene type assay.For β 1AR, be described in the variation at coding nucleotide 145 and 1165 places, its corresponding amino acid 49 and 389.These allelotrope are named as β 1-Ser49, β 1-Gly49 and β 1-Arg389 and β 1-Gly389.All dna samples have all successfully been carried out the gene type assay of β 1AR-389 locus and 1030 increments originally successfully carried out the gene type assay of β 1AR-49.

3. statistical analysis

Main terminal point is complete former cause death, because the hospital care of judging by the terminal point council (BESTTrial Investigators, 2001) that causes in heart failure and the associating terminal point of dead or hospital care in heart failure.The last 389 amino acids variants of β 1AR are considered to suppose influences the oligogene of beta blocker effect type.Form with mean value ± SD writes down the successive clinical variable, and compares by t-check or Wilcoxon rank test.Implement relatively according to ratio form record sort variable and by card square or the accurate method of inspection of Fisher ' s.Make up the accumulation survival curve by Kaplan-Meier method (Kaplan et al., 1958) and SAS (r) proprietary software (version 6.12.Cary, NC:SAS Institute, 1996).Use the Cox proportional hazards regression models to check according to specifying the therapeutic action of genotype fractionated.Adjust the result according to the specified β 1-Gly49 of age, sex, race and Yi polymorphism.Because number relatively is limited and formerly hypothesis be based on cell, transgenosis and other to people's research (Kaplan et al., 1958) and SAS (r) proprietary software (version 6.12.Cary, NC:SAS Institute, 1996; Perez et al., 2003); With Wagoner et al., 2002), think that P value＜0.05 has significance, does not adjust at multiple comparisons.

B. result

Result from transfectional cell and transgenic mice promotes the patient from BEST is carried out gene type assay, BEST is the test of a beta blocker bucindolol treatment III-IV level heart failure patient, it comprises placebo part (BEST Trial Investigators, 2001).Most of population statistics and baseline clinical characteristics are the patient who participates in the research of DNA subgroup and do not have do not have significant difference between the patient of participation.Especially it should be noted that is not having difference aspect age, sex, NYHA classification and the cause of disease in heart failure.Baseline heart rate (1.3bpm), systolic pressure (+1.7mmHg), body weight (+1.9kg), notice DNA subgroup participator the small and clinical inapparent difference between the non-participator aspect LVEF (+0.9%) and the non-white's percentage (5%).The overall gene frequency of Arg389 is 67%, this with in the general population (Mason et al., 1999) and in test group in heart failure (Smal et al., 2002) report that the gene frequency about this polymorphism is similar.

The patient's who divides into groups according to main hypothetical gene type (β 1AR-389) and processing feature is provided in table 2.For age, sex, race, the cause of disease in heart failure, NYHA classification or baseline LVEF, between group, there is not difference by placebo, bucindolol treatment or genotype classification.As finding, the number of the Gly389 individuality that isozygotys is relatively little (52 of placebo, 42 of bucindolol group).Thus, Arg homozygote and Gly carrier (having one or two Gly allelotrope) are compared.Be divided into 4 groups according to treatment and genotype subsequently, each group is all formed (table 2) by＞200 objects.

Table 2

	Placebo N=525				Bucindolol group N=515
										The N=236 that Arg isozygotys	The N=237 of Arg/Gly heterozygosis	The N=52 that Gly isozygotys	Gly carrier N=289	The N=257 that Arg isozygotys	The N=216 of Arg/Gly heterozygosis	The N=42 that Gly isozygotys	Gly carrier N=258
									Age-mean value (standard error)	60.5(11.8)	60.3(12.4)	59.6(13.2)	60.2(12.5)	59.8(11.8)	61.6(12.0)	56.6(14.2)	60.8(12.5)
Sex-N (%) masculinity femininity	187(79％) 49(21％)	183(77％) 54(23％)	42(81％) 10(19％)	225(78％) 64(22％)	206(80％) 51(20％)	173(80％) 43(20％)	34(81％) 8(19％)	207(80％) 51(20％)
									The non-Black people Black people of race-N (%)	212(90％) 24(10％)	182(77％) 55(23％)	33(63％) 19(37％)	215(74％) 74(26％)	215(84％) 42(16％)	165(76％) 51(24％)	26(62％) 16(38％)	191(74％) 67(26％)
The non-ischemia of nosetiology-N (%) ischemia	145(61％) 91(39％)	137(58％) 100(42％)	34(65％) 18(35％)	171(59％) 118(41％)	138(54％) 119(46％)	129(60％) 87(40％)	23(55％) 19(45％)	152(59％) 106(41％)
									Functional classification-the N of NYHA (%) III IV	223(94％) 13(6％)	217(92％) 20(8％)	48(92％) 4(8％)	265(92％) 24(8％)	242(94％) 15(6％)	194(90％) 22(10％)	36(86％) 6(14％)	230(89％) 28(11％)
LVEF%-mean value (standard error)	23.3(7.0)	24.2(7.1)	23.6(6.8)	24.1(7.0)	23.4(7.2)	23.7(7.1)	22.6(6.9)	23.5(7.1)

According to the baseline characteristic of patient among treatment and the genotype fractionated BEST, data are mean value ± SD.

Be presented in Fig. 5 and 6 according to the survival rate of β 1AR-389 genotype fractionated with the patient of placebo and bucindolol treatment.The individuality of adjusting according to age, sex and race relatively shows, compare with Arg389 patient with placebo treatment, with the Arg389 patient of isozygotying of bucindolol treatment have increase survival rate (risk compares=0.62,95%CI=0.40-0.96, P=0.03).Therefore, the survival rate raising that is caused by bucindolol in Arg389 patient totals over placebo 38%.This same relatively demonstration of carrying out in the Gly carrier, (P=0.57), this indication does not have therapeutic response to bucindolol for risk ratio=0.90,95%CI=0.62-1.30 not have difference in the survival curve.β 1AR genotype is reacted also have a significant effect (Fig. 5) to the hospital care in heart failure at bucindolol.Compare with the Arg389 patient of isozygotying with placebo treatment, in homologous genes type patient, observe with bucindolol treatment the reduction aspect the hospital care (risk is than=0.64,95%CI=0.46-0.88, P=0.006).Aspect hospital care, compare with placebo, the Gly389 carrier do not show from medicine, obtain benefit (risk is than=0.86,95%CI=0.64-1.15, P=0.298).For hospital care in heart failure or dead combined results, compare with placebo, the favourable therapeutic action (Fig. 5 and 7) that bucindolol is relevant is conspicuous (risk ratio=0.66 to Arg389 patient, 95%CI=0.50-0.88, P=0.004), but in the Gly389 carrier bucindolol treatment and placebo relatively do not have notable difference (risk is than=0.87,95%CI=0.67-1.11, P=0.250).Adjustment at 49 variants (β 1-Ser49, β 1-Gly49) does not obviously influence top any result.According to so adjusting (comprising age, sex and race), be 0.62 to the risk ratio of the homozygotic survival rate of β 1-Arg389,95%CI=0.40-0.97, P=0.035; To β 1-Gly389 carrier do not have obvious therapeutic action (risk is than=0.89,95%CI=0.61-1.30, P=0.56).Similarly, at 49 adjustment the risk ratio of hospital care is not had observable influence: for the Arg389 homozygote, risk is than=0.63,95%CI=0.45-0.86, P=0.007; For the Gly389 carrier, risk ratio=0.85,95%CI=0.63-1.14, P=0.54.Combined results for and hospital care dead according to β 1-389 fractionated is not made amendment according to β 1-49 genotype yet.

Embodiment 4

In some BEST patients with the relevant mortality risk of anti-sympathetic nerve effect

Part as BEST test core scheme, measurements of systematicness vein norepinephrine is one of strong basis line prediction of mortality ratio, and 1.8 and 1.6 times of increases with the mortality ratio risk are relevant respectively by single argument and multivariate analysis demonstration Ln norepinephrine.Astoundingly, as following demonstration, the variation of norepinephrine in the time of 3 months has complex relationship with the mortality ratio that depends on the treatment group.In the bucindolol group, but not in the placebo treatment group, a considerable amount of patients (test population 18%) demonstrate noradrenaline levels and reduce, and this risk of mortality ratio subsequently with 1.7 times higher is relevant.

Most of but the not all adrenergic activity that studies show that is main determining factor (Cohn et al., 1984 of chronic heart failure (CHF) final result; Kaye et al., 1995; Isnard et al., 2000; Rockman et al., 1989).In addition, heart adrenergic activity is first neurohormone mark, along with its level in object of left ventricle dysfunction uprises (Runquist et al., 1997).These observationss have constituted beta blocker treatment basis of heart failure key element (Bristow, 2000).

On the other hand, it is a kind of important compensation mechanism that adrenergic is supported in the failure heart, is used to keep static myocardial function in normal relatively scope (Port et al., 2001).When the suprarenin motility reduces in the chronic heart failure object fast, myocardial function can variation (Gaffneyet al., 1963), and treatment can increase serious adverse effects when the suprarenin motility significantly reduces, comprise mortality ratio (Cohn et al., 2003; Swedberg et al., 2002).Show that based on these observationss adrenergic active " anti-sympathetic nerve " pharmacology reduces the mode that can significantly be different from β-blocking-up influences natural medical history in heart failure.

Although in many CHF outcome researches and clinical trial, investigated baseline adrenergic activity (Benedict et al., 1996; Swedberg et al., 1996; Francis et al., 1993; Anand et al., 2003), have only the object of relative minority (hundreds of is individual usually) in these researchs (Anand et al., 2003), to be investigated up to date.In addition, only in two other tests (Swedberg et al., 1990; Anand et al., 2003) investigate as the relation of the time behavior of the norepinephrine of the potential determinative of natural medical history in and never organize, investigated in the research of placebo at the extensive CHF that used potent antiadrenergic agent.Therefore, active baseline values of adrenergic and variation in BEST, have been investigated to the influence of clinical effectiveness and the interaction of bucindolol (a kind of beta blocker) and clinical effectiveness with anti-sympathetic nerve characteristic.

A. method

1. clinical protocol

BEST scheme and main result (Mason et al., 1999 had before been described; Small et al., 2002).Because when initial in the delay of determining on the program, so test start beginning in back 6 months all at random among the patient for the norepinephrine blood sample collection.As a result of, 2126 in 2708 arbitrary object of BEST have at least baseline norepinephrine sample to be collected and to measure.

2. the collection of norepinephrine sample and measurement

Baseline, 3 and 12 months the time by inserting No. 21 butterfly pins in the arm vein and object is lain on the back extracted peripheral vein NE sample in indoor 30 minutes in peace and quiet.Abandon and begin 3ml blood most, extract subsequently 5ml blood and transfer in the precooling 5ml pipe that contains EDTA at once.In 30 minutes, separated plasma is also freezing at-70 ℃.(LabCorp, Raritan NJ), are stored in-85 ℃ and detect at this sample in 3 weeks will to place sample on the dry ice to transport to centralab in per 3 months.By using Bio-Rad HPLC method (Bio-Rad Laboratories Hercules, HPLC-electrochemical detection method measuring N E CA).Quality control comprise from second preserve the pipe to have＜200pg/ml or＞all samples of 2000pg/ml initial value measure again; And measure known amount with conventional procedure (per 20 samples).

3. statistical method

, represent with ratio or per-cent with mean value and standard deviation (SD) expression for continuous data for grouped data.Continuous data is used T-check or Wilcoxon rank test, grouped data application card square or Fisher are accurately checked (Fisher ' s exact test).The alpha levels of use 0.05 (two tails are adjusted) is used to indicate significance,statistical.

The associating terminal point of survival rate and mortality ratio+CHF hospital care is predicted in the level of norepinephrine or the variation in the time of 3 months when being applied in baseline.Absolute data and log translation data are carried out initial analysis.Because the asymmetry of noradrenaline levels is used natural logarithm (Ln) data converted in multivariate Cox proportional hazards regression models.

Use is divided into 3 groups based on the method (Kalbfleisch et al., 1980) of maximum likelihood with the variation of norepinephrine and is used for predicting mortality ratio or mortality ratio+CHF hospital care.Such division methods has been found the optimal segmentation of norepinephrine value, and it makes the possibility maximization of the Cox proportional hazard model of gained.In addition, use flexible cubic spline analysis (flexible cubic spline analysis) (Green et al., 1994) to determine shape and the significance level of the variation of norepinephrine 3 months the time to the relation of survival rate.

B. result

1. study colony

Baseline is demographic in the object that has obtained baseline norepinephrine data does not at least have different (BEST Trial Investigators, 2001) with the colony descriptive data with full-fledged research colony.

2. norepinephrine data

Baseline suprarenin mean value is 501 ± 316pg/ml in placebo (n=1061), is 529 ± 370pg/ml (p=0.061 is to placebo) in bucindolol group (n=1065).The paired t of (p=0.0002) analyzes during by (p=0.0085) and 12 months 3 months the time, norepinephrine demonstrates statistics in placebo increases significantly, yet shows in the time of 3 months that in the bucindolol group trend (p=0.067) that obviously reduces (p=0.0001) and showed reduction in the time of 12 months (Fig. 8).Change in the time of 3 months (p＜0.0001) between the group of norepinephrine and in the time of 12 months (p＜0.0001) have very high significance statistically.With respect to the variation in the placebo, reduce by 19% and 13% respectively during at 3 months and 12 months the time at the norepinephrine of bucindolol group.

3. the baseline norepinephrine is as the prediction of the associating terminal point of mortality ratio or mortality ratio+CHF hospital care

Fig. 9 has described the risk ratio for general mortality rate according to baseline norepinephrine value, and wherein by being divided into four parts, and the one or four/a part of designated risk is 1.0 than (HR) comparatively speaking.For whole group (full entry person) with for each treatment group, along with the increase of four/part, mortality risk has the property of carrying out increase.Associating terminal point for mortality ratio+CHF hospital care has obtained analog result (Figure 10).

Table 3 has provided the potential modification that the single argument of baseline norepinephrine and multivariate analysis and other schemes have been predesignated mortality ratio.The single argument HR (95% fiducial limit) that the Ln norepinephrine produces is 1.82 (1.58-2.09), p＜0.001.In multivariate analysis, the Ln norepinephrine is one of the strongest anticipated mortality device.

4. the variation of norepinephrine is as the predictor of mortality ratio or mortality ratio+CHF hospital care associating terminal point

The variation of the four/part of norepinephrine in the time of 3 months is presented in the table 4 with the mortality ratio subsequently or the relation of mortality ratio+CHF hospital care, and wherein HR is the calculated value that changes with respect to the one or four/part.Is identical in order to keep norepinephrine variation/quartile in placebo and bucindolol group, implements the quartile analysis from the cut-point that derives from whole group.This produces four/part (first and second) and two four/parts (third and fourth) that norepinephrine increases that two norepinephrines reduce.Norepinephrine changes in the absolute change of pg/ml with from the % of baseline value lists in the table 4.Because the anti-sympathetic nerve effect of bucindolol, have more bucindolol patient in the one or four/part and more placebo patients in four or four/part.

As what in table 4, can see, for norepinephrine absolute change contrast mortality ratio, placebo demonstrates the trend that risk increases in the 4th/one or four/part, and (HR is 1.38, p=0.099), and with respect to the one or four/part second or three or four/part in mortality ratio do not produce the trend of difference.For mortality ratio+CHF hospital care, four: the one or four/part has 1.46 remarkable HR (p=0.011) in placebo.Comparatively speaking, for arbitrary clinical effectiveness, the bucindolol group does not demonstrate the trend that risk increases in four: the one or four/part, but with respect to the one or four/risk of part mortality ratio in three or four/part reduce (HR0.66, p=0.046) and the trend (p=0.22) of mortality ratio+risky reduction of CHF hospital care in three: the one or four/part.

For mortality ratio and mortality ratio+CHF hospital care both, change the trend of risky increase or risky increase in three: the first of placebo and four: the one or four/part for the % of norepinephrine.Comparatively speaking, in the bucindolol group, the the 3rd or the 4th with respect in one or four/part for this trend that does not have for arbitrary clinical endpoint HR to increase, and similar to the norepinephrine absolute change, HR in three: the one or four/part (0.77) has the trend (p=0.21) of reduction.

Table 4 has also provided with bucindolol: the treatment group HR that placebo is represented, this is each norepinephrine four/part absolute for basis or that % changes.For mortality ratio, in absolute change (HR=0.63) or the % variation (HR=0.56) of the three or four/part for norepinephrine, bucindolol: the HR of placebo obviously＜whole (bucindolol is compared mortality ratio and reduced with placebo).For mortality ratio+hospital care, HR also obviously reduces in four or four/part, observe similar pattern.Compare with mortality ratio, for mortality ratio+CHF hospital care, the two or four/a part of at bucindolol: produce almost the significantly increase of (p=0.067) about absolute change among the placebo HR, and change the increase (HR=1.39) that produces remarkable (p=0.021) about %.

To change relevant treatment relevant difference mortality ratio risk in order further exploring, to use method (Bristow, 1984) based on possibility with norepinephrine.As showing among Figure 11, the object of 11 placebo and the object of 153 bucindolol groups have been identified in the probability analysis that separates in each treatment group, and these objects have subsequently the high risk of mortality ratio respectively, and (HR 3.31, p=0.004; HR 1.69, and norepinephrine reduces p=0.002) and in the time of 3 months.Norepinephrine reduces in placebo 〉=783pg/ml in these risk group, reduction 〉=244.5pg/ml in the bucindolol group.The subgroup that norepinephrine increased when Figure 11 also illustrated in two treatment groups at 3 months is had higher mortality ratio risk by evaluation.

Because provide the largest optimization of the prediction cut point that the norepinephrine that mortality ratio increases changes based on the method for possibility, we utilize and use the contrary less differentiation match (Fowler and Bristow, 1985) of closing of flexible cubic spline.The best-fit of this method is U-shaped, the nonlinear curve with 5 nodes and 3DOF, is respectively 13.2 (p=0.0042) and 11.1 (p=0.011) and 32.5 (p＜0.0001) for the card square value of bucindolol treatment group, placebo treatment group and whole group (all participants).

5. the feature of the object that increases relevant norepinephrine increase with the mortality ratio risk or reduce

Compare with intermediate change group separately in contrast, list in table 5 in the feature of the mortality ratio excessive risk subgroup of norepinephrine variation range two ends evaluation by analytical method based on possibility.153 objects that have a bucindolol subgroup that reduces than high mortality risk and norepinephrine through evaluation have high baseline noradrenaline levels and during at 3 months norepinephrine on average reduce 529pg/ml.With seldom or do not have norepinephrine to change that (intermediate change control group 44pg/ml) is compared, and these objects also have lower LVEFs and RVEFs and higher heart rate.Have the IV level object that 153 bucindolol treatment targets that obvious norepinephrine reduces also have higher proportion, and compare with the intermediate change group and to have the trend that contrasts non-black race towards more black race.Have 79% to be classified as the heart reason in the 52 routine death that in these 153 objects, take place, and wherein 63%, 27% and 2% by respectively owing to sudden cardiac death, pump failure and myocardial infarction.By contrast, increase the relevant subgroup than the high mortality risk (n=137) with having of bucindolol treatment with norepinephrine and have lower baseline RVEF, similar baseline LVEF, still comparing with the intermediate change group had obviously less LVEF to increase in the time of 3 months.In this subgroup, IV level % and non-black race/black race distributes and middle groups does not have difference.In this subgroup, 35 examples in the 43 routine death are because cardiovascular, but minority is paroxysmal (34% pair 51% pump failure and 6% be because myocardial infarction).

C. discuss

Baseline norepinephrine data validation and the former report of expansion from the BEST test about positive relationship between adrenergic activation levels and the bad clinical effectiveness.The data of baseline norepinephrine show that this parameter is the potent predictor of clinical effectiveness, is identified as it in CHF colony.Be astoundingly, in BEST, can do not treated and significantly reduction by the risk increase that higher baseline noradrenaline levels causes by antiadrenergic drug, because in bucindolol and placebo treatment group, along with the increase of norepinephrine four/part value, the risk of mortality ratio or mortality ratio+CHF hospital care is than also increasing gradually.A kind of possibility for this shortage bucindolol provide protection in described higher baseline norepinephrine four/part is, has CHF and the anti-sympathetic nerve effect that at utmost takes place in the object of myocardial dysfunction in late period.

On the other hand, demonstrate in the time of 3 months that bucindolol demonstrates the clinical protection effect in four/part of the active patient who increases of adrenergic.In four/part that norepinephrine reduces, do not observe this reduction of clinical endpoint.In fact, for mortality ratio+CHF hospital care, two or four/part that norepinephrine reduces demonstrates the evidence that risk increases in bucindolol treatment patient.And, when four of 3 monthly variation of norepinephrine/part and the or four/part (it has maximum reduction) when comparing, the relation table of three: the one or a four/part reveals, for the bucindolol group but not for the placebo evidence that mortality ratio increases in one or four/part.Showing among the patient that norepinephrine reduces these promptings of bucindolol side effect in the time of 3 months impels other analysis is carried out in the anti-sympathetic nerve effect of this unique beta blocker.

Compare with placebo, bucindolol made norepinephrine reduce by 19% in the time of 3 months.This with causing that norepinephrine reduces by 24% relatively and can compare by maincenter sympatholytic moxonidine during at 3 months in the MOXCON test (Cohn et al., 2003).With the same in MOXCON, as if the risk increase of the anti-sympathetic nerve effect of bucindolol with unfavorable clinical effectiveness is relevant, especially for sudden death.Except the evidence in four/part of norepinephrine reduction discussed above, based on the Analysis and Identification of possibility have in the bucindolol group 18% have tangible norepinephrine reduce (＞224pg/ml), their mortality risk has 1.65 times of increases, yet has only 1% to be accredited as the mortality ratio risk with increase and the remarkable reduction of norepinephrine is arranged in the placebo treatment group.This is analyzed and has also disclosed mortality risk increase in the patient that norepinephrine increases, but bucindolol is quantitatively similar with placebo-treated patients.Confirmed that by flexible cubic spline fitting process the two ends of norepinephrine variation range 3 months the time have the mortality risk of increase, this has produced the significant U-shaped curve of statistics for bucindolol and placebo treatment group.

Be in the subgroup that the mortality ratio risk increases the bucindolol treatment target with norepinephrine reduction identifies according to probability analysis, comprise patient than heart failure in late period (IV is to the III level), higher baseline noradrenaline levels, lower LV and RV function and black race Bi Fei black race's larger proportion trend.Therefore the anti-sympathetic nerve of bucindolol acts in the object subclass that may rely on the active serious myocardial dysfunction of supporting heart function of adrenergic and may cause adverse consequences, but this mechanism is not by our data acknowledgement, and other explanation is possible.

The clinical testing data of only previously disclosed relation about systemic adrenergic activity change and consequence is from CONSENSUS (Swedberg et al., 1990), wherein neurohormone has nothing to do in the variation in 6 whens week and consequence in 239 objects, and Val-HeFT (Anand et al., 2003), but the absolute change when wherein norepinephrine was at 4 months in 4301 patients can not the % variation prediction placebo-and valsartan-treatment group in the difference of mortality ratio subsequently.Yet,, between the increase of norepinephrine abswolute level and mortality ratio or mortality ratio+CHF hospital care risk increase, found positive relationship with different among the Val-HeFT.The main new discovery of this research is that the active reduction of adrenergic can both be relevant with the unfavorable clinical consequences in the chronic heart failure colony with increase.Bucindolol shows the effect that alleviates of these risks, and is opposite with the risk that is caused by baseline norepinephrine measured before the treatment beginning, and the undesirable action that norepinephrine increases can be treated and is removed by using antiadrenergic drug jointly.

In a word, measured peripheral vein noradrenaline levels is assessed from the BEST test, studies show that systemic adrenergic is active comprehensively, late among the CHF, 1) the baseline norepinephrine is unfavorable clinical consequences but is not the predictor of therapeutic response, 2) increase and the reduction of norepinephrine in the time of 3 months all indicates negative consequence, with 3) bucindolol alleviates the risk that norepinephrine increases, but the clinical risk that makes the patient of some type place norepinephrine to reduce by its anti-sympathetic nerve characteristic.

Table 3. multivariate analysis baseline NE

Concomitant variable	Risk is than (95%CI)	The P value
			Ln NE is as single argument	1.82 (1.58-2.09)	＜0.001
	Multivariate analysis
			Ln NE CAD, (CAD is to non-CAD) LVEF, (≤20% couple＞20%) race, (Black people are to non-Black people) sex, (male sex is to the women) NYHA, (IV is to III)	1.61 (1.40-1.85) 1.68 (1.42-2.01) 1.46 (0.25-1.71) 1.26 (1.04-1.50) 1.04 (0.84-1.28) 1.61 (1.28-2.01)	＜0.001 ＜0.001 ＜0.001 0.016 0.724 ＜.001

Table 4

In placebo and bucindolol group, the variation of norepinephrine (NE) in the time of 3 months is to the effect of mortality ratio (M) or mortality ratio+CHF hospital care (M+H) subsequently; And the therapeutic action compared with placebo of bucindolol, according to norepinephrine change four/part, risk than and (95% fiducial interval)

NE changes	Change the mortality ratio risk ratio of four/part value according to NE with respect to the one or four/part value			Crude death rate (%) and change the risk ratio of the bucindolol/placebo of four/part value according to NE
								Absolute value pg/ml	The second/the first	The three/the first	The four/the first	First	Second	The 3rd	The 4th
Placebo (P): M M+H	0.98 (0.65- 1.48) 1.21 (0.89- 1.64)	1.01 (0.68- 1.51) 1.11 (0.82- 1.50)	1.38 (0.94- 2.03) 1.46 (1.09- 1.96)	24.5	27.2	27.5.	37.5.
								Bucindolol (B): M M+H	0.99 (0.70- 1.41) 1.00 (0.75- 1.32	0.66 (0.43- 0.99) 0.83 (0.61- 1.12)	1.15 (0.80- 1.65) 1.10 (0.82- 1.47)	26.1	25.7	17.7 *	28.6
B/P： M M+H	-	-	-	0.96 (0.65- 1.43) 1.19 0.88- 1.61)	0.98 (0.68- 1.43) 1.30 (0.98- 1.72)	0.63 (0.41- 0.96) 0.58 (0.43- 0.78)	0.80 (0.57- 1.14) 0.74 (0.56- 0.98)
								% changes
Placebo (P): M M+H	1.21 (0.80- 1.84) 1.27 (0.93- 1.72)	1.42 (0.96- 2.10) 1.33 (0.99- 1.78)	1.37 (0.92- 2.04) 1.35 (1.00- 1.81)	23.1	27.3	32.1	29.2
								Bucindolol (B): M M+H	1.14 (0.80- 1.62) 1.18 (0.89- 1.56)	0.77 (0.51- 1.16) 0.91 (0.67- 1.24)	1.19 (0.83- 1.71) 1.18 (0.88- 1.58)	25.2	26.0	18.8	29.1
B/P： M M+H	-	-	-	1.03 (0.69- 1.54) 1.14 (0.84- 1.54)	0.98 (0.68- 1.42) 1.39 (1.05- 1.85)	0.56 (0.38- 0.84) 0.65 (0.48- 0.88)	0.90 0.63- 1.29) 0.66 (0.50- 0.88)

Four/part is: the absolute value pg/ml that NE changes, first part＜-144 (placebo n=155, bucindolol n=268); Second section-144 is to＜-9 (placebo n=206, bucindolol n=214), and third part-9 is to 111 (placebo n=236, bucindolol n=186), the 4th part＞111 (placebo n=248, bucindolol n=173); %NE changes, first part＜-30.2 (placebo n=160, bucindolol n=262), second section-30.2 is to＜-2.5 (placebo n=198, bucindolol n=223), third part-2.5 is to 31.1 (placebo n=240, bucindolol n=181), the 4th part＞31.1 (placebo n=247, bucindolol n=175). ^*, p＜0.05 pair one or four/part, Fisher accurately checks

Table 5

(Inter) NE in the middle of the definite subgroup contrast of possibility with increase risk changes the Demographics of subgroup, and (NE, pg/ml) variation (Δ) of reduction (Redxn) or increase (Incr) is relevant for wherein said increase risk and norepinephrine.NE represents with pg/ml; Data are represented with mean value ± SD; ^*, p＜0.05 couple Inter; ^#, p＜0.10 couple Inter

Parameter	Placebo			Bucindolol
							Redxn (ΔNE≤ -783)	Inter (ΔNE＞- 783， ≤362)	Incr (ΔNE ＞362)	Redxn (ΔNE≤ -244.5)	Inter (ΔNE- 244.5，≤ +145)	Incr (ΔNE ＞145)
	Number of objects baseline NE; The NE of pg/ml in the time of 3 months changes; The NE of pg/ml in the time of 12 months changes; Pg/ml death toll (%) age (year) sex (%M/F) race's (the non-Black people/Black people of %) NYHA level (%III/IV) CHF duration; Month; Median teiology (% Ischemic/ischemic) baseline heart rate (HR; BPM) HR of the variation of the HR in the time of 3 months in the time of 12 months changes systaltic BP (SBP; MmHg) SBP that changes in the time of 12 months of the SBP in the time of 3 months changes LVEF, the as% of EF unit (EFU)) LVEF that changes in the time of 12 months of LVEF in the time of 3 months changes	n＝11 1500 *±405 -1024 *±220 -667 *±528 6 (55％) *63.6 ±9.8 64/36 73/27 82/18 73.0 #45/55 79.2 ±12.4 -5.5 ±17.0 -10.7 #±13.2 111 ±20 4.7 ±13.8 5.6 ±19.5 22.8 ±6.6 0.2 ±6.4 5.7 ±11.9	n＝762 464 ±245 -16 ±188 45 ±268 200 (26％) 60.3 ±11.9 80/20 80/20 92/8 39.0 42/58 81.6 ±12.8 -2.3 ±12.5 -2.6 ±13.5 118 ±18 0.0 15.4 0.7 ±18.1 23.1 ±7.2 2.3 ±6.6 3.3 ±8.7	n＝72 514 ±328 642 * ±335 297 * ±560 34 (47％) * 64.7 * ±10.8 82/18 82/18 83/17 * 36.0 29/71 * 78.3 * ±12.3 1.0 * ±13.3 -1.8 ±13.5 116 ±19 -0.1 ±18.1 1.9 ±21.0 23.3 ±7.6 0.6 * ±7.2 1.4 ±7.6	n＝153 932 * ±544 -529 * ±458 -349 * ±347 52 (34％) * 60.1 ±12.2 79/21 74/26 # 86/14 * 36.0 46/54 85.5 * ±13.8 -13.6 * ±14.8 -12.6 * ±14.4 116 ±19 -0.7 ±18.2 2.7 ±20.1 20.1 * ±8.0 7.0 # ±8.4 8.8 ±9.2	n＝551 422 ±189 -44 ±103 17.5 ±216 114 (21％) 60.7 ±12.1 81/19 80/20 93/7 36.0 42/58 81.0 ±13.0 -9.7 ±12.4 -7.9 ±13.5 118 ±18 -0.5 ±15.7 0.7 ±18.0 24.1 ±7.0 5.7 ±7.9 7.3 ±10.4							n＝137 409 ±199 326 * ±244 161 * ±246 43 (31％) * 62.0 ±12.7 82/18 77/23 93/7 31.0 31/69 * 80.6 ±13.7 -7.1 * ±12.8 -8.1 ±14.6 120 ±18.2 -4.7 * ±16.4 -0.9 ±16.8 23.3 ±6.7 4.2 * ±7.0 7.1 ±8.8

Embodiment 5

The popularity of A2C-adrenergic receptor heredity variant in BEST

Following table provides with the result who is reported by Liggett group (Small et al., 2002) at first and compares α in BEST _2c-adrenergic receptor heredity variant (WT/WT=homozygous wildtype, WT/DEL=heterozygote, the DEL/DEL=α that isozygotys _2cDel322-325) popularity (%).By using primer covers described disappearance through pcr amplification α _2cThe zone of AR sequence is also moved amplified reaction and is assessed sample from BEST differentiating to have or do not have subsequently on the gel of 12 base pair differences in length between the product of this disappearance.

Table 6

Research	Non-Black people			Black people			Whole group
											WT/WT	WT/DEL	DEL/DEL	WT/WT	WT/DEL	DEL/DEL	WT/WT	WT/DEL	DEL/DEL
BEST	91.6	8.2	0.2	33.8	47.8	18.4	80.0	16.1	3.9
										Small，et al CHF	86.4	6.2	7.4	29.5	17.9	52.6	58.5	11.9	29.6
Small, contrast	94.3	3.8	1.9	34.5	48.8	16.6	67.7	23.8	8.5

As seen in the last table, the frequency of the frequency of α 2cDel322-325 allelotrope in black race colony in the non-black race colony is 0.423 to 0.043 (p＜0.0001) in BEST.Secondly, in black race, the different height of frequency (0.615, p＜0.0001) among the frequency of α 2cDel322-325 allelotrope in BEST and the black race CHF patient in Small etal, but similar to the frequency of black race in Small et al contrast (0.411, p=.85).These differences may reflect at Small et al. (n=78 name Black people CHF patient, 84 Black people's contrasts) tests used relative little sample size in (n=207 name Black people in the research of DNA subgroup) with BEST.

Only supposed α in the people _2cThe Del322-325 polymorphism is relevant with the suprarenin motility of increase, and does not have direct surveys.

For a possible cause of the little difference of baseline norepinephrine between α 2cDel322-325 homozygote and the contrast of α 2c wild-type of in this suggestion, directly setting forth be, systematicness vein norepinephrine is not the good index that substitutes of heart suprarenin motility, and in chronic heart failure, the variation of heart suprarenin motility can take place under the situation that lacks systemic norepinephrine variation.

The result who changes when baseline and 3 months according to the norepinephrine of α 2c acceptor type is presented in the table 7:

Table 7. norepinephrine (NE), (n) of mean value ± SD and BEST test is according to α 2c acceptor type; * p＜0.05 pair placebo changes

α 2c acceptor type	Baseline NE, pg/ml	Change (pg/ml) at 3 months NE
							Placebo	Bucindolol	All
α 2c wild-type homozygote or heterozygote	479±264(710)	12±274(305)	-50 *±227(304)	-19±254(609)
					α 2cDel322-325 homozygote	521±350(161)	51±323(60)	-153 *±468(67)	-57±417(127)

As seen in table 7, in BEST the baseline values of systemic vein NE or change strong support above-mentioned about α 2cDel322-325 acceptor variant to the power actuated hypothesis of baseline adrenergic; Higher baseline norepinephrine has only the trend of non-significance to help α 2cDel322-325 homozygote during for 3 months.On the other hand, can find out at an easy rate in table 7 that the norepinephrine that is caused by bucindolol is reduced in will be much larger than in α 2c wild-type homozygote or heterozygote in the α 2cDel322-325 homozygote.

Embodiment 6

Embodiment 3 and 4 expansion

A. material and method

1. stripped people's ventricle research

Obtain the heart of non-depletion from local potential donor that can not transplanted heart because physics or abo blood group are incompatible.Obtain depleted heart from the late period that causes by ischemia or non-ischemia dilated cardiomyopathy of experience heart transplantation the heart failure patient.The Demographics of described heart is provided in the result.Assess contractile response { 1755 isolating, a people's girder that stimulates as previously mentioned; A-c}.Girder of the same size (1-2 * 6-8mm) 36 ℃, use 95%O ₂-5%CO ₂Being fixed on 80ml muscle in the pH7.45 thyrode moral solution (Tyrode solution) of air-blowing bathes in the chamber.After the balance, L _Maximum75% tension force is applied in each independent girder.Stimulate by 10% 5-ms pulse applied field above threshold subsequently, and balance after, use prescribed concentration and increased in per 5 minutes apply the full dose-effect curve of dosage drafting to Racemic isoproterenol, bucindolol or xamoterol.In the test with the transduction of Forskolin enhancing signal e, and f}, before implementing dose-effect curve 15-20 minute, with 10 ^-6This adenylyl cyclase activator of M puts on tissue bath, and makes it to realize the stability of tension force reaction.To stimulate tension force (mN/mm ²) deduct the heart contraction tension force that baseline tension force calculates every kind of dosage.Calculate maximum tension, produce 50% maximum activity tension force (EC by non-linear replicate measurement covariance analysis ₅₀) Racemic isoproterenol concentration and rate of curve.Use identifies respectively that about the significant negative or positive rate of curve of the statistics of integrated data the variable force (inotropic) of negative or positive acts on, and detects the rate of curve difference between each genotype group by interacting.According to described in embodiment 3 and 4, using statistical method.

2. transfectional cell, radioligand combination, cAMP detect

With the construct stable transfection Chinese hamster inoblast of former description, thereby make its expressing human β respectively ₁-Arg389 or β ₁-Gly389 acceptor.Use 1 μ M Proprasylyte to determine non-specific binding as described, by using ¹²⁵I-cyanopindolol ( ¹²⁵I-CYP) radioligand is in conjunction with determining β ₁AR expresses and to the affinity of bucindolol.According to [ ³H]-two kinds of clones that the VITAMIN B4 method uses as directed two kinds of acceptors to have equal expression level implement full cell cAMP accumulation research.At 37 ℃, attached cell is exposed to carrier (basis), the bucindolol of 10 μ M norepinephrines or 10 μ M norepinephrines and prescribed concentration was kept 15 minutes.

B. result

1. exsomatize contractile response and β of people's ventricle ₁The AR genotype is relevant

In these researchs, at endogenous expression, lack and exist under the condition of ventricular failure, utilize the right ventricle girder that separates from human heart to use respective organization to determine the effect of genotype to contraction.The LVEF of pre-outer planting (pre-explant) is 0.61+0.13 for Arg in non-depleted group and is 0.53+0.15 for Gly, and is 0.21+0.11 for Arg in the depletion group and is 0.17+0.07 for Gly.6 among among 11 depleted Arg patients 5 and 11 the depleted Gly patients have the ischemia dilated cardiomyopathy, and all other failure heart patients are non-ischemia dilated cardiomyopathies.The age of non-failure heart is: Arg39 ± 16 years old, Gly 43 ± 20 years old (p=0.64).Age for failure heart is Arg48 ± 15 years old, Gly 54 ± 8 years old (p=0.26).To distribute be 3 male sex and 8 women and 5 male sex and 6 women in Gly in Arg to sex in non-failure heart group.To distribute be 8 male sex and 3 women and 2 male sex and 9 women in Gly in Arg to sex in failure heart.Figure 12 shows be from non-depletion and depleted human heart, take off and according to β ₁Heart contraction tension force to Racemic isoproterenol in the AR-389 genotype fractionated right ventricle girder reacts.In non-failure heart, has response difference between the genotype, wherein β ₁Higher (13 ± 2.5 couples of 5.2 ± 1.4mN/mm of the homozygotic maximum tension of-Arg389 ²β ₁-Gly389 carrier, P=0.01).Importantly, observe in the girder from failure heart that this identical phenotype has even bigger allele-specific relative different: the tension force that maximum Racemic isoproterenol stimulates is for β ₁-Arg389 is 9.4 ± 1.9mN/mm ²With for β ₁-Gly389 is 2.4 ± 0.60mN/mm ²(P=0.008).

Second group of 23 failure hearts is used to assess bucindolol, β ₁{ d} and the Racemic isoproterenol inotropic action in isolating right ventricle girder of-AR selectivity partial agonist xamoterol.The LVEF average out to 0.18 ± 0.09 of this group, it has 10 non-ischemias and 13 ischemia dilated cardiomyopathies.Mean age is 52 ± 11 years old, and 20 male sex and 3 women are arranged.13 in 23 hearts is the Arg389 homozygote, and 10 is Gly carrier (all heterozygotes).Between Arg homozygote and Gly carrier, aspect LVEF, age, the myocardosis cause of disease and sex, there is not difference.In 8 of 23 hearts, implement bucindolol and xamoterol test; Other 15 hearts or implemented xamoterol or implemented the bucindolol dose-effect curve, and do not use other medicament.All 23 hearts have all been implemented full Racemic isoproterenol dose-effect curve.

Result displayed is closely similar among the result of Racemic isoproterenol docs-effect and Figure 12 between two kinds of genotype groups.In unshowned data, in helping the genotypic docs-effect of Arg/Arg, notable difference is arranged, wherein by interactional check being shown difference and maximum value that rate of curve has a highly significant (p＜0.001) have difference (Arg, xxxx; Gly, yyyy, p＜0.05).As seen in fig. 3, bucindolol all produces negative inotropic action (be negative slope in two groups, the p value all＜0.01 is checked non-significant interaction between the rate of curve) in two genotype groups separately.When having the Forskolin pre-treatment, the Arg heart keeps negative rate of curve (P＜0.05), but the slope of Gly dose-effect curve and 0 does not have difference (p=0.25).When lacking Forskolin (Figure 13 C), xamoterol produces positive inotropic action in the Arg heart, but produces negative inotropic action (two slope p values all＜0.05) in the Gly girder.When carrying out pre-treatment with Forskolin, xamoterol all produces positive inotropic action in two kinds of genotype groups (be positive rate of curve in two kinds of genotype, p=＜0.05), wherein the Arg/Arg heart has bigger inotropic action, this be 3 * 10 at xamoterol dosage ^-8M and 10 ^-7Has significance during the baseline comparison of M.

2. the functional antagonistic action that NE stimulates cAMP in transfectional cell

In these researchs, utilize Arg389 (123 ± 19) and Gly389 (137 ± 16) people β ₁The AR expression level equates (fmol/mg, cell n=4).The basal level of cAMP is 72 ± 8.5 and 59 ± 9.1fmol/ hole.The initial cAMP accumulation test of existence when the bucindolol of 10 μ M do not demonstrate bucindolol and have the evidence that endogenous is intended sympathetic activity (ISA) on arbitrary acceptor.For the audit function antagonistic action, make the agonist norepinephrine of cellular exposure lacking or exist under the situation of bucindolol of different concns in 10 μ M, and definite cAMP level.As shown in figure 13, compare with Gly389, Arg389 shows the bigger cAMP hormesis for agonist when lacking bucindolol, and this representative is the main phenotype { 998} of two kinds of acceptors as previously mentioned.Although the Arg389 acceptor has significantly greatly NE-mediation hormesis, bucindolol effectively antagonism this effect.The cAMP that is caused by bucindolol generates the absolute difference that reduces for expressing β ₁The cell of-Arg389 is bigger: compare with 115 ± 23fmol/ml in the Gly389 cell, bucindolol in the Arg389 cell, cause the maximum of 435 ± 80fmol/ml cAMP reduce (P=0.008, n=4).The usefulness of not finding bucindolol for described effect has difference (to be respectively EC ₅₀=46 ± 4.5 and 35 ± 11nM, P=0.94, n=4).In addition, exist ¹²⁵I-CYP competition is in conjunction with in the research, to the affinity of bucindolol at β ₁-Arg389 (pK _i=9.6 ± 0.04) and β ₁-Gly389 acceptor (pK _i=9.6 ± 0.11, there is not difference between n=3).These discoveries show that bucindolol can antagonism β ₁The reaction of-Arg389 enhanced.

3. to the mechanism of the favourable treatment of Arg389 genotype

Typically the adrenergic activity of identifying by elevation system vein noradrenaline levels is increased in the impaired function in heart failure of support in the heart failure patient, but helps development { h} in heart failure.Observe this complex relationship between adrenergic activity and result in BEST, wherein the increase of baseline norepinephrine is relevant independently with adverse consequences, but the adrenergic activatory significantly reduces and the relevant { i} of mortality ratio increase.Do not resemble other and be used for treating beta blocker in heart failure, bucindolol has potent anti-sympathetic nerve characteristic, and the patient that 18% usefulness bucindolol is treated in BEST shows the excessive reduction of norepinephrine in the time of 3 months and is attended by 1.7 times of increases of mortality ratio risk subsequently, and { i}, this makes the people wander back to the mortality in said patients for the treatment of with pure sympatholytic moxonidine in the MOXCON test increases { j}.Do not understood fully although anti-sympathetic nerve effect increases the reason of mortality ratio risk, it may support relevant with the alpha 2 adrenergic-mediated contractility to failure heart of forfeiture.Therefore Arg homozygote patient may be than the forfeiture of the potential better tolerance norepinephrine of Gly carrier signal, because as shown in Figure 12 and 13, even low-level catecholamine agonist also causes the increase of power in the Arg homozygote.The another kind of mechanism that the Arg homozygote can obtain the treatment advantage of bucindolol treatment may be to disadvantageous β ₁The antagonistic action greatly of-AR signal is as suggested in Fig. 3.Can obtain more favourable bucindolol result of treatment in order to determine that any mechanism can be explained with respect to the Gly carrier in the Arg homozygote, norepinephrine changed the influence (table 8) to mortality ratio when the contriver had compared baseline norepinephrine and 3 months.As what in table 8, can see, Arg homozygote contrast Gly carrier reduces than increasing with the baseline norepinephrine about the risk of mortality ratio, and the Arg homozygote that this prompting is treated for bucindolol along with the increase of suprarenin motility has the advantage of development gradually.For the variation in the norepinephrine analysis, formerly be accredited as with respect to norepinephrine obviously reduce (reduce by three months the time in treatment＞244pg/ml) and the group that the mortality ratio risk increases, have very for a short time or do not have norepinephrine to change (244 to 145pg/ml) and do not have reference group that the mortality ratio risk increases and since norepinephrine increase (＞145pg/ml) make in the group that the mortality ratio risk increases, the contriver has compared the mortality ratio that the Arg homozygote contrasts the Gly carrier.As seen in table 8, in cause the subgroup that the mortality ratio risk increases owing to excessive anti-sympathetic nerve effect, the Arg homozygote there is not advantage (group 1); 1.07 risk than showing that the advantage for the Gly carrier can be ignored in this group.On the other hand, for the baseline norepinephrine, along with the norepinephrine that increases gradually 3 months the time rises to certain position, the risk ratio decreases, and described position is in the group that norepinephrine increases gradually to be because mortality ratio has 64% reduce relatively preferably (p=0.08) for the homozygotic benefit of Arg.These norepinephrines change and baseline data shows, for β ₁The Arg homozygotic state of AR, bucindolol treatment advantage are to be directly related to the active degree of adrenergic, and not directly related with the provide protection of avoiding anti-sympathetic nerve effect.

Table 8

Measuring norepinephrine (NE) (pg/ml, the patient of bucindolol treatment in BEST DNA research n=439)

The NE group	Mortality ratio risk ratio, Arg/Arg: Gly carrier	95％C.l.	The Coxp value	The # incident
					BSLNE(n)
64-356(146)	0.90	0.37，2.22	0.82	19
					358-545(144)	0.74	0.37，1.51	0.41	31
546-2571(149)	0.68	0.32，1.47	0.33	29
					*NE in the time of 3 months changes (n)
Group 1,＜-244 (70)	1.07	0.41，2.78	0.89	17
					Group 2 ,-244to 145 (248)	0.82	0.43，1.59	0.56	36
Group 3,＞145 (54)	0.36	0.11，1.15	0.08	14

The BSL=baseline. ^*, the NE in the bucindolol group when 3 months (mos) changes with the survival rate result is relevant subsequently; Cut-point obtains from whole group according to previous disclosed probability analysis.According to group 2 comparative analysis, the mortality ratio of group has 1.69 times of (p＜.05) increase, and organize 3 mortality ratio and have 1.65 times (p＜0.05) to increase.

Embodiment 7

Analyzing adjuncts from the BEST test

In chronic heart failure, that the activation of adrenergic nerve system has is dual, the result (Figure 14) of opposition on the surface.On the one hand, the heart that the adrenergic that continues activates to depletion provides important support, comes this support of pharmacological elimination to increase mortality ratio (Bristow et al., 2004 by sympatholytic; Cohn et al., 2003).On the other hand, chronic beta-adrenergic stimulates and is myocardium characteristic of disease, and the blocking-up of B-adrenergic receptor can improve the phenotype and the clinical effectiveness of DCM (dilated cardiomyopathy).The challenge of antiadrenergic drug energy therapy is not disturb the adrenergic support basically, yet has but suppressed detrimental action.Reversible, strategy that mass action/competitive beta blocker is initial with low dosage are successful in the balance of handling this fragility, and be especially true in the heart failure patient in late period not too.

Be clear that in BEST bucindolol has met with the difficulty (Anderson etal., 2003) owing to the support that has exceedingly reduced in IV level patient faces in the balance antiadrenergic drug can the process of therapy effect.With the IV level patient of bucindolol treatment 6 months at begin treatment, have on the associating terminal point of dead or hospital care in heart failure that statistics is significant, 1.7 times of increases, however III level patient do not have this early stage detrimental action and totally have (p=0.0001) of highly significant, 22% incidence reduces.Have the subgroup patient of big anti-sympathetic nerve reaction in IV level patient, to exceed ratio to bucindolol, with expected the same because this subgroup has very high baseline noradrenaline levels (Figure 15).The subgroup patient who bucindolol is had big anti-sympathetic nerve reaction also has the LV and the RV function of deterioration, with the same (Figure 16) of being expected.

The experience degree of depth anti-sympathetic nerve effect in BEST (norepinephrine reduces 〉=and mortality ratio in 244.5pg/ml) the subgroup (18% in the treatment group) has 1.7 times increase.This subgroup result is similar with the result (Cohn et al., 2003) among the patient who accepts " pure " sympatholytic moxonidine in the MOXCON test.Appearing to have two kinds of pharmacogenetics modes can be used for protecting the patient to avoid such detrimental action: 1) they have the α of wild-type _2cAcceptor, it is one of the limiting factor (s) that discharges of norepinephrine (Bristow 2000); 2) they have β ₁The high function variant of acceptor infers that this variant can allow them to resist the forfeiture of norepinephrine signal.Therefore, for wild-type α _2cAR or β ₁The prescreen that AR-389Arg/Arg exists has differentiated that mortality ratio reduces by the patient colony of 29% (p=0.031) or 38% (p=0.030) respectively.

Demonstrate in black race's subgroup have specific drugs genome spectrum in the patient of quite big quantity, treatment does not have effect and the trend that obtains unfavorable result is arranged described patient to bucindolol.Black race is for α _2cThe afunction variant of acceptor has the gene frequency of much higher (near 10 times), and it relates in the 3rd kytoplasm of receptor protein the disappearance (Small etal., 2000) of 322-325 amino acids in the ring.Expection α _2cThis afunction among the AR will increase the suprarenin motility, because α ₂The normal presynaptic adrenergic restraining effect of acceptor has been subjected to infringement (Brum et al.2002).Object among the BEST and be the heterozygosis or the (α that isozygotys especially to this genetic mutation _2cThe AR-Del322-325 carrier) black race has the trend that the general norepinephrine increases when baseline, and bucindolol is had very large anti-sympathetic nerve reaction (Figure 17).In fact, in BEST near 61% black race to as if α _2cAR-Del322-325 carrier, non-by contrast people from black race are 8% (Figure 18).At α _2cIn the AR-Del322-325 carrier object, mortality ratio has 10% increase in bucindolol treatment group, by contrast at wild-type α _2cMortality ratio reduces by 29% (p=0.031) (Figure 19) in the AR object.

It is to have β that another kind is avoided the mode of the anti-sympathetic nerve effect of bucindolol ₁The 389Arg/Arg variant (Figure 15) of the high functionality of AR (Mason et al., 1999), it accounts for 47% in BEST colony.As shown in Figure 20 and the embodiment 4, for β ₁The AR individuality is that 389Arg/Arg (Arg homozygote) or 389Gly carrier may be the main determining factors to the beta-2-agonists reaction, even whether more important than existence in heart failure.Expect β ₁The AR-389Arg/Arg individuality has relative resistance to sympatholytic detrimental action, even because the norepinephrine of lower level will produce relative intensive variable force reaction (Figure 20).As shown in figure 19, in fact as if just so.Figure 21 shows at α _2cAmong the AR-Del322-325 carrier patient, β ₁The existence of AR-389Arg/Arg acceptor makes β ₁AR-389Gly carrier mortality ratio increases by 36% and changes mortality ratio reduction by 18% into.

Except the cardiac muscle of avoiding being produced by anti-sympathetic nerve effect suppresses β ₁The AR-389Arg/Arg genotype given to bucindolol bigger, " super-reaction " (Fig. 5-7).This may be because the β of high functionality ₁The AR-389Arg/Arg variant has been given myocardosis greatly.Because it is higher functional, the degree absolute that suppresses the bucindolol signal transduction is bigger, and this is interpreted as being of value to more potentially β ₁AR-389Arg/Arg genotype patient.

As shown in Figure 21 and Figure 20, the patient with 389Arg/Arg acceptor variant (the non-significance of the reduction of mortality ratio 38% (p=0.030) contrast mortality ratio in the Gly carrier reduces by 10%) and at α in whole group _2c(mortality ratio reduces the non-significance of 40% (p=0.037) contrast mortality ratio in the Gly carrier and reduces by 22%) has bigger mortality ratio reduction reaction to bucindolol among the AR wild-type patient.Therefore, whether there is super-reaction marker β ₁The AR-389Arg/Arg genotype is the main determining factor of bucindolol reaction in the heart failure patient in late period.Figure 22 understands that for example using genetic mutation to define subgroup reduces the progress with increase curative effect for mortality ratio in BEST.As a comparison, only another gone into to organize relatively that the U.S. patient's of a large amount of (＞500) beta blocker mortality ratio test in heart failure (MERIT-HF) also is indicated among Fig. 7.Just as can be seen, mortality ratio reduces from inapparent 10% be increased to 80% α whole group among the BEST _2cAmong the AR wild-type patient 29%, be increased to 47% β ₁Among the AR-389Arg/Arg patient 38%, be increased to 40% be α _2cThe AR wild-type is again β ₁Among the AR-389Arg/Arg patient 40%.By contrast, the risk ratio for metoprolol CR/XL among the U.S. patient who goes into group at MERIT-HF is 1.05 (Wedel et al., 2001).

Although may be from the β of BEST generation ₁AR-389 genotype specific data is generally effective for all beta blockers, but has good reason to think that these the possibility of result only are applied to bucindolol.At first, as discussed above, β ₁The AR-389Arg/Arg genotype has been avoided sympatholytic detrimental action, and bucindolol is unique for anti-sympathetic nerve effect in being used for treating beta blocker in heart failure.Secondly, in order to maximize the β of inhibition via high functionality ₁The signal of AR-389Arg/Arg acceptor, in fact bucindolol can be used for reducing norepinephrine and blocking acceptor, has only bucindolol to have this property combination.At last, can think in BEST the genetic mutation data that produce by bucindolol only late, be effective in the heart failure of III-IV level.There is sufficient reason can believe that bucindolol will be to have α in the heart failure in late period (NYHA level I-II) not too _2cThe AR-Del322-325 carrier adds β ₁The medicament selection of the object of AR-389Arg/Arg combination because anti-sympathetic nerve effect and myocardial function support forfeiture not too are problems, and has α in this type of patient colony _2cAR-Del/Del+ β ₁The increase (Small et al., 2002) that risk in heart failure has 10 times takes place in the individuality of AR-389Arg/Arg haplotype.Bucindolol may be last methods of treatment to these patients, (tackles α thus because it reduces norepinephrine _2cThe allelic influence of AR-Del322-325) and the blocking-up β ₁AR.

The arguement that whether has intrinsic sympathomimetic activity (ISA) in the literature relevant for bucindolol in human heart, this specific character have been used to explain the result of bucindolol BEST test.Although bucindolol has ISA clearly in the rodent cardiac muscle, contriver's broad research shows that bucindolol has the active while of extremely low inverse agonist without any ISA in the functional human myocardium of ventricle.As at Bristow et al., shown in 1998, go up bucindolol at Holter (Holtermonitoring) and do not increase the heart rate at night, this is considered to the most responsive indication of ISA in the human heart and is a kind of check (Xameterol Study Group, 1990 that can be easy to identify the ISA of xamoterol or celiprolol; Silke et al., 1997).In addition, the contriver has implemented extensive studies in isolating human heart prepared product, and bucindolol does not demonstrate the evidence of ISA in non-depletion (Figure 23 and 24) or depleted human heart (Figure 25 and 26).In fact, when with the pretreated prepared product of Forskolin, in the heart of depletion, carvedilol provides more positive variable force signal (need increase signal transduction for detecting weak ISA) (Figure 26) than bucindolol.In BEST, at high functionality β ₁The more favourable reaction of bucindolol further proves and opposes the ISA of bucindolol in people's ventricle cardiac muscle in the AR-389 Arg/Arg variant.

Embodiment 8

The BEST test is heavily visited

The expection hypothesis about two kinds of adrenergic receptor polymorphisms has been checked in the inferior research of DNA that implement, BEST in 1040 patients, based on (Mialet et al. in modular system, 2003) (Small et al. or in epidemiological study in heart failure, 2002) achievement, the therapeutic action of described receptor polymorphisms and bucindolol have potential and interact.These two kinds of adrenergic receptor genetic mutations demonstrate the gene frequency of otherness black race in to colony of non-black race, and find that they have influenced treatment result significantly in BEST.At first be α _2cDEL 322-325 polymorphism, a kind of genetic mutation of loss function, it increases the suprarenin motility and it is subject to the influence of the excessive anti-sympathetic nerve effect of bucindolol.As α _2cDEL 322-325 carrier and with the patient of bucindolol treatment 3 months the time norepinephrine on average be reduced to 153 ± 57 (SEM) pg/ml, by contrast at α with the bucindolol treatment _2cNorepinephrine is reduced to 50 ± 13pg/ml (p=0.008) among the WT/WT patient.In BEST this excessive anti-sympathetic nerve reaction and statistics significantly, the increase relevant (Bristow et al., 2004) of 1.7 times of mortality ratio.There are two to be subject to the clinical of significant anti-sympathetic nerve function influence or demography subgroup, IV level patient and black race, the mortality ratio risk of having only these two subgroups in BEST is than＞1.0 (BEST Writing Committee, 2001).α _2cThe allelic frequency of DEL 322-325 is 0.42 black race, is 0.04 (p＜0.0001) in non-black race.As shown in the hurdle 3 of table 9, in BEST for α _2c-adrenergic receptor is that the patient's of homozygous wildtype (WT/WT) full reason mortality ratio has 30% reduction (p=0.031) and 41% reduction (p=0.004) is arranged on cardiovascular mortality, yet the patient as DEL322-325 polymorphism carrier has 9% increase (p=NS) on full reason mortality ratio, and 3% increase (p=NS) is arranged on cardiovascular mortality.Substantially, 80% people comprises 24% black race in the BEST test, is α _2cWT/WT, the black race comprising 34%.Therefore the simple screening that DEL 322-325 polymorphism is existed and only treat in the U.S. population 85% for α _2c-adrenergic receptor is the American of homozygous wildtype, anti-sympathetic nerve effect/mortality ratio risk of removing bucindolol is increased, and strengthen its result of treatment.As following institute unfolded, as α _2cIf they have β DEL 322-325 carrier's patient ₁389Arg/Arg genotype (prevalence rate of this genotype in BEST is 0.32 in black race and is 0.51 in non-black race) just can be treated with bucindolol, therefore based on the non-black race's of 12% black race and 88% ethnic per-cent, the colony that is fit to for bucindolol is the 85%+6%=91% of total U.S. colony in heart failure.In addition, surpassing 50% black race (34%+21%=55%) can treat with bucindolol according to genotype screening.

Discovery can influence the bucindolol result of treatment in BEST other adrenergic receptor polymorphism is β ₁389Arg/Gly SNP wherein compares with there being Gly allelotrope (" Gly carrier " hurdle 6, table 9), and the variant of Arg/Arg higher function has been given " super-reaction " to bucindolol (table 9, hurdle 5).In the data bag of meeting on March 28 in 05 year, provide evidence, the 389Arg/Arg variant demonstrates higher signal transduction functionality than Gly carrier variant, and doctor's Liggett group discloses evidence, 389Arg allelotrope has bigger influence (Mialetet al., 2003) than 389Gly to myocardosis.As what in table 9, can see, full reason mortality ratio as 389Arg/Arg patient in BEST has 38% reduction (p=0.030), and because the cardiovascular mortality of bucindolol has 46% reduction, separately mortality ratio is respectively 10% and 22% (two p=NS) in as 389Gly carrier's patient by contrast.Because there is not evidence to show that carvedilol (Smallet al., 2002) or metoprolol CR/XL (White et al., 2003) are at β ₁389Arg/Arg and β ₁Have significant differentiation therapeutic response between the 389Gly carrier, so bucindolol has β ₁Advantageous effect among the patient of 389Arg/Arg acceptor gene variant may be owing to reduce the associating of norepinephrine signal and receptor competition antagonistic action.This aspect on, doctor's Bristow group produces evidence based on physiological and molecule/biomarker data, show that the heart failure patient for the treatment of with the beta blocker of full therapeutic dose demonstrates evidence (the Lowes etal. of lasting myocardium adrenergic signal, 2001), and therefore has β ₁The reduction of norepinephrine can provide extra benefit among the patient of 389Arg/Arg high functionality acceptor variant.

In addition, be β ₁389Arg/Arg is again α _2c-WT/WT (row 8, table 9) patient's full reason mortality ratio has 40% reduction (p=0.037), cardiovascular property mortality ratio has 47% reduction (p=0.020), and in mortality ratio+hospital care in heart failure, has 39% reduction (p=0.002), perhaps than in whole BEST group or in the opposite two times of types of homology, have bigger therapeutic response.On this aspect, table 9 intermediate hurdles 11 show in BEST to be α _2cDEL 322-325 is again β ₁389Gly carrier's patient's full reason mortality ratio has 35% increase, and has 36% increase on the CV mortality ratio.Obvious explanation for these adverse effects is to have low-function 389Gly carrier β ₁The heart failure patient in late period of-adrenergic receptor is impatient at and α _2cThe excessive anti-sympathetic nerve reaction that DEL carrier state is relevant.What by contrast, be characterised in that youngster's naphthol amine to lower concentration has a kickback has high functionality (389Arg/Arg) β ₁The patient of-acceptor does not have this detrimental action (table 9, hurdle 9) to overall or cardiovascular property mortality ratio.Although patient relatively in a small amount and incident have been got rid of at { α _2cDEL 322-325 and β ₁The 389Gly carrier } significance,statistical of arbitrary mortality ratio terminal point in the subgroup, when problem was patient safety, these found to support them on science and importance clinically with the consistence of the molecular pharmacology of anti-sympathetic nerve data and adrenergic receptor polymorphism.

In BEST, β ₁The 389Arg/Arg gene frequency is 0.72 non-black race, but has only 0.57 (p＜0.0001) in black race.Therefore, black race has the allelotrope (α that mortality ratio increases influence that is subject to of upper frequency in BEST _2cDEL 322-325) and " super-reaction " β of lower frequency ₁389Arg/Arg allelotrope.These two kinds of hereditary differences may help the trend (BEST Writing Committee, 2001) towards unfavorable result in this demography group in U.S. from black race.

The pharmacogenomics subgroup research of BEST is used perspective hypothesis based on the expection pharmacology interaction of bucindolol and specificity adrenergic receptor polymorphism, and described polymorphism is in before being investigated widely in model and robot system.Therefore, believe these pharmacogenomicses hypothesis than standard, the deutero-subgroup of running back over the past analysis is more effective.

Be included in down from the pharmacogenetics data of BEST analysis of experiments.The colony of agreeing DNA subgroup research and pharmacogenetics analysis does not have difference with the whole baseline characteristic of organizing.In addition, there is not evidence to show gene dosage effect for arbitrary polymorphism; The effect of heterozygote is with homozygotic identical or bigger.Therefore, suppose α _2cDEL 322-325 and β ₁389Gly allelotrope is as dominant negative.

Table 9

Terminal point	The whole group of BEST bucindolol (n=2708) mean value f/u 2.0 years	BEST α 2c WT/WT bucindolol (n=829/1036) mean value f/u 2.0 years	BEST α 2c DEL carrier's bucindolol (n=207/1036) mean value f/u 2.0 years	BEST β 1 389Arg/Arg bucindolol (n=493/1040) mean value f/u 2.0 years	BEST β 1 389 Gly carrier bucindolol (n=547/1040) mean value f/u 2.0 years
						Mortality ratio	0.90；860Ev (0.78，1.02) p＝0.10	0.70；155Ev (0.51，0.97) p＝0.031	1.09；37Ev (0.57，2.08) p＝0.79	0.62；82Ev (0.40，0.96) p＝0.030	0.90；111Ev (0.62，1.30) p＝0.57
The CV mortality ratio	0.86；731Ev (0.74，0.99) p＝0.040	0.59；130Ev (0.42，0.85) p＝0.004	1.03；32Ev (0.52，2.07) p＝0.92	0.54；66Ev (0.33，0.89) p＝0.015	0,78；97Ev (0.52，1.18) p＝0.24
						Mortality ratio+HF hospital care	0.81；1421Ev (0.73，0.90) p＜0.0001	0.72；341Ev (0.59，0.90) p＝0.003	0.89；84Ev (0.58，1.37) p＝0.60	0.66；190Ev (0.50，0.88) p＝0.004	0.87；236Ev (0.62，1.30) p＝0.25
The HF hospital care	0.78；1045Ev (0.69，0.88)； p＜0.001	0.74；267Ev (0.58，0.95) p＝0.016	0.76；67Ev (0.47，1.24) p＝0.27	0.64；154 (0.48，0.90) p＝0.006	0.86；181Ev (0.64，1.15) p＝0.30
						Terminal point	BEST β 1 389Arg/Arg+α 2c WT/WT bucindolol (n=418/1036) mean value f/u2.0	BEST β 1 389Arg/Arg+α 2cDEL carrier bucindolol (n=73/1036) mean value f/u 2.0 years	BEST β 1 389 Gly carrier+α 2c WT/WT bucindolol (n=411/1036) mean value f/u 2.0 years	BEST β 1 389Gly carrier+DEL carrier bucindolol (n=134/1036) mean value f/u 2.0 years	MERIT-HF US Pts metoprolol CR/XL (n=1071/3991) mean value f/u 1.0 years
Mortality ratio	0.60；69Ev (038，0.97) p＝0.037	0.71；13Ev (0.24，2.11) p＝0.53	0.82；86Ev (0.54，1.26) p＝0.37	135；24Ev (0.61，3.02) p＝0.46	1.05；100Ev 0.71，1.56 p＝NS
						The CV mortality ratio	0.53；56Ev (0.31，0.90) p＝0.020	0.58；73Ev (0.17，2.02) p＝0.39	0.67；411Ev (0.42，1.07) p＝0.09	1.36；22Ev (0.59，3.15) p＝0.47	～0.96；90Ev p＝NS
Mortality ratio+HF hospital care	0.61；156Ev (0.44，0.84) p＝0.002	0.85；34Ev (0.43，1.69) p＝0.64	0.86；185Ev (0.64，1.16) p＝0.32	0.81；50Ev (0.46，1.43) p＝0.47	-0.84；200Ev (0.61，1.12) p＝NS
						The HF hospital care	0.59；126Ev (.41，.84) p＝0.004	0.81；73Ev (0.38，1.72 p＝0.59	0.93；411Ev (0.67，1.29) p＝0.66	0.62；134Ev (0.32，1.21) p＝0.16	NA

The effect of comparing beta blocker and placebo in the unique available/disclosed data of the purpose treatment mortality ratio test of in coming comfortable U.S. heart failure patient, implementing

The Ev=incident; NA, unavailable

Embodiment 9

Additional studies: design I

Because the reason of ethics can not be implemented the placebo-controlled trial of relevant beta blocker in the heart failure patient that is caused by primary or secondary dilated cardiomyopathy.This allows non-bad effect (non-inferiority) or superiority (superiority) research resist active beta blocker contrast as design option, perhaps comes the bucindolol reaction between the icp gene variant as an alternative.Non-bad effect design alternative seems and will be excluded and (have only MERIT-HF to have pharmacogenomics subgroup research (Small et al. in testing in the other III phase owing to lacking acceptor gene variant data for any other medicament, 2002), and because too little and can not obtain any significant conclusion).The research and design I that shows in Figure 27 is a kind of possible research approach, and its time that is applied to death or hospital care in heart failure compares the associating of genotype target and bucindolol and the non-targeted therapies of metoprolol CR/XL as main terminal point.In design I, if be β for the patient of bucindolol random choose ₁The AR-389Gly carrier, they will not enter research, because based on the BEST data, can not fully guarantee further assessment at the reaction of bucindolol.These patients will change registration into and enter the group of obtaining the minimum level information that may be defined as life state, and no matter they with selected which kind of therapeutic modality of its treatment doctor treat.The main of design I relatively will be at the β with the bucindolol treatment ₁Carry out between AR-389Arg/Arg patient and all genotype patients with the non-targeted therapies treatment of metoprolol CR/XL (TOPROL XL).The big young pathbreaker of total random sample for design I is 900, has 662 objects to participate in main result relatively.

Embodiment 10

Additional studies: design II

In the research and design II (Figure 28) of additional studies design, symptoms exhibited and heart failure in late period (are strengthened by hospital care history in heart failure in the previous year; Therefore, allow the II level patient when screening to enter test) and the patient of LVEFs≤0.35 (BEST tests the general remark of colony) carry out under carrier's state (or heterozygote or homozygote), whether lacking α _2cThe preliminary screening of 322-325 DEL polymorphism.This restricted most of anti-sympathetic nerve detrimental action that is caused by bucindolol of having got rid of, this mainly shows as in BEST (risk than 1.09, hurdle 4, table 9) and mortality ratio increases in MOXCON trend, and is expected at pure α _2cUnder homozygous wildtype (WT/WT) the adrenergic receptor background in the remaining genotype for the treatment of bucindolol compare the advantage that will have a little with metoprolol CR/XL.

The main terminal point of this test is the non-bad effect of relative metoprolol CR/XL (TOPROL-XL), and that wherein uses in MERIT-HF test (Hjalmarson et al.2000) measurement adds 95% upper confidence limit (UCL) of the time of hospital care terminal point risk ratio in heart failure to mortality ratio.Although the MERIT-HF test comprises all genotype, having only 208 (5.2%) in 3991 patients that go into to organize is black race.α _2c322-325 DEL carrier in black race's object, account for considerable number (in BEST test, α _2c322-325 DEL gene frequency is 0.423 in black race, be 0.043 in non-black race, and the popularity of DEL carrier in black race is 66%, is 8% in non-black race).Therefore, the MERIT-HF test has the α near 90% _2cWild-type, or have can with the design II in the suitable genotype of planning studies colony of institute.In addition, because the not anti-sympathetic nerve effect of metoprolol CR/XL, so have no reason to be expected at α _2cWild-type is to producing the otherness reaction to metoprolol CR/XL between the DEL carrier.For bucindolol: 95% upper confidence limit of the risk ratio of metoprolol CR/CL (" the non-bad effect in limit ") is determined (Hasselblad et al., 2001) { (UCL bucindolol: metoprolol CR/XL) according to formula ^*(UCL metoprolol CR/XL: placebo)≤1.0}; For metoprolol: placebo uses the UCL 0.80 of the whole group of MERIT-HF to produce x ^*0.80≤1.0, or the non-bad effect x in limit≤1.25.Subsequent value 1.25 is reduced to 1.16, thereby provides bigger determinacy to non-bad effect.1.16 target UCL also be the value that is obtained when amplifying MERIT-HF risk ratio/UCL when from the observed value of 0.69/0.80 to 1.0 risks.Estimate that for the probability of bilateral α=≤ 0.05 (powerestimate) is 85% (by bucindolol to β than determining from 0.90 calculated risk subsequently ₁-389 Arg/Arg good inhibitory effect and/or its myocardium beta 2-receptor blocking effect obtain bucindolol at α _2cSlight advantage in the WT/WT colony).

The support (table 9) that the patient provides bucindolol to surpass the advantage of metoprolol CR/XL is gone into to organize by the U.S. of MERIT-HF, and their mortality ratio has 5% increase and mortality ratio+HF hospital care that 16% reduction is arranged when described patient treats with metoprolol CR/XL.By contrast, the mortality ratio of whole group has 10% reduction (p=0.10) and mortality ratio+HF hospital care that 19% reduction (p＜0.0001) is arranged in BEST, however α _2cThe wild-type mortality in said patients has 30% reduction (p=0.031) and mortality ratio to add the HF hospital care 28% reduction (p=0.003) is arranged.The risk ratio be divided by (0.72/0.84) produce 0.86 calculated risk ratio, and bucindolol is 1.00-0.86 or 0.14 to the effect size (effect size) of metoprolol CR/XL.Therefore must reasonably be expected at α _2cHelp 10% difference of bucindolol in the WT/WT type U.S. colony to metoprolol CR/XL.By proposed standard among Figure 28, no matter the risk ratio of bucindolol/metoprolol, if UCL＜1.16, conclusion will be to be α 100% so _2cThe late period of-adrenergic receptor homozygous wildtype in the HF colony for the effect of the time that arrives mortality ratio or HF hospital care bucindolol be not inferior to metoprolol CR/XL.

The purpose of test design second section is that the evidence that provides other proves, as secondary endpoints, the bucindolol of pharmacogenomics target is better than the metoprolol CR/XL of non-target.At this, the colony for the treatment of with bucindolol is β ₁-389 Arg/Arg patients, they also are α simultaneously _2cWild-type.47% of whole group in BEST (all objects) is β ₁-389 Arg/Arg, same 50% is α _2cWild-type patient.As shown in the table 9, β ₁-389 Arg/Arg+ α _2cThe dual-gene type of wild-type shows 40% reduction (p=0.037) on mortality ratio, 39% reduction (p=0.002) is arranged in mortality ratio+HF hospital care and 41% reduction (p=0.004) is arranged in the HF hospital care.For bucindolol: the calculated risk ratio of mortality ratio+HF hospital care of metoprolol CR/XL (MERIT U.S. data) will be 0.61/0.84 or 0.73.β in the bucindolol treatment of comparing with metoprolol CR/XL ₁-389 Arg/Arg+ α _2cUse 27% effect size among the dual-gene type patient of wild-type, produce 81% probability calculation result.Therefore in the suggestion test that Figure 28 shows, for the marginal non-bad effect of main terminal point is conservatively based on the whole group of result of MERIT-HF, however for mainly with the probability calculation result of secondary endpoints on the basis of relevant bucindolol of U.S. colony and metoprolol CR/XL real data, comprised the expection advantage of bucindolol in selected genotype.Support for the exactness of this comparison is in the DNA subgroup research of BEST test, the β of usefulness placebo treatment ₁-389 Arg/Arg patients and the β that uses placebo treatment ₁-389 Gly carrier patients have equal incident rate (incidence) (Fig. 6 and 7).In other words, as shown in Figure 7, at β with the bucindolol treatment ₁More favourable incident rate is owing to therapeutic action, with β fully among-389 Arg/Arg patients ₁The preferable natural medical history of-389 Arg/Arg patients has nothing to do.This one-level secondary endpoints will provide further evidence to the doctor, show that the genotype targeted therapies of bucindolol produces better clinical effectiveness than the non-targeted therapies with the beta blocker of ratifying in heart failure.

In test design shown in Figure 28, the colony for the treatment of with bucindolol equally also is α _2cThe β of wild-type ₁-389 Arg/Arg patients.In BEST in the whole group 47% is β ₁-389 Arg/Arg, the 50%th, α _2cWild-type patient.As shown in table 9, β ₁-389 Arg/Arg+ α _2cThe dual-gene type of wild-type shows on mortality ratio 40% reduction (p=0.037), and adding in mortality ratio has 39% reduction (p=0.002) in the HF hospital care, and 41% reduction (p=0.004) is arranged in the HF hospital care.For bucindolol: the calculated risk ratio that the mortality ratio of metoprolol CR/XL (MERIT U.S.data) adds the HF hospital care will be 0.61/0.84 or 0.73.With respect to the β of metoprolol CR/XL in the bucindolol treatment ₁-389 Arg/Arg add α _2cUse 27% big or small 81% the probability calculation result that produces of effect among the dual-gene type patient of wild-type.Therefore in suggestion test shown in Figure 28, for the marginal non-bad effect of main terminal point is conservatively based on the whole group of result of MERIT-HF, however for mainly with the probability calculation result of secondary endpoints in U.S. colony, comprised the expection advantage of bucindolol in selected genotype on the basis of relevant bucindolol and metoprolol CR/XL real data.

Embodiment 11

Additional studies: design III

Be that this therapeutic action that provides metoprolol CR/XL to surpass placebo has 36% to keep under 1.16 the situation in the non-bad effect in limit, it is sufficient inadequately being considered to for the proof effect, so propose another kind of design.1.14 UCL will with 90% probability keep metoprolol CR/XL surpass placebo therapeutic action 50%, this is considered to acceptable.Therefore, thus design shown in Figure 28 has been adjusted with 90% probability and has realized this target (Figure 29).By sample size is increased to 1600 and realized that in the mode that changed into 1: 1 from 2: 1 by the randomization that makes between bucindolol and the metoprolol CR/XL on the less degree statistics probability that needs to reduce UCL increases from 1300.Equally, in the reaction to factor feedback (agency feedback), we have added another secondary endpoints, at β ₁Bucindolol is to metoprolol CR/XL among-389 Arg/Arg patients.Predictive role size based on 25% estimates it is 71% for the probability of this secondary endpoints, yet based on 27% predictive role size, the probability of another secondary endpoints is estimated it is 88%.Because at β ₁Limited in the-389Arg/Arg colony about the data of metoprolol CR/XL, thus 25% the former to act on size be what be difficult to estimate; Only data available shows that the therapeutic action of comparing metoprolol CR/XL with placebo has very little or not enhancing (White et al., 2003).Be based on the U.S. MERIT-HF data of above-mentioned discussion for 27% effect size of another secondary endpoints.

Should think that aforementioned specification is the illustrative explanation to the principle of the invention.In addition, because will be easy to the present invention is made many modifications and variation, therefore can not require the present invention fully is limited to above-mentioned explanation and process for those skilled in the art.Therefore, all suitable modifications and equivalent can be required to drop in the scope of the invention of claims qualification.Word " comprises " when using in this specification sheets and subsequently claim, when " comprising ", " containing ", its meaning is the existence that is to illustrate institute's features set forth, integral body (integers), component or step, but does not get rid of existence or add one or more other features, integral body, component, step or its set.

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Sequence table

＜110〉Si Difen .B. Li Gete

Michael. Bristol

＜120〉use the methods of treatment of bucindolol based on gene target

<130>ARCA：003US

＜140〉the unknown

<141>2005-09-14

<150>60/609/689

<151>2004-09-14

<150>60/610,706

<151>2004-09-17

<160>8

<170>PatentIn Ver.2.1

<210>1

<211>1723

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(87)..(1520)

<400>1

tgctacccgc gcccgggctt ctggggtgtt ccccaaccac ggcccagccc tgccacaccc 60

cccgcccccg gcctccgcag ctcggc atg ggc gcg ggg gtg ctc gtc ctg ggc 113

Met Gly Ala Gly Val Leu Val Leu Gly

1 5

gcc tcc gag ccc ggt aac ctg tcg tcg gcc gca ccg ctc ccc gac ggc 161

Ala Set Glu Pro Gly Asn Leu Ser Ser Ala Ala Pro Leu Pro Asp Gly

10 15 20 25

gcg gcc acc gcg gcg cgg ctg ctg gtg ccc gcg tcg ccg ccc gcc tcg 209

Ala Ala Thr Ala Ala Arg Leu Leu Val Pro Ala Set Pro Pro Ala Ser

30 35 40

ttg ctg cct ccc gcc agc gaa agc ccc gag ccg ctg tct cag cag tgg 257

Leu Leu Pro Pro Ala Ser Glu Ser Pro Glu Pro Leu Ser Gln Gln Trp

45 50 55

aca gcg ggc atg ggt ctg ctg atg gcg ctc atc gtg ctg ctc atc gtg 305

Thr Ala Gly Met Gly Leu Leu Met Ala Leu Ile Val Leu Leu Ile Val

60 65 70

gcg ggc aat gtg ctg gtg atc gtg gcc atc gcc aag acg ccg cgg ctg 353

Ala Gly Asn Val Leu Val Ile Val Ala Ile Ala Lys Thr Pro Arg Leu

75 80 85

cag acg ctc acc aac ctc ttc atc atg tcc ctg gcc agc gcc gac ctg 401

Gln Thr Leu Thr Asn Leu Phe Ile Met Set Leu Ala Set Ala Asp Leu

90 95 100 105

gtc atg ggg ctg ctg gtg gtg ccg ttc ggg gcc acc atc gtg gtg tgg 449

Val Met Gly Leu Leu Val Val Pro Phe Gly Ala Thr Ile Val Val Trp

110 115 120

ggc cgc tgg gag tac ggc tcc ttc ttc tgc gag ctg tgg acc tca gtg 497

Gly Arg Trp Glu Tyr Gly Ser Phe Phe Cys Glu Leu Trp Thr Ser Val

125 130 135

gac gtg ctg tgc gtg acg gcc agc atc gag acc ctg tgt gtc att gcc 545

Asp Val Leu Cys Val Thr Ala Ser Ile Glu Thr Leu Cys Val Ile Ala

140 145 150

ctg gac cgc tac ctc gcc atc acc tcg ccc ttc cgc tac cag agc ctg 593

Leu Asp Arg Tyr Leu Ala Ile Thr Ser Pro Phe Arg Tyr Gln Ser Leu

155 160 165

ctg acg cgc gcg cgg gcg cgg ggc ctc gtg tgc acc gtg tgg gcc atc 641

Leu Thr Arg Ala Arg Ala Arg Gly Leu Val Cys Thr Val Trp Ala Ile

170 175 180 185

tcg gcc ctg gtg tcc ttc ctg ccc atc ctc atg cac tgg tgg cgg gcg 689

Ser Ala Leu Val Ser Phe Leu Pro Ile Leu Met His Trp Trp Arg Ala

190 195 200

gag agc gac gag gcg cgc cgc tgc tac aac gac ccc aag tgc tgc gac 737

Glu Ser Asp Glu Ala Arg Arg Cys Tyr Asn Asp Pro Lys Cys Cys Asp

205 210 215

ttc gtc acc aac cgg gcc tac gcc atc gcc tcg tcc gta gtc tcc ttc 785

Phe Val Thr Asn Arg Ala Tyr Ala Ile Ala Ser Ser Val Val Ser Phe

220 225 230

tac gtg ccc ctg tgc atc atg gcc ttc gtg tac ctg cgg gtg ttc cgc 833

Tyr Val Pro Leu Cys Ile Met Ala Phe Val Tyr Leu Arg Val Phe Arg

235 240 245

gag gcc cag aag cag gtg aag aag atc gac agc tgc gag cgc cgt ttc 881

Glu Ala Gln Lys Gln Val Lys Lys Ile Asp Ser Cys Glu Arg Arg Phe

250 255 260 265

ctc ggc ggc cca gcg cgg ccg ccc tcg ccc tcg ccc tcg ccc gtc ccc 929

Leu Gly Gly Pro Ala Arg Pro Pro Ser Pro Ser Pro Ser Pro Val Pro

270 275 280

gcg ccc gcg ccg ccg ccc gga ccc ccg cgc ccc gcc gcc gcc gcc gcc 977

Ala Pro Ala Pro Pro Pro Gly Pro Pro Arg Pro Ala Ala Ala Ala Ala

285 290 295

acc gcc ccg ctg gcc aac ggg cgt gcg ggt aag cgg cgg ccc tcg cgc 1025

Thr Ala Pro Leu Ala Asn Gly Arg Ala Gly Lys Arg Arg Pro Ser Arg

300 305 310

ctc gtg gcc cta cgc gag cag aag gcg ctc aag acg ctg ggc atc atc 1073

Leu Val Ala Leu Arg Glu Gln Lys Ala Leu Lys Thr Leu Gly Ile Ile

315 320 325

atg ggc gtc ttc acg ctc tgc tgg ctg ccc ttc ttc ctg gcc aac gtg 1121

Met Gly Val Phe Thr Leu Cys Trp Leu Pro Phe Phe Leu Ala Asn Val

330 335 340 345

gtg aag gcc ttc cac cgc gag ctg gtg ccc gac cgc ctc ttc gtc ttc 1169

Val Lys Ala Phe His Arg Glu Leu Val Pro Asp Arg Leu Phe Val Phe

350 355 360

ttc aac tgg ctg ggc tac gcc aac tcg gcc ttc aac ccc atc atc tac 1217

Phe Asn Trp Leu Gly Tyr Ala Asn Ser Ala Phe Asn Pro Ile Ile Tyr

365 370 375

tgc cgc agc ccc gac ttc cgc aag gcc ttc cag gga ctg ctc tgc tgc 1265

Cys Arg Ser Pro Asp Phe Arg Lys Ala Phe Gln Gly Leu Leu Cys Cys

380 385 390

gcg cgc agg gct gcc cgc cgg cgc cac gcg acc cac gga gac cgg ccg 1313

Ala Arg Arg Ala Ala Arg Arg Arg His Ala Thr His Gly Asp Arg Pro

395 400 405

cgc gcc tcg ggc tgt ctg gcc cgg ccc gga ccc ccg cca tcg ccc ggg 1361

Arg Ala Ser Gly Cys Leu Ala Arg Pro Gly Pro Pro Pro Ser Pro Gly

410 415 420 425

gcc gcc tcg gac gac gac gac gac gat gtc gtc ggg gcc acg ccg ccc 1409

Ala Ala Ser Asp Asp Asp Asp Asp Asp Val Val Gly Ala Thr Pro Pro

430 435 440

gcg cgc ctg ctg gag ccc tgg gcc ggc tgc aac ggc ggg gcg gcg gcg 1457

Ala Arg Leu Leu Glu Pro Trp Ala Gly Cys Asn Gly Gly Ala Ala Ala

445 450 455

gac agc gac tcg agc ctg gac gag ccg tgc cgc ccc ggc ttc gcc tcg 1505

Asp Ser Asp Ser Ser Leu Asp Glu Pro Cys Arg Pro Gly Phe Ala Ser

460 465 470

gaa tcc aag gtg tag ggcccggcgc ggggcgcgga ctccgggcac ggcttcccag 1560

Glu Ser Lys Val

475

gggaacgagg agatctgtgt ttacttaaga ccgatagcag gtgaactcga agcccacaat 1620

cctcgtctga atcatccgag gcaaagagaa aagccacgga ccgttgcaca aaaaggaaag 1680

tttgggaagg gatgggagag tggcttgctg atgttccttg ttg 1723

<210>2

<211>477

<212>PRT

＜213〉people

<400> 2

Met Gly Ala Gly Val Leu Val Leu Gly Ala Ser Glu Pro Gly Asn Leu

1 5 10 15

Ser Ser Ala Ala Pro Leu Pro Asp Gly Ala Ala Thr Ala Ala Arg Leu

20 25 30

Leu Val Pro Ala Ser Pro Pro Ala Ser Leu Leu Pro Pro Ala Ser Glu

35 40 45

Ser Pro Glu Pro Leu Ser Gln Gln Trp Thr Ala Gly Met Gly Leu Leu

50 55 60

Met Ala Leu Ile Val Leu Leu Ile Val Ala Gly Asn Val Leu Val Ile

65 70 75 80

Val Ala Ile Ala Lys Thr Pro Arg Leu Gln Thr Leu Thr Asn Leu Phe

85 90 95

Ile Met Ser Leu Ala Ser Ala Asp Leu Val Met Gly Leu Leu Val Val

100 105 110

Pro Phe Gly Ala Thr Ile Val Val Trp Gly Arg Trp Glu Tyr Gly Ser

115 120 125

Phe Phe Cys Glu Leu Trp Thr Ser Val Asp Val Leu Cys Val Thr Ala

130 135 140

Ser Ile Glu Thr Leu Cys Val Ile Ala Leu Asp Arg Tyr Leu Ala Ile

145 150 155 160

Thr Ser Pro Phe Arg Tyr Gln Ser Leu Leu Thr Arg Ala Arg Ala Arg

165 170 175

Gly Leu Val Cys Thr Val Trp Ala Ile Ser Ala Leu Val Ser Phe Leu

180 185 190

Pro Ile Leu Met His Trp Trp Arg Ala Glu Ser Asp Glu Ala Arg Arg

195 200 205

Cys Tyr Asn Asp Pro Lys Cys Cys Asp Phe Val Thr Asn Arg Ala Tyr

210 215 220

Ala Ile Ala Ser Ser Val Val Ser Phe Tyr Val Pro Leu Cys Ile Met

225 230 235 240

Ala Phe Val Tyr Leu Arg Val Phe Arg Glu Ala Gln Lys Gln Val Lys

245 250 255

Lys Ile Asp Ser Cys Glu Arg Arg Phe Leu Gly Gly Pro Ala Arg Pro

260 265 270

Pro Ser Pro Ser Pro Ser Pro Val Pro Ala Pro Ala Pro Pro Pro Gly

275 280 285

Pro Pro Arg Pro Ala Ala Ala Ala Ala Thr Ala Pro Leu Ala Asn Gly

290 295 300

Arg Ala Gly Lys Arg Arg Pro Ser Arg Leu Val Ala Leu Arg Glu Gln

305 310 315 320

Lys Ala Leu Lys Thr Leu Gly Ile Ile Met Gly Val Phe Thr Leu Cys

325 330 335

Trp Leu Pro Phe Phe Leu Ala Asn Val Val Lys Ala Phe His Arg Glu

340 345 350

Leu Val Pro Asp Arg Leu Phe Val Phe Phe Asn Trp Leu Gly Tyr Ala

355 360 365

Asn Ser Ala Phe Asn Pro Ile Ile Tyr Cys Arg Ser Pro Asp Phe Arg

370 375 380

Lys Ala Phe Gln Gly Leu Leu Cys Cys Ala Arg Arg Ala Ala Arg Arg

385 390 395 400

Arg His Ala Thr His Gly Asp Arg Pro Arg Ala Ser Gly Cys Leu Ala

405 410 415

Arg Pro Gly Pro Pro Pro Ser Pro Gly Ala Ala Ser Asp Asp Asp Asp

420 425 430

Asp Asp Val Val Gly Ala Thr Pro Pro Ala Arg Leu Leu Glu Pro Trp

435 440 445

Ala Gly Cys Asn Gly Gly Ala Ala Ala Asp Ser Asp Ser Ser Leu Asp

450 455 460

Glu Pro Cys Arg Pro Gly Phe Ala Ser Glu Ser Lys Val

465 470 475

<210>3

<211>1434

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(1434)

<400>3

atg ggc gcg ggg gtg ctc gtc ctg ggc gcc tcc gag ccc ggt aac ctg 48

Met Gly Ala Gly Val Leu Val Leu Gly Ala Ser Glu Pro Gly Asn Leu

1 5 10 15

tcg tcg gcc gca ccg ctc ccc gac ggc gcg gcc acc gcg gcg cgg ctg 96

Ser Ser Ala Ala Pro Leu Pro Asp Gly Ala Ala Thr Ala Ala Arg Leu

20 25 30

ctg gtg ccc gcg tcg ccg ccc gcc tcg ttg ctg cct ccc gcc agc gaa 144

Leu Val Pro Ala Ser Pro Pro Ala Ser Leu Leu Pro Pro Ala Ser Glu

35 40 45

agc ccc gag ccg ctg tct cag cag tgg aca gcg ggc atg ggt ctg ctg 192

Ser Pro Glu Pro Leu Ser Gln Gln Trp Thr Ala Gly Met Gly Leu Leu

50 55 60

atg gcg ctc atc gtg ctg ctc atc gtg gcg ggc aat gtg ctg gtg atc 240

Met Ala Leu Ile Val Leu Leu Ile Val Ala Gly Asn Val Leu Val Ile

65 70 75 80

gtg gcc atc gcc aag acg ccg cgg ctg cag acg ctc acc aac ctc ttc 288

Val Ala Ile Ala Lys Thr Pro Arg Leu Gln Thr Leu Thr Asn Leu Phe

85 90 95

atc atg tcc ctg gcc agc gcc gac ctg gtc atg ggg ctg ctg gtg gtg 336

Ile Met Ser Leu Ala Ser Ala Asp Leu Val Met Gly Leu Leu Val Val

100 105 110

ccg ttc ggg gcc acc atc gtg gtg tgg ggc cgc tgg gag tac ggc tcc 384

Pro Phe Gly Ala Thr Ile Val Val Trp Gly Arg Trp Glu Tyr Gly Ser

115 120 125

ttc ttc tgc gag ctg tgg acc tca gtg gac gtg ctg tgc gtg acg gcc 432

Phe Phe Cys Glu Leu Trp Thr Ser Val Asp Val Leu Cys Val Thr Ala

130 135 140

agc atc gag acc ctg tgt gtc att gcc ctg gac cgc tac ctc gcc atc 480

Ser Ile Glu Thr Leu Cys Val Ile Ala Leu Asp Arg Tyr Leu Ala Ile

145 150 155 160

acc tcg ccc ttc cgc tac cag agc ctg ctg acg cgc gcg cgg gcg cgg 528

Thr Ser Pro Phe Arg Tyr Gln Ser Leu Leu Thr Arg Ala Arg Ala Arg

165 170 175

ggc ctc gtg tgc acc gtg tgg gcc atc tcg gcc ctg gtg tcc ttc ctg 576

Gly Leu Val Cys Thr Val Trp Ala Ile Ser Ala Leu Val Ser Phe Leu

180 185 190

ccc atc ctc atg cac tgg tgg cgg gcg gag agc gac gag gcg cgc cgc 624

Pro Ile Leu Met His Trp Trp Arg Ala Glu Ser Asp Glu Ala Arg Arg

195 200 205

tgc tac aac gac ccc aag tgc tgc gac ttc gtc acc aac cgg gcc tac 672

Cys Tyr Asn Asp Pro Lys Cys Cys Asp Phe Val Thr Asn Arg Ala Tyr

210 215 220

gcc atc gcc tcg tcc gta gtc tcc ttc tac gtg ccc ctg tgc atc atg 720

Ala Ile Ala Ser Ser Val Val Ser Phe Tyr Val Pro Leu Cys Ile Met

225 230 235 240

gcc ttc gtg tac ctg cgg gtg ttc cgc gag gcc cag aag cag gtg aag 768

Ala Phe Val Tyr Leu Arg Val Phe Arg Glu Ala Gln Lys Gln Val Lys

245 250 255

aag atc gac agc tgc gag cgc cgt ttc ctc ggc ggc cca gcg cgg ccg 816

Lys Ile Asp Ser Cys Glu Arg Arg Phe Leu Gly Gly Pro Ala Arg Pro

260 265 270

ccc tcg ccc tcg ccc tcg ccc gtc ccc gcg ccc gcg ccg ccg ccc gga 864

Pro Ser Pro Ser Pro Ser Pro Val Pro Ala Pro Ala Pro Pro Pro Gly

275 280 285

ccc ccg cgc ccc gcc gcc gcc gcc gcc acc gcc ccg ctg gcc aac ggg 912

Pro Pro Arg Pro Ala Ala Ala Ala Ala Thr Ala Pro Leu Ala Asn Gly

290 295 300

cgt gcg ggt aag cgg cgg ccc tcg cgc ctc gtg gcc ctg cgc gag cag 960

Arg Ala Gly Lys Arg Arg Pro Ser Arg Leu Val Ala Leu Arg Glu Gln

305 310 315 320

aag gcg ctc aag acg ctg ggc atc atc atg ggc gtc ttc acg ctc tgc 1008

Lys Ala Leu Lys Thr Leu Gly Ile Ile Met Gly Val Phe Thr Leu Cys

325 330 335

tgg ctg ccc ttc ttc ctg gcc aac gtg gtg aag gcc ttc cac cgc gag 1056

Trp Leu Pro Phe Phe Leu Ala Asn Val Val Lys Ala Phe His Arg glu

340 345 350

ctg gtg ccc gcc ggc ccc ttc gtc ttc ttc aac tgg ctg ggc tac gcc 1104

Leu Val Pro Asp Arg Leu Phe Val Phe Phe Asn Trp Leu Gly Tyr Ala

355 360 365

aac tcg gcc ttc aac ccc atc atc tac tgc cgc agc ccc gac ttc cgc 1152

Asn Ser Ala Phe Asn Pro Ile Ile Tyr Cys Arg Ser Pro Asp Phe Arg

370 375 380

aag gcc ttc cag cga ctg ctc tgc tgc gcg cgc agg gct gcc cgc cgg 1200

Lys Ala Phe Gln Arg Leu Leu Cys Cys Ala Arg Arg Ala Ala Arg Arg

385 390 395 400

cgc cac gcg acc cac gga gac cgg ccg cgc gcc tcg ggc tgt ctg gcc 1248

Arg His Ala Thr His Gly Asp Arg Pro Arg Ala Ser Gly Cys Leu Ala

405 410 415

cgg ccc gga ccc ccg cca tcg ccc ggg gcc gcc tcg gac gac gac gac 1296

Arg Pro Gly Pro Pro Pro Ser Pro Gly Ala Ala Ser Asp Asp Asp Asp

420 425 430

gac gat gtc gtc ggg gcc acg ccg ccc gcg cgc ctg ctg gag ccc tgg 1344

Asp Asp Val Val Gly Ala Thr Pro Pro Ala Arg Leu Leu Glu Pro Trp

435 440 445

gcc ggc tgc aac ggc ggg gcg gcg gcg gac agc gac tcg agc ctg gac 1392

Ala Gly Cys Asn Gly Gly Ala Ala Ala Asp Ser Asp Ser Ser Leu Asp

450 455 460

gag ccg tgc cgc ccc ggc ttc gcc tcg gaa tcc aag gtg tag 1434

Glu Pro Cys Arg Pro Gly Phe Ala Ser Glu Ser Lys Val

465 470 475

<210>4

<211>477

<212>PRT

＜213〉people

<400>4

Met Gly Ala Gly Val Leu Val Leu Gly Ala Ser Glu Pro Gly Asn Leu

1 5 10 15

Ser Sar Ala Ala Pro Leu Pro Asp Gly Ala Ala Thr Ala Ala Arg Leu

20 25 30

Leu Val Pro Ala Ser Pro Pro Ala Ser Leu Leu Pro Pro Ala Ser Glu

35 40 45

Ser Pro Glu Pro Leu Ser Gln Gln Trp Thr Ala Gly Met Gly Leu Leu

50 55 60

Met Ala Leu Ile Val Leu Leu Ile Val Ala Gly Asn Val Leu Val Ile

65 70 75 80

Val Ala Ile Ala Lys Thr Pro Arg Leu Gln Thr Leu Thr Asn Leu Phe

85 90 95

Ile Met Ser Leu Ala Ser Ala Asp Leu Val Met Gly Leu Leu Val Val

100 105 110

Pro Phe Gly Ala Thr Ile Val Val Trp Gly Arg Trp Glu Tyr Gly Ser

115 120 125

Phe Phe Cys Glu Leu Trp Thr Ser Val Asp Val Leu Cys Val Thr Ala

130 135 140

Ser Ile Gln Thr Leu Cys Val Ile Als Leu Asp Arg Tyr Leu Ala Ile

145 150 155 160

Thr Ser Pro Phe Arg Tyr Gln Ser Leu Leu Thr Arg Ala Arg Ala Arg

165 170 175

Gly Leu Val Cys Thr Val Trp Ala Ile Ser Ala Leu Val Ser Phe Leu

180 185 190

Pro Ile Leu Met His Trp Trp Arg Ala Glu Ser Asp Glu Ala Arg Arg

195 200 205

Cys Tyr Asn Asp Pro Lys Cys Cys Asp Phe Val Thr Asn Arg Ala Tyr

210 215 220

Ala Ile Ala Ser Ser Val Val Ser Phe Tyr Val Pro Leu Cys Ile Met

225 230 235 240

Ala Phe Val Tyr Leu Arg Val Phe Arg Glu Ala Gln Lys Gln Val Lys

245 250 255

Lys Ile Asp Ser Cys Glu Arg Arg Phe Leu Gly Gly Pro Ala Arg Pro

260 265 270

Pro Ser Pro Ser Pro Ser Pro Val Pro Ala Pro Ala Pro Pro Pro Gly

275 280 285

Pro Pro Arg Pro Ala Ala Ala Ala Ala Thr Ala Pro Leu Ala Asn Gly

290 295 300

Arg Ala Gly Lys Arg Arg Pro Ser Arg Leu Val Ala Leu Arg Glu Gln

305 310 315 320

Lys Ala Leu Lys Thr Leu Gly Ile Ile Met Gly Val Phe Thr Leu Cys

325 330 335

Trp Leu Pro Phe Phe Leu Ala Asn Val Val Lys Ala Phe His Arg Glu

340 345 350

Leu Val Pro Asp Arg Leu Phe Val Phe Phe Asn Trp Leu Gly Tyr Ala

355 360 365

Asn Ser Ala Phe Asn Pro Ile Ile Tyr Cys Arg Ser Pro Asp Phe Arg

370 375 380

Lys Ala Phe Gln Arg Leu Leu Cys Cys Ala Arg Arg Ala Ala Arg Arg

385 390 395 400

Arg His Ala Thr His Gly Asp Arg Pro Arg Ala Ser Gly Cys Leu Ala

405 410 415

Arg Pro Gly Pro Pro Pro Ser Pro Gly Ala Ala Ser Asp Asp Asp Asp

420 425 430

Asp Asp Val Val Gly Ala Thr Pro Pro Ala Arg Leu Leu Glu Pro Trp

435 440 445

Ala Gly Cys Asn Gly Gly Ala Ala Ala Asp Ser Asp Ser Ser Leu Asp

450 455 460

Glu Pro Cys Arg Pro Gly Phe Ala Ser Glu Ser Lys Val

465 470 475

<210>5

<211>1958

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(39)..(1427)

<400>5

ccggctccag gagggacggc gtagctcgcg ggaggacc atg gcg tcc ccg gcg ctg 56

Met Ala Ser Pro Ala Leu

1 5

gcg gcg gcg ctg gcg gtg gcg gca gcg gcg ggc ccc aat gcg agc ggc 104

Ala Ala Ala Leu Ala Val Ala Ala Ala Ala Gly Pro Asn Ala Ser Gly

10 15 20

gcg ggc gag agg ggc agc ggc ggg gtt gcc aat gcc tcg ggg gct tcc 152

Ala Gly Glu Arg Gly Ser Gly Gly Val Ala Asn Ala Ser Gly Ala Ser

25 30 35

tgg ggg ccg ccg cgc ggc cag tac tcg gcg ggc gcg gtg gca ggg ctg 200

Trp Gly Pro Pro Arg Gly Gln Tyr Ser Ala Gly Ala Val Ala Gly Leu

40 45 50

gct gcc gtg gtg ggc ttc ctc atc gtc ttc acc gtg gtg ggc aac gtg 248

Ala Ala Val Val Gly Phe Leu Ile Val Phe Thr Val Val Gly Asn Val

55 60 65 70

ctg gtg gtg atc gcc gtg ctg acc agc cgg gcg ctg cgc gcg cca cag 296

Leu Val Val Ile Ala Val Leu Thr Ser Arg Ala Leu Arg Ala Pro Gln

75 80 85

aac ctc ttc ctg gtg tcg ctg gcc tcg gcc gac atc ctg gtg gcc acg 344

Asn Leu Phe Leu Val Ser Leu Ala Ser Ala Asp Ile Leu Val Ala Thr

90 95 100

ctg gtc atg ccc ttc tcg ttg gcc aac gag ctc atg gcc tac tgg tac 392

Leu Val Met Pro Phe Ser Leu Ala Asn Glu Leu Met Ala Tyr Trp Tyr

105 110 115

ttc ggg cag gtg tgg tgc ggc gtg tac ctg gcg ctc gat gtg ctg ttt 440

Phe Gly Gln Val Trp Cys Gly Val Tyr Leu Ala Leu Asp Val Leu Phe

120 125 130

tgc acc tcg tcg atc gtg cat ctg tgt gcc atc agc ctg gac cgc tac 488

Cys Thr Ser Ser Ile Val His Leu Cys Ala Ile Ser Leu Asp Arg Tyr

135 140 145 150

tgg tcg gtg acg cag gcc gtc gag tac aac ctg aag cgc aca cca cgc 536

Trp Ser Val Thr Gln Ala Val Glu Tyr Asn Leu Lys Arg Thr Pro Arg

155 160 165

cgc gtc aag gcc acc atc gtg gcc gtg tgg ctc atc tcg gcc gtc atc 584

Arg Val Lys Ala Thr Ile Val Ala Val Trp Leu Ile Ser Ala Val Ile

170 175 180

tcc ttc ccg ccg ctg gtc tcg ctc tac cgc cag ccc gac ggc gcc gcc 632

Ser Phe Pro Pro Leu Val Ser Leu Tyr Arg Gln Pro Asp Gly Ala Ala

185 190 195

tac ccg cag tgc ggc ctc aac gac gag acc tgg tac atc ctg tcc tcc 680

Tyr Pro Gln Cys Gly Leu Asn Asp Glu Thr Trp Tyr Ile Leu Ser Ser

200 205 210

tgc atc ggc tcc ttc ttc gcg ccc tgc ctc atc atg ggc ctg gtc tac 728

Cys Ile Gly Ser Phe Phe Ala Pro Cys Leu Ile Met Gly Leu Val Tyr

215 220 225 230

gcg cgc atc tac cga gtg gcc aag ctg cgc acg cgc acg ctc agc gag 776

Ala Arg Ile Tyr Arg Val Ala Lys Leu Arg Thr Arg Thr Leu Ser Glu

235 240 245

aag cgc gcc ccc gtg ggc ccc gac ggt gcg tcc ccg act acc gaa aac 824

Lys Arg Ala Pro Val Gly Pro Asp Gly Ala Ser Pro Thr Thr Glu Asn

250 255 260

ggg ctg ggc gcg gcg gca ggc gca ggc gag aac ggg cac tgc gcg ccc 872

Gly Leu Gly Ala Ala Ala Gly Ala Gly Glu Asn Gly His Cys Ala Pro

265 270 275

ccg ccc gcc gac gtg gag ccg gac gag agc agc gca gcg gcc gag agg 920

Pro Pro Ala Asp Val Glu Pro Asp Glu Ser Ser Ala Ala Ala Glu Arg

280 285 290

cgg cgg cgc cgg ggc gcg ttg cgg cgg ggc ggg cgg cgg cga gcg ggc 968

Arg Arg Arg Arg Gly Ala Leu Arg Arg Gly Gly Arg Arg Arg Ala Gly

295 300 305 310

gcg gag ggg ggc gcg ggc ggt gcg gac ggg cag ggg gcg ggg ccg ggg 1016

Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly Gln Gly Ala Gly Pro Gly

315 320 325

gcg gct gag tcg ggg gcg ctg acc gcc tcc agg tcc ccg ggg ccc ggt 1064

Ala Ala Glu Ser Gly Ala Leu Thr Ala Ser Arg Ser Pro Gly Pro Gly

330 335 340

ggc cgc ctg tcg cgc gcc agc tcg cgc tcc gtc gag ttc ttc ctg tcg 1112

Gly Arg Leu Ser Arg Ala Ser Ser Arg Ser Val Glu Phe Phe Leu Ser

345 350 355

cgc cgg cgc cgg gcg cgc agc agc gtg tgc cgc cgc aag gtg gcc cag 1160

Arg Arg Arg Arg Ala Arg Ser Ser Val Cys Arg Arg Lys Val Ala Gln

360 365 370

gcg cgc gag aag cgc ttc acc ttt gtg ctg gct gtg gtc atg ggc gtg 1208

Ala Arg Glu Lys Arg Phe Thr Phe Val Leu Ala Val Val Met Gly Val

375 380 385 390

ttc gtg ctc tgc tgg ttc ccc ttc ttc ttc agc tac agc ctg tac ggc 1256

Phe Val Leu Cys Trp Phe Pro Phe Phe Phe Ser Tyr Ser Leu Tyr Gly

395 400 405

atc tgc cgc gag gcc tgc cag gtg ccc ggc ccg ctc ttc aag ttc ttc 1304

Ile Cys Arg Glu Ala Cys Gln Val Pro Gly Pro Leu Phe Lys Phe Phe

410 415 420

ttc tgg atc ggc tac tgc aac agc tcg ctc aac ccg gtc atc tac acg 1352

Phe Trp Ile Gly Tyr Cys Asn Ser Ser Leu Asn Pro Val Ile Tyr Thr

425 430 435

gtc ttc aac cag gat ttc cgg cga tcc ttt aag cac atc ctc ttc cga 1400

Val Phe Asn Gln Asp Phe Arg Arg Ser Phe Lys His Ile Leu Phe Arg

440 445 450

cgg agg aga agg ggt ttg cgg cag tga ctcgcacccg tctgggaatc 1447

Arg Arg Arg Arg Gly Phe Arg Gln

455 460

ctggacagct ccgcgctcgg ggctgggcag aaggggcggc ccggacgggg gagctttccc 1507

agagacccgg ggatggattg gcctccaggg cgcaggggag ggtgcggcag ggcaggagct 1567

tggcagagag atagccgggc tccagggagt ggggaggaga gagggggaga cccctttgcc 1627

ttcccccctc agcaaggggc tgcttctggg gctccctgcc tggatccagc tctgggagcc 1687

ctgccgaggt gtggctgtga ggtcagggtt ttagagagca gtggcagagg tagcccccta 1747

aatgggcaag caaggagccc cccaaagaca ctaccactcc ccatccccgt ctgaccaagg 1807

gctgacttct ccaggaccta gtcggggggt ggctgccagg gggcaaggag aaagcaccga 1867

caatctttga ttactgaaag tatttaaatg tttgccaaaa acaacagcca aaacaaccaa 1927

actattttct aaataaacct ttgtaatcta a 1958

<210>6

<211>462

<212>PRT

＜213〉people

<400>6

Met Ala Ser Pro Ala Leu Ala Ala Ala Leu Ala Val Ala Ala Ala Ala

1 5 10 15

Gly Pro Asn Ala Ser Gly Ala Gly Glu Arg Gly Ser Gly Gly Val Ala

20 25 30

Asn Ala Ser Gly Ala Ser Trp Gly Pro Pro Arg Gly Gln Tyr Ser Ala

35 40 45

Gly Ala Val Ala Gly Leu Ala Ala Val Val Gly Phe Leu Ile Val Phe

50 55 60

Thr Val Val Gly Asn Val Leu Val Val Ile Ala Val Leu Thr Ser Arg

65 70 75 80

Ala Leu Arg Ala Pro Gln Asn Leu Phe Leu Val Ser Leu Ala Ser Ala

85 90 95

Asp Ile Leu Val Ala Thr Leu Val Met Pro Phe Ser Leu Ala Asn Glu

100 105 110

Leu Met Ala Tyr Trp Tyr Phe Gly Gln Val Trp Cys Gly Val Tyr Leu

115 120 125

Ala Leu Asp Val Leu Phe Cys Thr Ser Ser Ile Val His Leu Cys Ala

130 135 140

Ile Ser Leu Asp Arg Tyr Trp Ser Val Thr Gln Ala Val Glu Tyr Asn

145 150 155 160

Leu Lys Arg Thr Pro Arg Arg Val Lys Ala Thr Ile Val Ala Val Trp

165 170 175

Leu Ile Ser Ala Val Ile Ser Phe Pro Pro Leu Val Ser Leu Tyr Arg

180 185 190

Gln Pro Asp Gly Ala Ala Tyr Pro Gln Cys Gly Leu Asn Asp Glu Thr

195 200 205

Trp Tyr Ile Leu Ser Ser Cys Ile Gly Ser Phe Phe Ala Pro Cys Leu

210 215 220

Ile Mat Gly Ier Val Tyr Ala Arg Ile Tyr Arg Val Ala Lys Leu Arg

225 230 235 240

Thr Arg Thr Leu Ser Glu Lys Arg Ala Pro Val gly Pro Asp Gly Ala

245 250 255

Ser Pro Thr Thr Glu Asn Gly Leu Gly Ala Ala Ala Gly Ala Gly Glu

260 265 270

Asn Gly His Cys Ala Pro Pro Pro Ala Asp Val Glu Pro Asp Glu Ser

275 280 285

Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Ala Leu Arg Arg Gly

290 295 300

Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly

305 310 315 320

Gln Gly Ala Gly Pro Gly Ala Ala Glu Ser Gly Ala Leu Thr Ala Ser

325 330 335

Arg Ser Pro Gly Pro Gly Gly Arg Leu Ser Arg Ala Ser Ser Arg Ser

340 345 350

Val Glu Phe Phe Leu Ser Arg Arg Arg Arg Ala Arg Ser Ser Val Cys

355 360 365

Arg Arg Lys Val Ala Gln Ala Arg Glu Lys Arg Phe Thr Phe Val Leu

370 375 380

Ala Val Val Met Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe Phe

385 390 395 400

Ser Tyr Ser Leu Tyr Gly Ile Cys Arg Glu Ala Cys Gln Val Pro Gly

405 410 415

Pro Leu Phe Lys Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser Ser Leu

420 425 430

Asn Pro Val Ile Tyr Thr Val Phe Asn Gln Asp Phe Arg Arg Ser Phe

435 440 445

Lys His Ile Leu Phe Arg Arg Arg Arg Arg Gly Phe Arg Gln

450 455 460

<210>7

<211>1377

<212>DNA

＜213〉people

<220>

<221>CDS

<222>(1)..(1377)

<400>7

atg gcg tcc ccg gcg ctg gcg gcg gcg ctg gcg gtg gcg gca gcg gcg 48

Met Ala Ser Pro Ala Leu Ala Ala Ala Leu Ala Val Ala Ala Ala Ala

1 5 10 15

ggc ccc aat gcg agc ggc gcg ggc gag agg ggc agc ggc ggg gtt gcc 96

Gly Pro Asn Ala Ser Gly Ala Gly Glu Arg Gly Ser Gly Gly Val Ala

20 25 30

aat gcc tcg ggg gct tcc tgg ggg ccg ccg cgc ggc cag tac tcg gcg 144

Asn Ala Ser Gly Ala Ser Trp Gly Pro Pro Arg Gly Gln Tyr Ser Ala

35 40 45

ggc gcg gtg gca ggg ctg gct gcc gtg gtg ggc ttc ctc atc gtc ttc 192

Gly Ala Val Ala Gly Leu Ala Ala Val Val Gly Phe Leu Ile Val Phe

50 55 60

acc gtg gtg ggc aac gtg ctg gtg gtg atc gcc gtg ctg acc agc cgg 240

Thr Val Val Gly Asn Val Leu Val Val Ile Ala Val Leu Thr Ser Arg

65 70 75 80

gcg ctg cgc gcg cca cag aac ctc ttc ctg gtg tcg ctg gcc tcg gcc 288

Ala Leu Arg Ala Pro Gln Asn Leu Phe Leu Val Ser Leu Ala Ser Ala

85 90 95

gac atc ctg gtg gcc acg ctg gtc atg ccc ttc tcg ttg gcc aac gag 336

Asp Ile Leu Val Ala Thr Leu Val Met Pro Phe Ser Leu Ala Asn Glu

100 105 110

ctc atg gcc tac tgg tac ttc ggg cag gtg tgg tgc ggc gtg tac ctg 384

Leu Met Ala Tyr Trp Tyr Phe Gly Gln Val Trp Cys Gly Val Tyr Leu

115 120 125

gcg ctc gat gtg ctg ttt tgc acc tcg tcg atc gtg cat ctg tgt gcc 432

Ala Leu Asp Val Leu Phe Cys Thr Ser Ser Ile Val His Leu Cys Ala

130 135 140

atc agc ctg gac cgc tac tgg tcg gtg acg cag gcc gtc gag tac aac 480

Ile Ser Leu Asp Arg Tyr Trp Ser Val Thr Gln Ala Val Glu Tyr Asn

145 150 155 160

ctg aag cgc aca cca cgc cgc gtc aag gcc acc atc gtc gcc gtg tgg 528

Leu Lys Arg Thr Pro Arg Arg Val Lys Ala Thr Ile Val Ala Val Trp

165 170 175

ctc atc tcg gcc gtc atc tcc ttc ccg ccg ctg gtc tcg ctc tac cgc 576

Leu Ile Ser Ala Val Ile Ser Phe Pro Pro Leu Val Ser Leu Tyr Arg

180 185 190

cag ccc gac ggc gcc gcc tac ccg cag tgc ggc ctc aac gac gag acc 624

Gln Pro Asp Gly Ala Ala Tyr Pro Gln Cys Gly Leu Asn Asp Glu Thr

195 200 205

tgg tac atc ctg tcc tcc tgc atc ggc tcc ttc ttc gcg ccc tgc ctc 672

Trp Tyr Ile Leu Ser Ser Cys Ile Gly Ser Phe Phe Ala Pro Cys Leu

210 215 220

atc atg ggc ctg gtc tac gcg cgc atc tac cga gtg gcc aag ctg cgc 720

Ile Met Gly Leu Val Tyr Ala Arg Ile Tyr Arg Val Ala Lys Leu Arg

225 230 235 240

acg cgc acg ctc agc gag aag cgc gcc ccc gtg ggc ccc gac ggt gcg 768

Thr Arg Thr Leu Ser Glu Lys Arg Ala Pro Val Gly Pro Asp Gly Ala

245 250 255

tcc ccg act acc gaa aac ggg ctg ggc gcg gcg gca ggc gca ggc gag 816

Ser Pro Thr Thr Glu Asn Gly Leu Gly Ala Ala Ala Gly Ala Gly Glu

260 265 270

aac ggg cac tgc gcg ccc ccg ccc gcc gac gtg gag ccg gac gag agc 864

Asn Gly His Cys Ala Pro Pro Pro Ala Asp Val Glu Pro Asp Glu Ser

275 280 285

agc gca gcg gcc gag agg cgg cgg cgc cgg ggc gcg ttg cgg cgg ggc 912

Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Ala Leu Arg Arg Gly

290 295 300

ggg cgg cgg cga gcg ggc gcg gag ggg ggc gcg ggc ggt gcg gac ggg 960

Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly

305 310 315 320

cag ggg gcg gct gag tcg ggg gcg ctg acc gcc tcc agg tcc ccg ggg 1008

Gln Gly Ala Ala Glu Ser Gly Ala Leu Thr Ala Ser Arg Ser Pro Gly

325 330 335

ccc ggt ggc cgc ctc tcg cgc gcc agc tcg cgc tcc gtc gag ttc ttc 1056

Pro Gly Gly Arg Leu Ser Arg Ala Ser Ser Arg Ser Val Glu Phe Phe

340 345 350

ctg tcg cgc cgg cgc cgg gcg cgc agc agc gtg tgc cgc cgc aag gtg 1104

Leu Ser Arg Arg Arg Arg Ala Arg Ser Ser Val Cys Arg Arg Lys Val

355 360 365

gcc cag gcg cgc gag aag cgc ttc acc ttt gtg ctg gct gtg gtc atg 1152

Ala Gln Ala Arg Glu Lys Arg Phe Thr Phe Val Leu Ala Val Val Met

370 375 380

ggc gtg ttc gtg otc tgc tgg ttc ccc ttc ttc ttc agc tac agc ctg 1200

Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe Phe Ser Tyr Ser Leu

385 390 395 400

tac ggc atc tgc cgc gag gcc tgc cag gtg ccc ggc ccg ctc ttc aag 1248

Tyr Gly Ile Cys Arg Glu Ala Cys Gln Val Pro Gly Pro Leu Phe Lys

405 410 415

ttc ttc ttc tgg atc ggc tac tgc aac agc tcg ctc aac ccg gtc atc 1296

Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser Ser Leu Asn Pro Val Ile

420 425 430

tac acg gtc ttc aac cag gat ttc cgg cga tcc ttt aag cac atc ctc 1344

Tyr Thr Val Phe Asn Gln Asp Phe Arg Arg Ser Phe Lys His Ile Leu

435 440 445

ttc cga cgg agg aga agg ggc ttc agg cag tga 1377

Phe Arg Arg Arg Arg Arg Gly Phe Arg Gln

450 455

<210>8

<211>458

<212>PRT

＜213〉people

<400>8

Met Ala Ser Pro Ala Leu Ala Ala Ala Leu Ala Val Ala Ala Ala Ala

1 5 10 15

Gly Pro Asn Ala Ser Gly Ala Gly Glu Arg Gly Ser Gly Gly Val Ala

20 25 30

Asn Ala Ser Gly Ala Ser Trp Gly Pro Pro Arg Gly Gln Tyr Ser Ala

35 40 45

Gly Ala Val Ala Gly Leu Ala Ala Val Val Gly Phe Leu Ile Val Phe

50 55 60

Thr Val Val Gly Ash Val Leu Val Val Ile Ala Val Leu Thr Ser Arg

65 70 75 80

Ala Leu Arg Ala Pro Gln Asn Leu Phe Leu Val Ser Leu Ala Ser Ala

85 90 95

Asp Ile Leu Val Ala Thr Leu Val Met Pro Phe Ser Leu Ala Asn Glu

100 105 110

Leu Met Ala Tyr Trp Tyr Phe Gly Gln Val Trp Cys Gly Val Tyr Leu

115 120 125

Ala Leu Asp Val Leu Phe Cys Thr Ser Ser Ile Val His Leu Cys Ala

130 135 140

Ile Ser Leu Asp Arg Tyr Trp Ser Val Thr Gln Ala Val Glu Tyr Asn

145 150 155 160

Leu Lys Arg Thr Pro Arg Arg Val Lys Ala Thr Ile Val Ala Val Trp

165 170 175

Leu Ile Ser Ala Val Ile Ser Phe Pro Pro Leu Val Ser Leu Tyr Arg

180 185 190

Gln Pro Asp Gly Ala Ala Tyr Pro Gln Cys Gly Leu Asn Asp Glu Thr

195 200 205

Trp Tyr Ile Leu Ser Ser Cys Ile Gly Ser Phe Phe Ala Pro Cys Leu

210 215 220

Ile Met Gly Leu Val Tyr Ala Arg Ile Tyr Arg Val Ala Lys Leu Arg

225 230 235 240

Thr Arg Thr Leu Ser Glu Lys Arg Ala Pro Val Gly Pro Asp Gly Ala

245 250 255

Ser Pro Thr Thr Glu Asn Gly Leu Gly Ala Ala Ala Gly Ala Gly Glu

260 265 270

Asn Gly His Cys Ala Pro Pro Pro Ala Asp Val Glu Pro Asp Glu Ser

275 280 285

Ser Ala Ala Ala Glu Arg Arg Arg Arg Arg Gly Ala Leu Arg Arg Gly

290 295 300

Gly Arg Arg Arg Ala Gly Ala Glu Gly Gly Ala Gly Gly Ala Asp Gly

305 310 315 320

Gln Gly Ala Ala Glu Ser Gly Ala Leu Thr Ala Ser Arg Ser Pro Gly

325 330 335

Pro Gly Gly Arg Leu Ser Arg Ala Ser Ser Arg Ser Val Glu Phe Phe

340 345 350

Leu Ser Arg Arg Arg Arg Ala Arg Ser Ser Val Cys Arg Arg Lys Val

355 360 365

Ala Gln Ala Arg Glu Lys Arg Phe Thr Phe Val Leu Ala Val Val Met

370 375 380

Gly Val Phe Val Leu Cys Trp Phe Pro Phe Phe Phe Ser Tyr Ser Leu

385 390 395 400

Tyr Gly Ile Cys Arg Glu Ala Cys Gln Val Pro Gly Pro Leu Phe Lys

405 410 415

Phe Phe Phe Trp Ile Gly Tyr Cys Asn Ser Ser Leu Asn Pro Val Ile

420 425 430

Tyr Thr Val Phe Asn Gln Asp Phe Arg Arg Ser Phe Lys His Ile Leu

435 440 445

Phe Arg Arg Arg Arg Arg Gly Phe Arg Gln

450 455

Claims (70)

1. assess the method that bucindolol is treated the patient for one kind, it comprises: obtain the sequence information about at least a polymorphism in patient's adrenergic receptor gene, wherein said information is the prediction to bucindolol curative effect in the patient.

2. according to the process of claim 1 wherein that the adrenergic receptor gene is β ₁AR or α _2cAR.

3. according to the method for claim 2, wherein the adrenergic receptor gene is β ₁AR.

4. according to the method for claim 2, wherein the adrenergic receptor gene is α _2cAR.

5. according to the method for claim 2, wherein said sequence information comprises or (i) at one or two β of patient ₁The sequence at 1165 Nucleotide places or (ii) at patient β in the encoding sequence in the AR allelotrope ₁Proteinic 389 amino acids of AR, wherein said individuality is just being considered to treat with bucindolol.

6. according to the method for claim 5, wherein the patient is at one or two β ₁The genotype at 1165 Nucleotide places is known in the allelic encoding sequence of AR.

7. according to the method for claim 5, wherein the patient is at two β ₁Genotype in the allelic encoding sequence of AR is known.

8. according to the method for claim 5, wherein at patient's β ₁389 amino acids are known in the AR protein.

9. according to the method for claim 5, also comprise obtaining biological specimen from the patient.

10. according to the method for claim 9, wherein said biological specimen is a blood sample.

11., also comprise obtaining relevant (i) or patient's medical history (ii) according to the method for claim 5.

12., wherein understand (i) or (ii) relate to definite (i) or (ii) according to the method for claim 5.

13. according to the method for claim 12, wherein at one or two β of patient ₁The sequence at 1165 Nucleotide places is determined in the allelic encoding sequence of AR.

14., determine that wherein sequence comprises chain termination sequencing, restrictive diges-tion, allele-specific polymeric enzyme reaction, single-strand conformation polymorphism analysis, hereditary bit analysis, temperature gradient gel elec-trophoresis (TGGE) or ligase chain reaction (LCR) according to the method for claim 13.

15. according to the method for claim 13, wherein the patient is determined to be in one or two β ₁1165 places have cytosine(Cyt) in the allelic encoding sequence of AR.

16. according to the method for claim 15, wherein the patient is determined to be in a β ₁1165 places have cytosine(Cyt) and at another β in the allelic encoding sequence of AR ₁1165 places have guanine in the allelic encoding sequence of AR.

17. according to the method for claim 12, patient β wherein ₁Proteinic 389 amino acids of AR are determined.

18., determine that wherein amino acid comprises application antibody, high pressure liquid chromatography or mass spectroscopy according to the method for claim 17.

19. according to the method for claim 17, wherein the patient is determined to be in β ₁389 places have arginine in the AR protein.

20. according to the method for claim 17, wherein the patient is determined to be in β ₁389 places have glycine in the AR protein.

21. according to the method for claim 17, wherein the patient is defined in some β ₁389 places have arginine and at other β in the AR protein ₁It in the AR protein glycine.

22., also comprise the report of preparing to comprise definite (i) or result (ii) according to the method for claim 12.

23. method according to claim 5, wherein the patient has the symptom of medical conditions or has been suffered from medical conditions by diagnosis, and described medical conditions comprises heart failure, dilated cardiomyopathy, ischemic heart disease, pheochromocytoma, migraine, irregular pulse, hypertension or anxiety disorder.

24. according to the method for claim 23, wherein the patient has the symptom of ischemic heart disease or has been diagnosed as ischemic heart disease.

25. according to the method for claim 24, wherein ischemic heart disease is stenocardia and/or myocardial infarction.

26. according to the method for claim 23, wherein the patient has symptom in heart failure or has been diagnosed as heart failure.

27. according to the method for claim 26, wherein heart failure is the heart failure in late period.

28. according to the method for claim 27, its middle and advanced stage heart failure is NYHA III or the heart failure of IV level.

29. according to the method for claim 5, also be included in understanding (i) or (ii) after prescribe or use bucindolol and give the patient, wherein the patient has the Arg389/Arg389 genotype.

30. according to the method for claim 5, also be included in understanding (i) or (ii) after will not be that the beta blocker of bucindolol is prescribed or is administered to the patient, wherein the patient does not have the Arg389/Arg389 genotype.

31., comprise that also understanding is at (iii) one or two α of patient according to the method for claim 5 _2cIn the 964-975 position place nucleotide sequence or (iv) patient's α in the allelic encoding sequence of AR _2cIn the proteinic 322-325 amino acids of the AR sequence whether disappearance is arranged.

32. according to the method for claim 31, also be included in and bucindolol prescribed after the understanding (iii) or (iv) or be administered to the patient, wherein the patient is α _2cThe homozygous wildtype of AR 322-325 disappearance.

33. according to the method for claim 31, also being included in after the understanding (iii) or (iv) will not be that the beta blocker of bucindolol is prescribed or is administered to the patient, wherein the patient is α _2cThe carrier of AR 322-325 disappearance.

34. according to the method for claim 3, wherein said sequence information comprises that whether (i) is at one or two α of patient _2c964-975 position nucleotide sequence has lacked or (ii) at patient's α in the AR allelotrope _2c322-325 amino acids sequence lacks in the AR protein.

35. according to the method for claim 34, wherein known patient is at its one or two α _2c964-975 position nucleotide sequence has disappearance in the AR allelotrope.

36. according to the method for claim 34, wherein known patient is at two α _2cHas disappearance in the AR allelotrope.

37. according to the method for claim 34, wherein known patient is at α _2cPlace, the proteinic 322-325 of AR position has disappearance.

38., also comprise obtaining biological specimen from the patient according to the method for claim 34.

39. according to the method for claim 38, wherein said biological specimen is a blood sample.

40., also comprise and obtain relevant (i) or patient's medical history (ii) according to the method for claim 34.

41., wherein understand (i) or (ii) relate to definite (i) or (ii) according to the method for claim 34.

42. according to the method for claim 41, wherein at one or two α of patient _2cNucleotide sequence described in the allelic encoding sequence of AR is lacked through definite.

43., determine that wherein sequence comprises chain termination sequencing, restrictive diges-tion, allele-specific polymeric enzyme reaction, single-strand conformation polymorphism analysis, hereditary bit analysis, temperature gradient gel elec-trophoresis (TGGE) or ligase chain reaction (LCR) according to the method for claim 42.

44. according to the method for claim 42, wherein the patient is defined in one or two α _2cThe disappearance that has 964-975 position Nucleotide in the allelic encoding sequence of AR.

45. according to the method for claim 44, wherein the patient is defined in a α _2cHas the disappearance of 964-975 position Nucleotide in the allelic encoding sequence of AR and at another α _2cThe disappearance that does not have 964-975 position Nucleotide in the allelic encoding sequence of AR.

46. according to the method for claim 41, wherein at α _2cThe disappearance of 322-325 amino acids is determined in the AR protein.

47., determine that wherein amino acid comprises application antibody, high pressure liquid chromatography or mass spectroscopy according to the method for claim 46.

48. according to the method for claim 46, wherein the patient is defined in α _2cThe disappearance that has the 322-325 amino acids in the AR protein.

49. according to the method for claim 46, wherein the patient is determined to be in any α _2cThe disappearance that does not have the 322-325 amino acids in the AR protein.

50. according to the method for claim 46, wherein the patient is confirmed as the α of the existing 322-325 of having amino acids disappearance _2cAR protein has the α with 322-325 amino acids disappearance again _2cAR protein.

51., also comprise the report of preparing to comprise definite (i) or result (ii) according to the method for claim 41.

52. method according to claim 34, wherein the patient has the symptom of medical conditions or has been suffered from medical conditions by diagnosis, and described medical conditions comprises heart failure, dilated cardiomyopathy, ischemic heart disease, pheochromocytoma, migraine, irregular pulse, hypertension or anxiety disorder.

53. according to the method for claim 52, wherein the patient has the symptom of ischemic heart disease or has been diagnosed as ischemic heart disease.

54. according to the method for claim 53, wherein ischemic heart disease is stenocardia and/or myocardial infarction.

55. according to the method for claim 52, wherein the patient has symptom in heart failure or has been diagnosed as heart failure.

56. according to the method for claim 55, wherein said heart failure is the heart failure in late period.

57. according to the method for claim 56, its middle and advanced stage heart failure is NYHA III or the heart failure of IV level.

58. according to the method for claim 34, also be included in understanding (i) or (ii) after prescribe or use bucindolol and give the patient, wherein the patient is the homozygous wildtype of Del322-325 polymorphism.

59. according to the method for claim 34, also be included in understanding (i) or (ii) after prescribe or use the beta blocker that is not bucindolol and give the patient, wherein the patient is the Del322-325 carrier.

60., also comprise and understanding or (i) at one or two β of patient according to the method for claim 34 ₁The sequence at 1165 Nucleotide places or (ii) patient's β in the allelic encoding sequence of AR ₁Proteinic 389 amino acids of AR, wherein said individuality is just being considered to treat with bucindolol.

61. according to the method for claim 60, also be included in and prescribe after the understanding (iii) or (iv) or use bucindolol and give the patient, wherein the patient is at its β ₁389 do not have glycine in the AR protein.

62. according to the method for claim 60, also be included in and prescribe after the understanding (iii) or (iv) or use the beta blocker that is not bucindolol and give the patient, wherein the patient is not the homozygote of Arg389 polymorphism.

63. a treatment has the patient's of the heart patient's condition method, it comprises uses the bucindolol of significant quantity or prescribes to the patient, but wherein the patient do not have a detection level be the β of glycine at 389 ₁AR protein.

64. a treatment has the patient's of the heart patient's condition method, it comprises uses the bucindolol of significant quantity or prescribes to the patient, and wherein the patient is at two β ₁1165 cytosine(Cyt)s in the AR allelotrope nucleotide coding sequence are homozygotes.

65. whether an assess heart failure patient will be to the method for bucindolol active responding, it comprises:

A) obtain indication i) one or two β of patient ₁The existence of polymorphism or ii) at β on 1165 coding sites in the allelic encoding sequence of AR ₁The information of the existence of the proteinic 389 amino acids place polymorphisms of AR; With

B) prescribe or use bucindolol.

66. the method with bucindolol treatment patient, it comprises:

A) obtain indication i) one or two β of patient ₁The existence of polymorphism or ii) at β on 1165 coding sites in the allelic encoding sequence of AR ₁The information of the existence of the proteinic 389 amino acids place polymorphisms of AR; With

B) or to described individuality open the bucindolol treatment prescription, wherein patient's genotype is β ₁The proteinic Arg389 homozygote of AR; Open the bucindolol prescription perhaps for described individuality, wherein patient's genotype is not β ₁The proteinic Arg389 homozygote of AR.

67. a treatment has the patient's of the heart patient's condition method, it comprises uses the bucindolol of significant quantity or prescribes to the patient, but wherein the patient does not have the α with 322-325 position disappearance of detection level _2cAR protein.

68. a treatment has the patient's of the heart patient's condition method, it comprise with significant quantity be not that the beta blocker of bucindolol is used or prescribed to the patient, wherein the patient is at α _2cAmong the AR Del322-325 carrier.

69. whether an assess heart failure patient will be to the method for bucindolol active responding, it comprises:

A) obtain indication i) at one or two α _2cThe disappearance of corresponding 964-975 position residue or ii) at α in the AR allelotrope _2cThe information of the disappearance of corresponding 322-325 position residue in the AR protein; With

B), so just prescribe or use bucindolol if described patient is the carrier of Del322-325 polymorphism.

70. the method with bucindolol treatment patient, it comprises:

A) obtain indication i) at one or two α _2cThe disappearance of 964-975 position nucleic acid or ii) at α in the AR allelotrope _2cThe information of the disappearance of corresponding 322-325 position residue in the AR protein; With

B) or to described individuality open the bucindolol treatment prescription, wherein patient's genotype is for α _2cDel322-325 is a homozygous wildtype in the AR protein; Open the bucindolol prescription perhaps for described individuality, wherein patient's genotype is for α _2cAR protein is the Del322-325 carrier.

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