WO2021169845A1 - 神经调节蛋白用于预防、治疗或延缓心力衰竭的方法和组合物 - Google Patents

神经调节蛋白用于预防、治疗或延缓心力衰竭的方法和组合物 Download PDF

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WO2021169845A1
WO2021169845A1 PCT/CN2021/076772 CN2021076772W WO2021169845A1 WO 2021169845 A1 WO2021169845 A1 WO 2021169845A1 CN 2021076772 W CN2021076772 W CN 2021076772W WO 2021169845 A1 WO2021169845 A1 WO 2021169845A1
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heart failure
neuregulin
treatment
probnp
nrg
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PCT/CN2021/076772
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English (en)
French (fr)
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周明东
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上海泽生科技开发股份有限公司
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Priority to AU2021225283A priority Critical patent/AU2021225283A1/en
Priority to US17/799,252 priority patent/US20230083149A1/en
Priority to JP2022550720A priority patent/JP2023514738A/ja
Priority to CA3172439A priority patent/CA3172439A1/en
Priority to KR1020227032863A priority patent/KR20220145870A/ko
Priority to EP21761756.2A priority patent/EP4115899A4/en
Priority to CN202180015952.9A priority patent/CN115515620A/zh
Publication of WO2021169845A1 publication Critical patent/WO2021169845A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1883—Neuregulins, e.g.. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/4756—Neuregulins, i.e. p185erbB2 ligands, glial growth factor, heregulin, ARIA, neu differentiation factor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/58—Atrial natriuretic factor complex; Atriopeptin; Atrial natriuretic peptide [ANP]; Brain natriuretic peptide [BNP, proBNP]; Cardionatrin; Cardiodilatin
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to the application of neuregulin in the preparation of a medicine for preventing, treating or delaying human heart failure, and a method of using the medicine for preventing, treating or delaying human heart failure.
  • the present invention provides a method for preventing, treating or delaying human heart failure by using a drug containing neuregulin in a special population suffering from heart failure.
  • the present invention provides a method for treating heart failure using a drug containing neuregulin. The method involves first testing the patient before treatment, and then providing appropriate treatment based on the test results. When the test result is in an optimal treatment range, the patient is suitable to treat heart failure by administering an effective dose of neuregulin.
  • Heart failure is a syndrome of cardiac insufficiency caused by various heart diseases.
  • the current drugs for the treatment of heart failure mainly focus on angiotensin converting enzyme (ACE) inhibitors. These vasodilators cause blood vessels to dilate, lower blood pressure and reduce the load on the heart.
  • ACE angiotensin converting enzyme
  • the percentage decrease in mortality after the use of ACE inhibitors is statistically different, the actual mortality rate is only an average decrease of 3%-4%, and there are some potential side effects.
  • Other options for preventing or treating heart failure also have corresponding limitations. For example, heart transplantation is obviously more expensive and more invasive than medical treatment, and it is further limited by the presence or absence of a donor heart.
  • the use of mechanical devices, such as a biventricular pacemaker is also invasive and relatively expensive. Therefore, due to the lack of current treatment methods, people need new treatment methods.
  • neuregulin neuroregulin
  • NRG neuregulin
  • HRG glial growth factor
  • GGF neuregulin
  • NDF new differentiation factor
  • the neuregulin family contains 4 members: NRG1, NRG2, NRG3, and NRG4 (Falls et al., Exp Cell Res. 284:14-30, 2003).
  • NRGs involve a series of biological reactions: stimulate breast cancer cells to differentiate and secrete milk protein; induce neural crest cells to differentiate into Schwann cells; stimulate skeletal muscle cells to synthesize acetylcholine receptors; and promote cardiomyocyte survival and DNA synthesis.
  • NRG1 plays an important role in the nervous system, heart, and breast.
  • NRG1 signaling plays a role in the development and function of other organ systems and the pathogenesis of human diseases, including schizophrenia and breast cancer. There are many isomers of NRG1.
  • mice studies on genetically mutated mice (knockout mice) show that different isoforms in the N-terminal region or epidermal growth factor (EGF) similar regions have different functions in vivo.
  • the present invention is based on neuregulin 1 ⁇ (NRG1 ⁇ ).
  • Neuromodulin 1 ⁇ is a transmembrane protein (Holmes et al., Science 256, 1205-1210, 1992).
  • the outer part of the membrane is the N-terminal, including the immunoglobulin-like domain (Ig-like domain) and the EGF-like domain (EGF-like domain), and the inner part is the C-terminal.
  • Ig-like domain immunoglobulin-like domain
  • EGF-like domain EGF-like domain
  • the extra-membrane part of neuregulin can be cleaved off by enzymes to be in a free state, which facilitates the binding of ErbB receptors on the surface of surrounding cells and activates the corresponding cell signal transmission.
  • ErbB receptor family is also divided into four categories, ErbB1, ErbB2, ErbB3 and ErbB4, all of which are transmembrane proteins with molecular weights around 180-185KD. Except for ErbB2, they all contain a ligand binding region at the N-terminus of the membrane; except for ErbB3, they all contain protein tyrosine kinase activity at the C-terminus of the membrane. Among them, ErbB1 is the receptor of epidermal growth factor, and both ErbB3 and ErbB4 are receptors of neuregulin. Among neuregulin receptors, only ErbB2 and ErbB4 are highly expressed in the heart (Yarden et al., Nat Rev Mol Cell Biol, 2: 127-137, 2001).
  • the inner membrane of the body is phosphorylated (Yarden et al., Nat Rev Mol Cell Biol, 2:127-137, 2001).
  • the phosphorylated membrane can further bind to a variety of signaling proteins in the cell, thereby activating the downstream ERK or AKT signaling pathway, causing a series of cellular responses: including stimulation or inhibition of cell proliferation, cell apoptosis, cell migration, and cell differentiation Or cell adhesion.
  • Neuromodulin is particularly important for the development of the heart (WO0037095, CN1276381, WO03099300, WO9426298, US6444642, WO9918976, WO0064400, Zhao et al., J. Biol. Chem. 273, 10261-10269, 1998).
  • the expression of neuregulin is mainly restricted to the endocardium, and then it is released into the surrounding cardiomyocytes through the paracrine pathway and binds to the extramembrane part of the protein tyrosine kinase receptor ErbB4 on the cell membrane. ErbB4 then forms with ErbB2 Heterodimer.
  • WO0037095 shows that a certain concentration of neuregulin can continuously activate the ERK signaling pathway, promote the growth and differentiation of cardiomyocytes, guide the reconstruction of the sarcomere and cytoskeleton of cardiomyocytes and cell adhesion, improve the structure of cardiomyocytes, and enhance the contraction of cardiomyocytes.
  • WO0037095 and WO003099300 also pointed out that neuromodulin can be used to detect, diagnose and treat various cardiovascular diseases.
  • Cardiac muscle function and management WO0037095; 2. New applications of growth factor neuromodulin and its analogues: CN1276381; 3. Neuregulin based methods and composition for treating cardiovascular diseases: WO03099300; 4. Zhao YY, Sawyer DR, Baliga RR, Opel DJ, Han X, Marchionni MA and Kelly RA.Neuregulins Promote Survival and Growth of Cardiac Myocytes.J.Biol.Chem.273, 10261-10269 ); 5. Methods for treating muscle disease and disorder: WO9426298; 6.
  • NRG-1 neurotrophic factor-1 ⁇
  • NRG-1 ⁇ neuregulin-1 ⁇
  • ErbB2 can form heterodimers with ErbB3 or ErbB4 and its affinity is higher than ErbB3 or ErbB4 homodimers.
  • the results of neurodevelopment studies suggest that the formation of the sympathetic nervous system requires a complete NRG-1 ⁇ , ErbB2 and ErbB3 signaling system. Targeted destruction of NRG-1 ⁇ , or ErbB2 or ErbB4 leads to embryonic death due to heart development defects.
  • the present invention provides a method for treating heart failure using drugs containing neuregulin. The method involves first testing the patient before treatment, and then providing appropriate treatment based on the test results. When the test result is in an optimal treatment range, the patient is suitable to treat heart failure by administering an effective dose of neuregulin.
  • the first aspect of the present invention provides a drug component containing an effective dose of neuregulin for the treatment of heart failure, and patients who receive the drug component treatment can achieve significant curative effects.
  • the efficacy is a significant reduction in mortality.
  • the therapeutic effect is a significant reduction in readmission treatment.
  • the efficacy is a reduction in the level of biomarkers that characterize improvement in chronic heart failure.
  • the drug component is administered to the patient through a course of introduction.
  • the introductory course of dosing regimen includes continuous administration of the pharmaceutical component for at least 3, 5, 7 or 10 days.
  • the administration of the drug component is maintained for at least 3, 6 or 12 months after the end of the administration regimen of the introductory course.
  • the maintenance course dosing regimen includes administration of the drug component every 3, 5, 7 or 10 days.
  • the heart failure patient treated is a female.
  • the treated heart failure patient is a female heart failure patient whose plasma level of NT-proBNP does not exceed 3000 fmol/ml before treatment and a male heart failure patient whose plasma level of NT-proBNP does not exceed 1600 fmol/ml before treatment patient.
  • the heart failure patient to be treated is a female heart failure patient whose plasma level of NT-proBNP before treatment does not exceed 3000 fmol/ml.
  • Another aspect of the present invention provides a method for improving survival rate or reducing mortality for patients with chronic heart failure, including administering a drug component containing an effective dose of neuregulin to patients with chronic heart failure.
  • the pharmaceutical component is administered to the patient through an introductory course.
  • the introductory course of administration regimen includes continuous administration of the pharmaceutical component for at least 3, 5, 7 or 10 days.
  • the administration of the drug component is maintained for at least 3, 6 or 12 months after the end of the administration regimen of the introductory course.
  • the maintenance course dosing regimen includes administration of the drug component every 3, 5, 7 or 10 days.
  • the heart failure patient treated is a female.
  • the treated heart failure patient is a female heart failure patient whose plasma level of NT-proBNP does not exceed 3000 fmol/ml before treatment and a male heart failure patient whose plasma level of NT-proBNP does not exceed 1600 fmol/ml before treatment patient.
  • the heart failure patient to be treated is a female heart failure patient whose plasma level of NT-proBNP before treatment does not exceed 3000 fmol/ml.
  • the drug can provide long-term curative effects for patients with chronic heart failure.
  • long-term efficacy refers to improved survival.
  • long-term efficacy refers to reducing the rate of readmissions.
  • long-term efficacy refers to the improvement (decrease) of biomarkers that can characterize the long-term prognosis of chronic heart failure.
  • the drug is administered to the patient through an introductory course.
  • the introductory course dosing regimen includes administering the drug for at least 3, 5, 7 or 10 consecutive days.
  • the patient is given maintenance dosing for at least 3, 6 or 12 months.
  • the maintenance course dosing regimen includes administration of the drug every 3, 5, 7 or 10 days.
  • the heart failure patient treated is a female.
  • the treated heart failure patient is a female heart failure patient whose plasma level of NT-proBNP does not exceed 3000 fmol/ml before treatment and a male heart failure patient whose plasma level of NT-proBNP does not exceed 1600 fmol/ml before treatment patient.
  • the heart failure patient to be treated is a female heart failure patient whose plasma level of NT-proBNP before treatment does not exceed 3000 fmol/ml.
  • a method for screening patients who are suitable for treating heart failure with neuregulin includes detecting the patient's plasma level of NT-proBNP.
  • the plasma level of NT-proBNP does not exceed 4000 fmol/ml, indicating that the patient is suitable for treating heart failure with neuregulin.
  • the NT-proBNP plasma level of 1600 fmol/ml to 4000 fmol/ml indicates that the patient is suitable for treating heart failure with neuregulin.
  • the plasma level of NT-proBNP does not exceed 1600 fmol/ml, indicating that the patient is suitable for treating heart failure with neuregulin.
  • female patients with heart failure whose plasma level of NT-proBNP does not exceed 3000 fmol/ml are suitable for treating heart failure with neuregulin.
  • female heart failure patients with NT-proBNP plasma levels not exceeding 3000 fmol/ml and male heart failure patients with NT-proBNP plasma levels not exceeding 1600 fmol/ml are suitable for treating heart failure with neuregulin.
  • any female heart failure patient with plasma level of NT-proBNP is suitable for treating heart failure with neuregulin.
  • a method for screening patients who are suitable for treating heart failure with neuregulin involves the use of the New York Heart Association (NYHA) heart function classification method to assess heart function.
  • NYHA Class II indicates that the patient is suitable for treating heart failure with neuregulin.
  • NYHA Class III indicates that the patient is suitable for treating heart failure with neuregulin.
  • Another aspect of the present invention provides a method of using neuregulin to treat chronic heart failure.
  • the method includes an evaluation procedure before treatment and a decision on whether the patient is suitable for neuregulin treatment based on the evaluation result.
  • the assessment program includes NYHA cardiac function ratings for patients with chronic heart failure.
  • the evaluation procedure includes measuring the plasma NT-proBNP or BNP level of each patient with chronic heart failure.
  • the diagnostic kit includes an immunoassay reagent for measuring the plasma level of NT-proBNP in patients with heart failure, where a level of not more than 4000 fmol/ml indicates that the patient is suitable for treating heart failure with neuregulin.
  • the NT-proBNP plasma level of 1600 fmol/ml to 4000 fmol/ml indicates that the patient is suitable for treating heart failure with neuregulin.
  • the plasma level of NT-proBNP does not exceed 1600 fmol/ml, indicating that the patient is suitable for treating heart failure with neuregulin.
  • female patients with heart failure whose plasma level of NT-proBNP does not exceed 3000 fmol/ml are suitable for treating heart failure with neuregulin.
  • N-terminal brain natriuretic peptide precursor (NT-proBNP) is used as a biomarker for diagnostic tests.
  • a level of no more than 4000 fmol/ml indicates that the patient is suitable for treating heart failure with neuregulin.
  • a level of 1600 fmol/ml to 4000 fmol/ml indicates that the patient is suitable for treating heart failure with neuregulin.
  • the plasma level of NT-proBNP does not exceed 1600 fmol/ml, indicating that the patient is suitable for treating heart failure with neuregulin.
  • female patients with heart failure whose plasma level of NT-proBNP does not exceed 3000 fmol/ml are suitable for treating heart failure with neuregulin.
  • the companion diagnostic kit includes a kit for detecting plasma NT-proBNP or BNP levels and instructions on how to use the kit and how to judge whether a patient is suitable for neuregulin treatment based on the test results.
  • a medicine kit in another aspect of the present invention, includes one or more containers containing an effective therapeutic dose of neuregulin.
  • the protein contained therein can be neuregulin alone or in a composition with other pharmaceutically acceptable substances.
  • the preferred pharmaceutical form can be combined with sterile physiological saline, glucose solution, buffer solution or other pharmaceutically acceptable sterile solutions.
  • the composition can be lyophilized or dried.
  • the kit of the present invention further includes a needle or syringe, which is preferably sterile packaged for injection, and/or has a packaged alcohol swab.
  • the kit of the present invention further includes instructions, and the instructions may optionally include a method for a doctor or a patient to use the pharmaceutical composition.
  • the kit of the present invention includes an effective dose of NRG and a diagnostic kit, which includes an immunoassay reagent for measuring the plasma level of NT-proBNP in patients with heart failure.
  • a drug-effective dose of neuregulin is administered to patients with chronic heart failure, and the plasma levels of NT-proBNP in these patients are within a suitable therapeutic range before treatment.
  • the suitable treatment range is no more than 4000 fmol/ml.
  • the suitable treatment range is between 1600 fmol/ml and 4000 fmol/ml.
  • the suitable treatment range is no more than 1600 fmol/ml.
  • the suitable treatment range for female patients with heart failure is no more than 3000 fmol/ml.
  • the plasma level is determined by immunoassay.
  • a drug-effective dose of neuregulin is administered to patients with chronic heart failure, and the specific level of cardiac function of these patients is assessed by the American Heart Association (NYHA) cardiac function classification method.
  • NYHA American Heart Association
  • the specific level of cardiac function is NYHA II.
  • the specific level of cardiac function is NYHA III.
  • neuromodulin or “neuregulin” or “NRG” refers to a protein or polypeptide capable of binding and activating ErbB2, ErbB3, ErbB4 or its heterologous or homodimers, including isoforms of neuregulin , EGF-like functional domains in neuregulin, polypeptides containing neuregulin EGF-like functional domains, mutants or derivatives of neuregulin, and other neuregulin-like gene products capable of activating the above-mentioned receptors.
  • Neuromodulin also includes NRG-1, NRG-2, NRG-3 and NRG-4 proteins, polypeptides, fragments, and complexes with NRG-like functions.
  • the neuregulin is a protein or polypeptide that can bind to and activate ErbB2/ErbB4 or ErbB2/ErbB3 heterodimer.
  • the neuregulin of the present invention is a fragment of the NRG-1 ⁇ 2 isoform, that is, a fragment of amino acids 177-237, which contains an EGF-like functional domain.
  • the amino acid sequence of this fragment is: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ (SEQ ID NO:1).
  • the neuromodulin used in the present invention can activate the above-mentioned receptors and regulate their biological functions, such as stimulating skeletal muscle cells to synthesize acetylcholine receptors; promoting the differentiation, survival and DNA synthesis of cardiomyocytes.
  • Neuromodulin also includes those neuregulin mutants with conservative mutations that do not substantially affect biological functions.
  • Those of ordinary skill in the art are well aware that the mutation of a single amino acid in a non-critical region generally does not cause changes in the biological function of the protein or polypeptide (see Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin /Cummings Pub.co.,p.224).
  • the neuromodulin used in the present invention can be isolated from natural sources, or obtained through recombinant technology, artificial synthesis or other means.
  • epidermal growth factor-like functional domain or “EGF-like analogous region” or “EGF-like functional domain” refers to the neuregulin gene that can bind and activate ErbB2, ErbB3, ErbB4 or its heterologous or homologous two.
  • Polypeptide fragments of the polymer and have structural similarities with the EGF receptor binding region described in the following references: WO 00/64400; Holmes et al., Science, 256:1205-1210 (1992); U.S. Patent Nos. 5,530,109 and 5,716,930; Hijazi et al., Int. J.
  • the EGF-like domain binds to and activates ErbB2/ErbB4 or ErbB2/ErbB3 heterodimers.
  • the EGF-like domain comprises amino acids of the receptor binding region of NRG-1.
  • the EGF-like domain refers to amino acids 177-226, 177-237, or 177-240 of NRG-1.
  • the EGF-like domain comprises amino acids of the receptor binding region of NRG-2. In certain embodiments, the EGF-like domain comprises amino acids of the receptor binding region of NRG-3. In certain embodiments, the EGF-like domain comprises amino acids of the receptor binding region of NRG-4. In some embodiments, the EGF-like functional domain includes the amino acid sequence described in US Patent 5,834,229: Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn.
  • the dosage form, dosage and route of administration of neuregulin, and better pharmaceutical composition form can be determined according to methods known in the art (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, 1997.4; Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Banga, 1999; Pharmaceutical Formulation Development of Peptides and Proteins, Hovgaard and Frkjr(Ed.),Francis, Inc. of Liposomes, Lasic and Papahadjopoulos(Ed.), Elsevier Science, 1998; Textbook of Gene Therapy, Jain, Hogrefe & Huber Publishers, 1998; Adenoviruses: Basic Biology to Gene Therapy, Vol.
  • the neuregulin can be formulated for oral administration, rectal administration, topical administration, inhalation administration, oral administration (such as sublingual administration), parenteral administration (such as subcutaneous injection, intramuscular injection, skin Intravenous injection or intravenous injection), transdermal administration or other suitable administration methods.
  • oral administration such as sublingual administration
  • parenteral administration such as subcutaneous injection, intramuscular injection, skin Intravenous injection or intravenous injection
  • transdermal administration or other suitable administration methods.
  • the most suitable route of administration depends on the nature and severity of the condition to be treated, as well as the nature of the special neuregulin protein used.
  • the neuregulin can be administered alone. Or more suitably, neuregulin may be co-administered with some pharmaceutically acceptable carriers or excipients. Any suitable pharmaceutically acceptable carrier or excipient can be used in the current method (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997
  • neuregulin alone or different preparations made by combining it with other media, carriers, and excipients are suitable for various appropriate administration routes, such as intracavernous sinus injection, subcutaneous injection, and intravenous injection , Intramuscular injection, intradermal injection, oral or oral administration.
  • the method can be a unit-dose injection, packed in an ampoule or multi-dose container, with preservatives added.
  • the dosage form can adopt suspension, solution, emulsion in water or oil medium, and can contain suspending agent, stabilizer and/or dispersing agent.
  • the active ingredient may be in powder form and dissolved in a suitable medium, such as sterile non-pyrogenic water or other solvents, before use.
  • local administration can be made into foam, gel, ointment, transdermal patch or paste, etc.
  • the pharmaceutically acceptable components and corresponding administration modes used in the present invention include, but are not limited to, the contents disclosed in U.S. Patent Nos. 5,736,154; 6,197,801B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374B1; and 5,686,102.
  • the dose of the drug used for treatment or prevention will vary depending on the severity of the patient and the route of administration.
  • the dosage, or frequency of administration also varies with the age, weight, condition, and response of each patient.
  • the attending doctor should know how and when to terminate, interrupt or adjust the therapeutic dose to a low dose based on toxicity or adverse reactions. On the contrary, if the clinical response is insufficient, the doctor should also know how and when to adjust the treatment to a high level.
  • Dosage forms include tablets, lozenges, cachets, dispersions, suspensions, solutions, capsules, patches, etc., see “Remington's Pharmaceutical Sciences”.
  • neuregulin alone is used as the active substance or with other drug carriers and excipients, such as ⁇ -cyclodextrin, 2-hydroxy-propyl- ⁇ -cyclodextrin, and is formulated according to common drugs Technology is combined into a mixture.
  • the required carrier form is also different.
  • parenteral dosage forms such as intravenous injection or perfusion
  • pharmaceutical vehicles well known to those skilled in the art, such as water, ethylene glycol, oil, buffer, sugar, preservatives, liposomes, etc.
  • Similar parenteral components include, but are not limited to 5% w/v glucose, physiological saline or other solutions.
  • the neuregulin protein alone, or its combination with other substances, is made into an intravenous injection solution and contained in a medicine bottle with a capacity ranging from 1ml to 2000ml. The volume of the diluent will not be used depending on the total dose administered.
  • the present invention also provides a kit to implement the dosing regimen of the present invention.
  • the kit includes one or more containers containing an effective therapeutic dose of neuregulin.
  • the protein contained therein can be neuregulin alone or in a composition with other pharmaceutically acceptable substances.
  • the preferred pharmaceutical form can be combined with sterile physiological saline, glucose solution, buffer solution or other pharmaceutically acceptable sterile solutions.
  • the composition can be lyophilized or dried.
  • the kit may optionally further contain a pharmaceutically acceptable solution, preferably a sterile solution, which is formulated with the composition when injection is required.
  • Typical pharmaceutically acceptable solutions are physiological saline and glucose solutions.
  • the kit of the present invention further includes a needle or syringe, which is preferably sterile packaged for injection, and/or has a packaged alcohol swab.
  • the instructions may optionally include the method for the doctor or patient to use the drug component.
  • treatment refers to any way that can improve or change the symptoms of discomfort, disorder, or disease.
  • the effect can be preventive, completely or partially preventing the occurrence of a certain disease or its symptoms, or therapeutic, partially or completely curing a certain disease and/or the adverse effects caused by the disease.
  • Treatment also includes any pharmaceutical use of the compositions described herein.
  • Heart failure refers to an abnormality of heart function. In this case, the heart cannot supply blood according to the needs of tissue metabolism.
  • Heart failure includes various disease states such as congestive heart failure, myocardial infarction, arrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic cardiomyopathy and myocarditis.
  • Heart failure can be caused by a variety of factors, including but not limited to ischemic, congenital, rheumatic, viral, toxic, or idiopathic heart failure.
  • Chronic cardiac hypertrophy is a significant pre-congestive heart failure disease state, which can cause cardiac arrest.
  • protein used herein has the same meaning as “polypeptide” and “peptide”.
  • long-term therapeutic effect refers to the therapeutic effect caused by treatment or intervention that may not be observed in the short term.
  • the long-term effect may be to improve survival, reduce the rate of hospitalization and retreatment, or improve biomarkers to characterize long-term prognosis.
  • the time period for observing the therapeutic effect is about 6 months. In some embodiments, the time period for observing the therapeutic effect is about 1 year. In some embodiments, the time period for observing the therapeutic effect is about 2 years. In other embodiments, the time period for observing the therapeutic effect is about 3 years, 5 years, 10 years or longer.
  • survival refers to the time or probability that the body maintains vitality or survival, and can be expressed in terms of survival time or survival rate.
  • Survival time refers to the time period from the beginning of diagnosis or treatment to the end of life.
  • Survival rate refers to the percentage of patients who survive the diagnosis or treatment within a period of time. For a single individual, prolonging survival time after treatment or intervention can be regarded as a curative effect. For a group of individuals or a large number of people, prolonging the average survival time or improving the survival rate is regarded as a curative effect.
  • re-admission treatment refers to the number or frequency of patients who need to go to the hospital for treatment within a certain period of time.
  • the admission may be caused by various conditions, or only by the same conditions being treated.
  • reducing the number of readmissions within a certain period of time can be regarded as curative.
  • reducing the total number of readmissions or the average time for treatment is regarded as a curative effect.
  • N-terminal brain natriuretic peptide or “NT-proBNP” refers to the inactive N-terminal brain natriuretic peptide (BNP) precursor fragment
  • pro-BNP is the precursor of BNP
  • BNP is a kind of hormone
  • Related active natriuretic peptides are mainly released by the cardiomyocytes of the left ventricular wall. Under the expansion and contraction of the myocardial wall, the pro-hormone proBNP is cleaved by protease to split into BNP and inactive NT-proBNP.
  • NT-proBNP In the daily management of suspected or confirmed heart failure, measuring the plasma levels of BNP and NT-proBNT is a very promising method. Most of the clinical applications of BNP and NT-proBNP have proposed their diagnostic characteristics, and there is more and more evidence to support the prognostic value of BNP and NT-proBNP. Since the half-life of NT-proBNP in the blood is 6 times that of BNP, it is a more widely used diagnostic or prognostic indicator of heart failure.
  • the level of NT-proBNP in plasma can be analyzed by commercial kits. For illustration and not limitation, commercial kits are from Roche or Biomedica.
  • Both BNP and NT-proBNP levels in plasma can be used for screening, diagnosing heart failure, and are very useful for establishing the prognosis of heart failure, because these two markers are typically elevated in patients with worsening conditions.
  • the present invention found that the level of BNP or NT-proBNP can indicate patients who are suitable for treating heart failure with neuregulin.
  • the "New York Heart Association” or “NYHA” cardiac function classification used here is a simple way to classify the degree of heart failure. It divides patients into 4 categories according to the degree of limitation of their physical activity. Restrictions or symptoms refer to normal breathing and varying degrees of dyspnea and/or angina: I, asymptomatic and unrestricted ordinary activity, such as dyspnea when walking, climbing stairs, etc.; II, mild symptoms (mild) Difficulty breathing and/or angina) and ordinary activities are slightly restricted; III, physical activity is significantly restricted, even in the case of less than normal activity, for example, short distance walking (20-100m), a sense of rest and fashion Comfortable; IV, severely restricts the amount of activity, even when resting, there are symptoms, most patients are bedridden.
  • I asymptomatic and unrestricted ordinary activity, such as dyspnea when walking, climbing stairs, etc.
  • II mild symptoms (mild) Difficulty breathing and/or angina) and ordinary activities are
  • activity unit or "EU” or “U” refers to the amount of standard product that can induce 50% of the maximum activity response.
  • the EC50 in order to determine the unit of activity of an active agent, the EC50 must be determined. For example, if the EC50 of a product is 0.1 ⁇ g, then this amount is 1 unit. Furthermore, if 1 ⁇ g of this product is used, 10EU (1/0.1) is used.
  • the EC50 can be determined by any method known in the art, including the method used by the inventors in the following examples. The determination of active units is important for the quality control of genetic engineering products and clinically used drugs, so that different drugs and/or different batches of products can be quantified with the same standard.
  • NRG-1 used in the present invention. It mainly uses NRG to bind and activate the ErbB3 and ErbB4 receptors on the cell surface, and indirectly lead to The characteristics of ErbB2 receptor phosphorylation (see Michael D. Sadick et al., 1996, Analytical Biochemistry, 235:207-214 and WO03/099300).
  • the method is called kinase receptor activation enzyme-linked immunosorbant assay (KIRA-ELISA), which mainly includes two separate microplates.
  • One of the microplates is used for cell culture, ligand stimulation, cell lysis and receptor lysis.
  • the other microplate is used for receptor capture and phosphorylation ELISA detection.
  • This method is used to detect the ErbB2 receptor activation level of the adherent breast cancer cell line MCF-7 induced by NRG protein.
  • Triton-X100 lysate to solubilize membrane proteins, in which the free ErbB2 receptor is captured by the ErbB2 specific antibody that does not cross-react with ErbB3 or ErbB4 in the ELISA well, and is detected by anti-phosphotyrosine ELISA
  • the phosphorylation level of ErbB2 receptor Prepare a repeatable standard curve, the EC50 value of heregulin ⁇ 1(177-244) is approximately 360pM.
  • KIRA-ELISA method and quantitative anti-phosphotyrosine Western Blot method it can be found that the results measured by these two methods are very closely related.
  • the analysis method described here can specifically quantify the level of ErbB2 tyrosine phosphorylation, which is the result of the interaction of the HRG protein with ErbB3 and/or ErbB4 receptors.
  • the amount of standard that can cause 50% of the maximum response is defined as 1 activity unit (1EU). Therefore, different drugs or different batches of products can be quantified using uniform standards.
  • Example 1 Randomized, double-blind, multi-center, standard treatment-based placebo parallel-controlled evaluation of the survival study of recombinant human neuregulin on patients with chronic heart failure (209 trial)
  • excipient of rhNRG-1 (250 ⁇ g/piece, does not contain the active ingredient of recombinant human neuregulin)
  • the entry criteria for clinical trials include patients with chronic heart failure aged 18-65 years, left ventricular ejection fraction (LVEF) ⁇ 40% (NYHA heart function class III or IV), and stable clinical symptoms (including clinical symptoms and signs) And receiving standard basic treatment for heart failure has reached the target dose or the maximum tolerated dose for more than 1 month).
  • the main exclusion criteria include acute myocardial infarction, hypertrophic cardiomyopathy, constrictive pericarditis, obvious valve disease or congenital heart disease, severe pulmonary hypertension, systolic blood pressure ⁇ 90mmHg or >160mmHg, severe ventricular arrhythmia, the first 6 Those who have undergone cardiac surgery or cerebrovascular accident within months, claustrophobic or pregnant women. All patients need to understand and sign an informed consent form.
  • Example 2 Multi-center, randomized, double-blind, placebo parallel-controlled evaluation of the survival study of recombinant human neuregulin on patients with chronic heart failure based on standard treatment (301 trial)
  • rhNRG-1 recombinant human neuregulin for injection
  • excipient of rhNRG-1 (250 ⁇ g/piece, does not contain the active ingredient of recombinant human neuregulin)
  • the inclusion criteria for clinical trials include patients with chronic heart failure aged 18-80 years, left ventricular ejection fraction (LVEF) ⁇ 40% (NYHA heart function class III or IV), and stable clinical symptoms (including clinical symptoms and signs) And receiving standard basic treatment for heart failure has reached the target dose or the maximum tolerated dose for more than 1 month).
  • the main exclusion criteria include acute myocardial infarction, hypertrophic cardiomyopathy, constrictive pericarditis, obvious valve disease or congenital heart disease, severe pulmonary hypertension, systolic blood pressure ⁇ 90mmHg or >160mmHg, severe ventricular arrhythmia, the first 6 Those who have undergone cardiac surgery or cerebrovascular accident within months, claustrophobic or pregnant women. All patients need to understand and sign an informed consent form.
  • Example 3 Multi-center, randomized, double-blind, placebo parallel-controlled evaluation of the survival study of recombinant human neuregulin on patients with chronic heart failure based on standard treatment (305 trial)
  • excipient of rhNRG-1 (250 ⁇ g/piece, does not contain the active ingredient of recombinant human neuregulin)
  • the entry criteria for clinical trials include patients with chronic heart failure aged 18-75 years, left ventricular ejection fraction (LVEF) ⁇ 40% (NYHA heart function class II or III), and stable clinical symptoms (including clinical symptoms and signs) And receiving standard basic treatment for heart failure has reached the target dose or the maximum tolerated dose for more than 1 month).
  • the main exclusion criteria include acute myocardial infarction, hypertrophic cardiomyopathy, constrictive pericarditis, obvious valve disease or congenital heart disease, severe pulmonary hypertension, systolic blood pressure ⁇ 90mmHg or >160mmHg, severe ventricular arrhythmia, the first 6 Those who have undergone cardiac surgery or cerebrovascular accident within months, claustrophobic or pregnant women. All patients need to understand and sign an informed consent form.
  • the combined analysis data of three independent trials involving all female subjects including NYHA II-IV patients with mild, moderate, and severe
  • the relative all-cause mortality of the subjects was reduced by more than 50%.
  • all subjects in the female medication group showed a survival benefit, consistent with the trend of the combined analysis of the three trials.
  • the combined analysis showed that the lower the baseline NT-proBNP level of subjects during the study period, the greater the survival benefit (relative reduction in all-cause mortality); the NT-proBNP level did not exceed 3000fmol/mL in the baseline period Among female subjects, the mortality rate of female subjects in the medication group was 0%, while the all-cause mortality rate in the placebo control group based on standard treatment was nearly 10%. The survival benefit of subjects was statistically significant (P ⁇ 0.001). Each independent trial also showed a survival benefit trend for female subjects consistent with the combined analysis.
  • LVEF left ventricular ejection fraction
  • N total analysis
  • n number of specific categories
  • NYHA Heart Failure Classification of the New York Society of Cardiology
  • LVEF left ventricular ejection fraction
  • N total analysis
  • n number of specific categories
  • NYHA Heart Failure Classification of the New York Society of Cardiology
  • the female subjects with NT-proBNP ⁇ 3000fmol/ml and male subjects with NT-proBNP ⁇ 1600fmol/ml in the three clinical trials were analyzed individually and collectively, as shown in Table 4.
  • the mortality results of the rhNRG-1 treatment group were significantly better than those of placebo.

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Abstract

本发明提供了纽兰格林蛋白在制备用于预防、治疗或延缓人类心力衰竭的药物中的应用，以及所述用于预防、治疗或延缓人类心力衰竭的药物的使用方法。该方法包含在治疗之前首先对患者进行测试，所述测试包括检测NT-proBNP或BNP的血浆水平，然后根据测试结果提供恰当的治疗。当测试结果在一个最佳治疗范围时，患者适合通过施用有效剂量的纽兰格林来治疗心力衰竭。

Description

神经调节蛋白用于预防、治疗或延缓心力衰竭的方法和组合物 技术领域
本发明涉及纽兰格林蛋白在制备用于预防、治疗或延缓人类心力衰竭的药物中的应用，以及所述用于预防、治疗或延缓人类心力衰竭的药物的使用方法。特别地，本发明提供了预防、治疗或延缓人类心力衰竭的方法，该方法是在患有心力衰竭的特殊人群中使用包含纽兰格林蛋白的药物。特别地，本发明提供了一种使用包含纽兰格林的药物治疗心力衰竭的方法。该方法包含在治疗之前首先对患者进行测试，然后根据测试结果提供恰当的治疗。当测试结果在一个最佳治疗范围时，患者适合通过施用有效剂量的纽兰格林来治疗心衰。
发明背景
心力衰竭(heart failure，HF)是各种心脏疾病导致心功能不全的一种综合征。目前治疗心衰的药物主要集中于血管紧张素转化酶(ACE)抑制剂，这些血管舒张剂引起血管扩张、降低血压并减少心脏负荷。虽然使用ACE抑制剂后死亡率的百分数下降是具有统计学差异的，但实际的死亡率仅平均下降3％-4％，并且还有一些潜在的副作用。其他预防或治疗心衰的选择亦有相应的局限。比如，心脏移植显然比药物治疗更昂贵且更具侵入性，并且还进一步受有无供体心脏的限制。使用机械装置，比如双心室心脏起搏器，同样具有侵入性并且比较昂贵。因此，由于当前治疗手段的不足，人们需要有新的治疗方法。
一种很有前途的新的治疗手段涉及给心衰患者或有心衰风险的患者施用神经调节蛋白(neuregulin，NRG，纽兰格林；heregulin，HRG)，又叫神经胶质生长因子(glial growth factor,GGF),neu分化因子(new differentiation factor,NDF),为分子量在44KD左右的糖蛋白，它们在细胞间传递信号，是酪氨酸激酶受体ErbB家族的配体。神经调节蛋白家族含4个成员：NRG1，NRG2，NRG3，NRG4(Falls et al.,Exp Cell Res.284:14-30,2003)。NRGs涉及一系列生物学反应：刺激乳腺癌细胞分化和分泌乳汁蛋白；诱导神经嵴细胞分化成Schwann细胞；刺激骨骼肌细胞合成乙酰胆碱受体；并且促进心肌细胞成活和DNA合成。用纽兰格林基因严重缺陷的纯合子小鼠胚胎做活体研究证明纽兰格林对于心脏和神经发育是必须的。NRG1在神经系统、心脏和乳腺中起着重要作用，还有证据显示NRG1信号传递在其他一些器官系统的发育、功能以及人类疾病(包括精神分裂症和乳腺癌)的发病机理中起作用。NRG1有很多异构体。对基因突变小鼠(基因敲除小鼠)的研究说明在N末端区或表皮生长因子(EGF)类似区不同的异构体，其在体功能也不一样。本发明是以神经调节蛋白1β(NRG1β)为基础的。
神经调节蛋白1β为一跨膜蛋白(Holmes et al.,Science 256,1205-1210,1992)。膜外部分是N末端，包括免疫球蛋白类似区(Ig-like domain)和EGF类似区(EGF-like domain)，膜内部分是C末端。在细胞外基质的金属蛋白酶作用下，神经调节蛋白的膜外部分可被酶切下来而呈游离状态，从而有利于和周围细胞表面的ErbB受体结合，激活相应的细胞信号传递。
ErbB受体家族也分为四类，ErbB1、ErbB2、ErbB3和ErbB4，它们都是跨膜蛋白，分子量在180-185KD附近。除ErbB2外，它们在膜外的N末端都含配体结合区；除ErbB3外，它们在膜内的C末端都含蛋白 酪氨酸激酶活性。其中ErbB1是表皮生长因子的受体，ErbB3和ErbB4都是神经调节蛋白的受体。在神经调节蛋白的受体中，只有ErbB2和ErbB4在心脏表达量较高(Yarden et al.,Nat Rev Mol Cell Biol,2:127-137,2001)。
当神经调节蛋白与ErbB3或ErbB4的膜外部分结合时，将引起ErbB3、ErbB4与其他ErbB受体(常常包括ErbB2)形成异源二聚体，或ErbB4自身形成同源二聚体，然后导致受体的膜内部分被磷酸化(Yarden et al.,Nat Rev Mol Cell Biol,2:127-137,2001)。磷酸化的膜内部分可进一步与细胞内的多种信号传递蛋白结合，从而激活下游ERK或AKT信号通路，引起一系列细胞反应：包括刺激或抑制细胞增殖、细胞凋亡、细胞迁移、细胞分化或细胞粘连。
神经调节蛋白对心脏的发育尤其重要(WO0037095，CN1276381，WO03099300，WO9426298，US6444642，WO9918976，WO0064400，Zhao et al.,J.Biol.Chem.273,10261-10269,1998)。在胚胎发育早期，神经调节蛋白的表达主要局限于心内膜，随后通过旁分泌途径释放到周围心肌细胞并与细胞膜上的蛋白酪氨酸激酶受体ErbB4膜外部分结合，ErbB4进而与ErbB2形成异源二聚体。ErbB4/ErbB2复合物的形成及激活对早期海绵样心脏形成小梁是必须的。神经调节蛋白、ErbB4和ErbB2三个蛋白基因中的任何一个缺失都会使胚胎没有小梁并在发育早期死于子宫。WO0037095显示一定浓度的神经调节蛋白可持续激活ERK信号通路，促进心肌细胞的生长及分化，引导心肌细胞和细胞粘连处肌节和细胞骨架的重建，改善心肌细胞的结构，增强心肌细胞的收缩。WO0037095及WO003099300还指出神经调节蛋白可用于检测、诊断和治疗各种心血管疾病。
下面列举了与本发明有关的一些现有技术文献：1.Cardiac muscle function and manipulation:WO0037095；2.生长因子神经调节蛋白及其类似物的新应用：CN1276381；3.Neuregulin based methods and composition for treating cardiovascular diseases:WO03099300；4.Zhao YY,Sawyer DR,Baliga RR,Opel DJ,Han X,Marchionni MA and Kelly RA.Neuregulins Promote Survival and Growth of Cardiac Myocytes.J.Biol.Chem.273,10261-10269(1998)；5.Methods for treating muscle diseases and disorder:WO9426298；6.Methods of increasing myotube formation or survival or muscle cell mitogenesis,differentiation or survival using a neuregulin:US6444642.7.Therapeutic methods comprising use of a neuregulin:WO9918976；8.Methods for treating congestive heart failure:WO0064400；9.Holmes WE,Sliwkowski MX,Akita RW,Henzel WJ,Lee J,Park JW,Yansura D,Abadi N,Raab H,Lewis GD,et al.Identification of heregulin,a specific activator p185erbB2.Science 256,1205-1210(1992)；10.Falls DL.Neuregulins:functions,forms and signalingstrategies.Experimental Cell Research,284,14-30(2003).11.Yarden Y,Sliwkowski X.Untangling the ErbB signaling Network.Nature Reviews:Molecular Cell Biology,2127-137(2001).
已有研究表明NRG-1的EGF样类似区，约50至64个氨基酸，足以结合并活化这些受体。以前的研究表明纽兰格林-1β(NRG-1β)能以高亲和力直接结合ErbB3和ErbB4。孤儿受体ErbB2能与ErbB3或ErbB4形成异源二聚体并且其亲和力比ErbB3或ErbB4同源二聚体要高。神经发育的研究结果提示交感神经系统的形成需要完整的NRG-1β、ErbB2和ErbB3信号传导系统。靶向破坏NRG-1β、或ErbB2或ErbB4后由于心脏发育缺陷而导致胚胎致死。最近的研究也突显了NRG-1β、ErbB2和ErbB4在心血管发育以及维持成年正常心脏功能方面具有重要作用。研究表明NRG-1β能增强成年心肌细胞的肌小节的组织结构。 施用一种重组的NRG-1βEGF样类似区能够显著改善或防止不同心衰动物模型的心肌功能的恶化，在临床试验中也有同样效果。但是如何使用这种产品或组合物，如何在一些亚人群上取得更好的治疗效果，还需要更多的研究。本发明提供了一种使用包含纽兰格林的药物治疗心力衰竭的方法。该方法包含在治疗之前首先对患者进行测试，然后根据测试结果提供恰当的治疗。当测试结果在一个最佳治疗范围时，患者适合通过施用有效剂量的纽兰格林来治疗心衰。
发明内容
A.发明概述
在纽兰格林治疗心衰的临床试验中，申请人发现通过纽约心脏协会(NYHA)心功能分级或者检测患者血浆中NT-proBNP或BNP水平筛选出来的心衰病人经纽兰格林治疗后能够取得显著的疗效。这些疗效包括死亡率显著下降。这些疗效包括再入院治疗显著减少。NRG还能够显著改善或防止不同心衰动物模型和临床试验受试者的心肌功能恶化。纽兰格林，纽兰格林多肽，纽兰格林突变体，纽兰格林衍生物,具有NRG样功能域的复合物或者具有纽兰格林类似活性的化合物都属于本发明的范畴。
本发明的第一方面，提供了一种用于治疗心力衰竭的包含有效剂量神经调节蛋白的药物组分，并且接受该药物组分治疗的患者能够取得显著疗效。在一些实施例中，疗效是死亡率显著下降。在一些实施例中，疗效是再入院治疗显著减少。在一些实施例中，疗效是表征慢性心力衰竭改善的生物标记物水平降低在一些实施例中，药物组分通过导入疗程施用于患者。在一些实施例中，导入疗程给药方案包括连续施用该药物组分至少3,5,7或10天。在一些优选的实施例中，导入疗程给药方案结束后，维持施用该药物组分至少3,6或12个月。在一些优选的实施例中，维持疗程给药方案包括每3,5,7或10天施用该药物组分。在一个优选的实施例中，治疗的心衰患者是女性。在一个优选的实施例中，治疗的心衰患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者和治疗前NT-proBNP的血浆水平不超过1600fmol/ml的男性心衰患者。在一个优选的实施例中，治疗的心衰患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者。
本发明的另一方面，为慢性心衰患者提供了一种提高生存率或降低死亡率的方法，包括对慢性心衰病人施用一种含有效剂量神经调节蛋白的药物组分。在一些实施例中，药物组分通过导入疗程施用于患者。在一些优选的实施例中，导入疗程给药方案包括连续施用该药物组分至少3,5,7或10天。在一些优选的实施例中，导入疗程给药方案结束后，维持施用该药物组分至少3,6或12个月。在一些优选的实施例中，维持疗程给药方案包括每3,5,7或10天施用该药物组分。在一个优选的实施例中，治疗的心衰患者是女性。在一个优选的实施例中，治疗的心衰患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者和治疗前NT-proBNP的血浆水平不超过1600fmol/ml的男性心衰患者。在一个优选的实施例中，治疗的心衰患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者。
本发明的另一方面，提供了纽兰格林蛋白在药物制备方面的应用。该药物能为慢性心力衰竭患者提供长期疗效。在一个实施例中，长期疗效指提高生存率。在一个实施例中，长期疗效指减少再入院率。在另一个实施例中，长期疗效指能够表征慢性心衰长期预后的生物标记物的改善(降低)。在一些实施例中，该药物通过导入疗程施用于患者。在一些优选的实施例中，导入疗程给药方案包括至少连续3,5,7或10 天施用该药物。在一些优选的实施例中，导入疗程给药方案结束后，对病人进行至少3,6或12个月的维持给药。在一些优选的实施例中，维持疗程给药方案包括每3,5,7或10天施用该药物。在一个优选的实施例中，治疗的心衰患者是女性。在一个优选的实施例中，治疗的心衰患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者和治疗前NT-proBNP的血浆水平不超过1600fmol/ml的男性心衰患者。在一个优选的实施例中，治疗的心衰患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者。
本发明的另一方面，提供了一种筛选适合用纽兰格林治疗心衰的患者的方法。该方法包括检测患者的NT-proBNP血浆水平。在一个实施例中，NT-proBNP血浆水平不超过4000fmol/ml表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，NT-proBNP血浆水平为1600fmol/ml到4000fmol/ml表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，NT-proBNP血浆水平不超过1600fmol/ml表明该患者适合用纽兰格林治疗心衰。在另一个优选的实施例中，NT-proBNP血浆水平不超过3000fmol/ml的女性心衰患者适合用纽兰格林治疗心衰。在另一个优选的实施例中，NT-proBNP血浆水平不超过3000fmol/ml的女性心衰患者和NT-proBNP血浆水平不超过1600fmol/ml的男性心衰患者适合用纽兰格林治疗心衰。在另一个优选的实施例中，任何NT-proBNP血浆水平的女性心衰患者适合用纽兰格林治疗心衰。
本发明的另一方面，提供了一种筛选适合用纽兰格林治疗心衰的患者的方法。该方法包括用纽约心脏协会(NYHA)心功能分级法来评估心脏功能。在一个实施例中，NYHA II级表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，NYHA III级表明该患者适合用纽兰格林治疗心衰。
本发明的另一方面，提供了一种使用纽兰格林治疗慢性心力衰竭的方法。该方法包括治疗前的评估程序和根据该评估结果决定患者是否适合接受纽兰格林治疗。在一些实施例中，评估程序包括慢性心衰患者的NYHA心脏功能评级。在另一个实施例中，评估程序包括检测每一个慢性心衰病人的血浆NT-proBNP或BNP水平。
本发明的另一方面，提供了一种诊断试剂盒，用来筛选适合用纽兰格林治疗的心衰患者。在一个实施例中，该诊断试剂盒包含用于测量心衰患者NT-proBNP血浆水平的免疫分析试剂，其中不超过4000fmol/ml的水平表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，NT-proBNP血浆水平为1600fmol/ml到4000fmol/ml表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，NT-proBNP血浆水平不超过1600fmol/ml表明该患者适合用纽兰格林治疗心衰。在另一个优选的实施例中，NT-proBNP血浆水平不超过3000fmol/ml的女性心衰患者适合用纽兰格林治疗心衰。
本发明的另一方面，为纽兰格林治疗慢性心衰提供了一个伴随的诊断测试。N-末端脑利钠肽前体(NT-proBNP)作为诊断测试的生物标记物。在一些实施例中，不超过4000fmol/ml的水平表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，1600fmol/ml到4000fmol/ml的水平表明该患者适合用纽兰格林治疗心衰。在另一个实施例中，NT-proBNP血浆水平不超过1600fmol/ml表明该患者适合用纽兰格林治疗心衰。在另一个优选的实施例中，NT-proBNP血浆水平不超过3000fmol/ml的女性心衰患者适合用纽兰格林治疗心衰。
本发明的另一方面，提供了一种能够确定心衰病人是否适合接受纽兰格林治疗的伴随诊断试剂盒。该 伴随诊断试剂盒包含一个检测血浆NT-proBNP或BNP水平的试剂盒和一个如何使用该试剂盒以及如何根据试验结果判断患者是否适合接受纽兰格林治疗的说明书。
本发明的另一方面，提供了一种药盒。这种药盒包括一个或多个含有效治疗剂量纽兰格林蛋白的容器，其中所含蛋白可为单独的纽兰格林蛋白，或与其他药物可接受物质形成组合物。优选的药物形式可与无菌生理盐水、葡萄糖溶液、缓冲溶液或其他药物可接受的无菌溶液结合。作为一种选择，组合物可被冻干或干燥。在一个实施例中，本发明的药盒进一步包括一个针头或注射器，其最好为无菌包装，用于注射，和/或有一个包装好的酒精棉块。在另一个实施例中，本发明的药盒进一步包括说明书，说明书可视情况包括医生或病人使用该药物组份的方法。在另一个优选的实施例中，本发明的药盒包含有效剂量的NRG和诊断试剂盒，该诊断试剂盒包含用于测量心衰患者NT-proBNP血浆水平的免疫分析试剂。
本发明的另一方面，对慢性心衰病人施用药物有效剂量的神经调节蛋白，治疗之前这些患者的NT-proBNP血浆水平处在合适的治疗范围内。在一个实施例中，合适的治疗范围是不超过4000fmol/ml。在另一个实施例中，合适的治疗范围是在1600fmol/ml至4000fmol/ml之间。在另一个实施例中，合适的治疗范围是不超过1600fmol/ml。在另一个优选的实施例中，女性心衰患者合适的治疗范围是不超过3000fmol/ml。在另一个优选的实施例中，通过免疫分析来测定血浆水平。
本发明的另一方面，对慢性心衰病人施用药物有效剂量的神经调节蛋白，这些患者的心功能具体等级是由美国心脏协会(NYHA)心功能分级方法所评定的。在一些实施例中，心功能的特定级别为NYHA II级。在一些实施例中，心功能的特定级别为NYHA III级。
B.定义
除另有定义，这里使用的所有科技术语与本发明所属技术领域的普通技术人员理解含义相同。所有专利文献、专利申请文献、公开的专利文献和其它出版物均作为参考。如本节阐述的定义与上述参考文献所述的定义不一致或相反时，以本节阐述的定义为准。
除非在上下文中明确表明，否则本说明书及其所附权利要求书中，所用的单数形式“一个”、“一种”和“该”包括相应的复数参考，并与“至少一个”或“一个或多余一个”可互换使用。
此处所用“神经调节蛋白”或“neuregulin”或“NRG”是指能够结合并激活ErbB2、ErbB3、ErbB4或其异源或同源二聚体的蛋白或多肽，包括神经调节蛋白的异构体、神经调节蛋白中的EGF样功能域、包含神经调节蛋白EGF样功能域的多肽、神经调节蛋白的突变体或衍生物以及其它能够激活上述受体的神经调节蛋白样的基因产物。神经调节蛋白还包括NRG-1，NRG-2，NRG-3和NRG-4蛋白、多肽、片段以及具有NRG样功能的复合物。优选的，神经调节蛋白是可以结合并激活ErbB2/ErbB4或ErbB2/ErbB3异源二聚体的蛋白或多肽。作为例子，但并非为了限制的目的，本发明的神经调节蛋白是NRG-1β2异构体的一个片段，即177-237位氨基酸片段，其中包含了EGF样功能域。该片段的氨基酸序列为：SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVMASFYKAEELYQ(SEQ ID NO:1)。本发明所用神经调节蛋白可以激活上述受体并调节它们的生物学功能，比如刺激骨骼肌细胞合成乙酰胆碱受体；促进心肌细胞的分化、存活以及DNA合成。神经调节蛋白还包括那些具有并不实质性影 响生物学功能的保守性突变的神经调节蛋白突变体。本技术领域中普通技术人员熟知，非关键区域的单个氨基酸的突变一般不会引起该蛋白或多肽的生物学功能的改变(参见Watson等人,Molecular Biology of the Gene,4th Edition,1987,The Bejacmin/Cummings Pub.co.,p.224)。本发明所用神经调节蛋白可以从天然的来源分离得到，或者通过重组技术、人工合成或其它手段得到。
此处所用“表皮生长因子样功能域”或“EGF样类似区”或“EGF样功能域”是指由neuregulin基因所编码的可以结合并激活ErbB2、ErbB3、ErbB4或其异源或同源二聚体的多肽片段，并且与下述参考文献中描述的EGF受体结合区域具有结构相似性：WO 00/64400；Holmes等，Science,256:1205-1210(1992)；美国专利5,530,109和5,716,930；Hijazi等，Int.J.Oncol.,13:1061-1067(1998)；Chang等，Nature，387:509-512(1997)；Carraway等，Nature，387:512-516(1997)；Higashiyama等，J.Biochem.,122:675-680(1997)；以及WO 97/09425。在某些实施方案中，EGF样功能域结合并激活ErbB2/ErbB4或ErbB2/ErbB3异源二聚体。在某些实施方案中，EGF样功能域包含NRG-1的受体结合区氨基酸。在某些实施方案中，EGF样功能域是指NRG-1的第177-226位、177-237位或177-240位氨基酸。在某些实施方案中，EGF样功能域包含NRG-2的受体结合区氨基酸。在某些实施方案中，EGF样功能域包含NRG-3的受体结合区氨基酸。在某些实施方案中，EGF样功能域包含NRG-4的受体结合区氨基酸。在某些实施方案中，EGF样功能域包含美国专利5,834,229中描述的氨基酸序列：Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro。
纽兰格林蛋白的剂型、剂量和给药途径、更好的药物组合物形式，能够根据本领域已知的方法来确定(参见例如Remington:The Science and Practice of Pharmacy,Alfonso R.Gennaro(Editor)Mack Publishing Company,1997.4；Therapeutic Peptides and Proteins:Formulation,Processing,and Delivery Systems,Banga,1999；Pharmaceutical Formulation Development of Peptides and Proteins,Hovgaard and Frkjr(Ed.),Taylor & Francis,Inc.,2000；Medical Applications of Liposomes,Lasic and Papahadjopoulos(Ed.),Elsevier Science,1998；Textbook of Gene Therapy,Jain,Hogrefe & Huber Publishers,1998；Adenoviruses:Basic Biology to Gene Therapy,Vol.15,Seth,Landes Bioscience,1999；Biopharmaceutical Drug Design and Development,Wu-Pong和Rojanasakul(Ed.),Humana Press,1999；Therapeutic Angiogenesis:From Basic Science to the Clinic,Vol.28,Dole等(Ed.),Springer-Verlag New York,1999)。
纽兰格林蛋白，可被制成口服给药、直肠给药、局部给药、吸入给药、口腔给药(如舌下给药)、非肠道给药(如皮下注射、肌肉注射、皮内注射或静脉注射)、经皮给药或者其他合适的给药方式的制剂。在所有给药方式中，最合适的给药途径取决于治疗状况的性质和严重程度，以及所使用特殊纽兰格林蛋白的性质。纽兰格林蛋白可以单独给药。或者更适宜的，纽兰格林蛋白可以与一些药物可接受的载体或赋形剂共同给药。任何适宜的药物可接受载体或赋形剂能够用于目前的方法(参见如Remington:The Science and Practice of Pharmacy,Alfonso R.Gennaro(Editor)Mack Publishing Company,April 1997)。
根据本发明，单独的纽兰格林蛋白或其与其他介质、载体、赋形剂结合所制成的不同制剂，适用于各种适当的给药途径，例如海绵窦内注射，皮下注射，静脉注射，肌肉注射，皮内注射，口服或口腔给药。方法可采用单位剂型的注射剂，装在安瓶或多剂量容器中，添加防腐剂。剂型可采用混悬液、溶液、在水或油介质中的乳剂，并且可包含悬浮剂、稳定剂和/或分散剂。或者，活性成分可以为粉末状，在使用前用 一种合适的介质，如无菌无热源水或其他溶剂溶解。本发明中局部给药可以制成泡沫，凝胶，药膏，透皮贴剂或糊剂等。
本发明所采用的药物可接受组分和相应的给药方式包括但不限于美国专利5,736,154；6,197,801B1；5,741,511；5,886,039；5,941,868；6,258,374B1；和5,686,102所公布的内容。
用于治疗或预防的药物剂量大小会由于患者的严重程度和给药途径的不同而不同。给药剂量，或给药频率，也因每个病人的年龄、体重、条件和反应不同而不同。
需要注意的是，主治医生应该知道如何及何时根据毒性或不良反应去终止、中断或调整治疗剂量至低剂量。相反的，如果临床反应不足，医生也应该知道如何及何时去调整治疗至高水平。
任何适合的给药途径都能够被使用。剂型包括片剂，锭剂，扁囊剂，分散剂，混悬剂，溶液，胶囊，贴剂等等，参见《Remington’s Pharmaceutical Sciences》。在实际应用中，单独的纽兰格林蛋白作为活性物质或其与其他药物载体、赋形剂，如β-环糊精，2-羟基-丙基-β-环糊精，按照常见的药物配制技术组合成混合物。根据给药途径不同，局部给药或非胃肠道用药，所需要的载体形式也不同。在制备非胃肠道给药剂型中，如静脉注射或灌注，可采用本领域技术人员所熟知的药物媒介，如水，乙二醇，油，缓冲液，糖，防腐剂，脂质体等。类似的非胃肠道组分包括，但不限于5％w/v葡萄糖，生理盐水或其他溶液。单独的纽兰格林蛋白，或其与其他物质形成的组合物，被制成静脉注射液盛于药瓶中，容量范围从1ml到2000ml不等。稀释液的容量将根据给药的总剂量不同而不用。
本发明也提供了一种药盒来实施本发明的给药方案。这种药盒包括一个或多个含有效治疗剂量纽兰格林蛋白的容器，其中所含蛋白可为单独的纽兰格林蛋白，或与其他药物可接受物质形成组合物。优选的药物形式可与无菌生理盐水、葡萄糖溶液、缓冲溶液或其他药物可接受的无菌溶液结合。作为一种选择，组合物可被冻干或干燥。在这种情况下，药盒可视情况进一步包含一种药物可接受的溶液，最好为无菌溶液，当需要注射时，与组合物配成溶液。典型的药物可接受溶液是生理盐水和葡萄糖溶液。
在另一个实施例中，本发明的药盒进一步包括一个针头或注射器，其最好为无菌包装，用于注射，和/或有一个包装好的酒精棉块。说明书可视情况包括医生或病人使用该药物组份的方法。
此处所用的“治疗”或“处理”是指可以使不适、紊乱或疾病的症状改善或向好的方向改变的任何方式。其效果可以是预防性的，完全或部分防止某一疾病或其症状发生，也可以是治疗性的，部分或完全治愈某一疾病和/或该疾病引起的不利影响。治疗还包括此处所述组合物的任何药物用途。
此处所用“心力衰竭”是指一种心脏功能的异常，在这种情况下，心脏不能按照组织代谢的需要进行供血。心力衰竭包括多种疾病状态比如充血性心力衰竭，心肌梗死，心律失常，家族性肥厚性心肌病，缺血性心脏病，特发性心肌病和心肌炎等之类的。心力衰竭可由多种因素导致，包括但不限于缺血性，先天性，风湿性，病毒性、毒性或特发性心力衰竭。慢性心脏肥大是一种显著的充血性心力衰竭前期的疾病状态，其可以引起心脏停搏。
除上下文另有说明，此处所用“蛋白”与“多肽”、“肽段”含义一致。
除上下文另有说明，此处所用“血浆”与“血清”含义一致。
除上下文另有说明，此处所用“不超过”是指“小于等于”。
此处所用“长期疗效”是指短期内可能无法观察到的由治疗或干预所引起的疗效。对于慢性心力衰竭患者而言，长期疗效可能是提高生存率，减少入院再治疗率，或是表征长期预后的生物标记物改善。在一些实施例中，观察疗效的时间期限约为6个月。在一些实施例中，观察疗效的时间期限约为1年。在一些实施例中，观察疗效的时间期限约为2年。在其他的实施例中，观察疗效的时间期限约为3年，5年，10年或更长。
此处所用“存活”是指机体保持活力或生存的时间或几率，可以用生存时间或生存率来表达。生存时间是指从诊断或治疗开始到生命结束的时间期限。生存率是指在诊断或治疗的一段时间期限内存活下来的患者百分比。对于单个个体来说，经治疗或干预后延长生存时间可被视为疗效。对于一组个体或大量人群来说，延长平均生存时间或提高生存率被视为疗效。
此处所用“再入院治疗”是指在一定时期内病人需要去医院治疗的次数或频率。入院可能是由各种条件引起的，或者仅由正在治疗中的同样条件引起的。对于每个个体来说，在一定时期内减少再入院治疗的次数可被认为是疗效。对于一组个体或大量人群来说，减少再入院治疗的总次数或平均时间被视为疗效。
此处所用“N端脑利钠肽”或“NT-proBNP”是指无活性的N端脑利钠肽(BNP)前体片段，pro-BNP是BNP的前体，BNP是一种与激素有关的活性钠尿肽，主要由左心室壁的心肌细胞释放。在心肌壁的伸缩作用下，前激素proBNP经过蛋白酶切，分裂成BNP和无活性的NT-proBNP。
在怀疑或确定心衰的日常处理中，测定BNP和NT-proBNT的血浆水平是非常有前景的手段。大多数有关BNP和NT-proBNP在临床上的应用提出了它们的诊断特性，并有越来越多的证据可以支持BNP和NT-proBNP的预后价值。由于NT-proBNP在血液中的半衰期是BNP的6倍，所以它是使用更广泛的心衰诊断或预后指标。血浆中的NT-proBNP水平可以由商业化药盒分析。为了举例说明而非限制，商业化药盒来自Roche或Biomedica公司。
BNP和NT-proBNP在血浆中的水平都可以用作筛选、诊断心衰，并且对建立心衰的预后非常有用，这是由于这两种标记物在病情恶化的患者身上典型升高。并且，本发明发现BNP或NT-proBNP水平可以指示适合用纽兰格林治疗心衰的患者。
此处所用“纽约心脏协会”或“NYHA”心脏功能分级是一种简单的划分心力衰竭程度的方法。它根据患者身体活动能力的限制程度将患者分为4类。限制或症状是指正常呼吸和不同程度的呼吸困难和/或心绞痛：I，无症状并且普通的活动量不受限制，例如当走路，爬楼梯等时呼吸困难；II,轻度症状(轻度呼吸困难和/或心绞痛)并且普通的活动受到轻度的限制；III,体力活动显著受限制，甚至在少于正常活动量的情况下，例如，短距离行走(20-100m)，休息时尚感舒适；IV，严重限制活动量，即使在休息时也有症状，大多数病人卧床不起。
此处所用“活性单位”或“EU”或“U”是指能诱导50％最大活性反应的标准产品的用量。换句话说，为了测定某一活性制剂的活性单位，必须测定EC50。例如，如果某一产品的EC50是0.1μg，则这个 量就是1个单位。进一步讲，如果使用了1μg该产品，就是使用了10EU(1/0.1)。可以用本领域已知的任何方法来测定EC50，包括下面的实施例中发明人所使用的方法。活性单位的测定对于基因工程产品和临床使用药物的质量控制是重要的，这样可以使不同药品和/或不同批次的产品以同样的标准来定量。
以下简要描述本发明中所采用的一种快速、敏感、高通量并定量的检测NRG-1生物活性的方法，其主要利用NRG可以结合并激活细胞表面的ErbB3和ErbB4受体，并间接导致ErbB2受体磷酸化的特性(参见Michael D.Sadick等,1996,Analytical Biochemistry,235:207-214以及WO03/099300)。
简而言之，该方法名为激酶受体活化酶联免疫吸附试验(kinase receptor activation enzyme-linked immunosorbant assay，KIRA-ELISA)，其主要包括两块分开的微孔板。其中一块微孔板进行细胞培养、配体刺激、细胞裂解和受体溶解。而另一微孔板则进行受体捕获和磷酸化ELISA检测。该方法用于检测NRG蛋白诱导的贴壁的乳腺癌细胞系MCF-7的ErbB2受体活化水平。用Triton-X100裂解液来溶解膜蛋白，其中游离的ErbB2受体被包被在ELISA孔中的与ErbB3或ErbB4无交叉反应的ErbB2特异性抗体捕获，并通过抗磷酸酪氨酸ELISA检测所捕获的ErbB2受体的磷酸化水平。制出有重复性的标准曲线，heregulin β 1(177-244)的EC50值大约为360pM。当相同的样品分别用KIRA-ELISA法和定量的抗磷酸酪氨酸Western Blot法检测ErbB2受体的磷酸化水平时，可以发现这两种方法所测的结果具有非常紧密的关联性。此处所描述的分析方法可具体地量化ErbB2酪氨酸磷酸化水平，该磷酸化是HRG蛋白同ErbB3和/或ErbB4受体作用的结果。
由于大多数基因工程药物是蛋白质或多肽类，它们的活性可通过其氨基酸序列或空间结构组成的活性中心所决定。蛋白质或多肽类的活性效价与它们的绝对量并不一致，所以不能像化学药物一样用重量单位来计量。然而，基因工程药物的生物活性一般同药效学和效价确定方法一致，该方法可以通过给定的生物活性确定其效价单位。因此，确定生物活性是定量活性物质过程的一部分，同时也是基因工程产物的质量控制的重要组成部分。生物活性标准的建立对于基因工程产物和临床用药的质量控制是相当重要的。
能引起最大反应50％的标准品的用量被定义为1个活性单位(1EU)。因此，不同的药物或不同批次的产品能用统一的标准来定量。
具体实施方式
实施例1：随机、双盲、多中心、标准治疗基础上的安慰剂并行对照评价重组人纽兰格林对慢性心力衰竭患者的生存研究(209试验)
为了评价注射用重组人纽兰格林(rhNRG-1，
Figure PCTCN2021076772-appb-000001
)对慢性心力衰竭的疗效，在中国的多个临床中心进行了双盲、多中心、标准治疗基础上的安慰剂并行对照的II期临床试验。共有351位心功能为NYHA III或IV级的慢性心力衰竭入组，并被随机分为安慰组或rhNRG-1组(0.6μg/kg)。各组之间在统计学和背景治疗方面无显著性差异。按照计划，病人在医院连续10天施用药物，第11天允许出院。从第3周到第25周做为门诊病人每周用药一次。采集病人治疗前，和每次用药后的血样。血浆NT-proBNP水平在核心实验室里检测(试剂盒来自Biomedica)。在该研究的第52周采集生存信息。
受试药物：
规格：rhNRG-1，61个氨基酸构成Neuregulin-1β2异构体的EGF类似功能域，分子量为7054Dal(1μg＝0.14nmol)。
安慰剂：
规格：rhNRG-1的赋形剂(250μg/支，不含重组人纽兰格林蛋白活性成分)
给药方案：
  1-10天 3-25周
剂量 0.6μg/kg/day rhNRG-1或安慰剂 0.8μg/kg/day rhNRG-1或安慰剂
给药途径 静脉滴注 静脉输注
疗程 每天10小时，连续10天 每周输注10分钟
临床试验的入组标准包括年龄为18-65岁、左心室射血分数(LVEF)≤40％的慢性心衰患者(NYHA心功能分级III或IV级)，临床症状稳定(包括临床症状、体征和接受心衰标准基础治疗已达目标剂量或最大耐受量1个月以上)。主要的排除标准包括急性心肌梗死，肥厚型心肌病，缩窄性心包炎，明显的瓣膜病变或先天性心脏病，重度肺动脉高压，收缩压<90mmHg或>160mmHg，严重室性心律失常，前6个月内有心脏外科治疗或脑血管意外者，幽闭恐惧症或怀孕女性。所用患者均需理解并签署知情同意书。
实施例2：多中心、随机、双盲、标准治疗基础上的安慰剂平行对照评价重组人纽兰格林对慢性心力衰竭患者的生存研究(301试验)
为了评价注射用重组人纽兰格林(rhNRG-1，
Figure PCTCN2021076772-appb-000002
)对慢性心力衰竭的疗效，在中国的多个临床中心进行了双盲、多中心、标准治疗基础上的安慰剂并行对照的III期临床试验。共有331位心功能为NYHA III或IV级的慢性心力衰竭入组，并被随机分为安慰组或rhNRG-1组(0.6μg/kg)或rhNRG-1组(1.0μg/kg)。各组之间在基线水平背景治疗方面无显著性差异。按照计划，病人在医院连续10天施用药物，第11天允许出院。从第3周到第25周做为门诊病人每周用药一次。采集病人治疗前，和每次用药后的血样。血浆NT-proBNP水平在核心实验室里检测(试剂盒来自Biomedica)。在该研究的第52周采集生存信息。
受试药物：
规格：rhNRG-1，61个氨基酸构成Neuregulin-1β2异构体的EGF类似功能域，分子量为7054Dal(1μg＝0.14nmol)。
安慰剂：
规格：rhNRG-1的赋形剂(250μg/支，不含重组人纽兰格林蛋白活性成分)
给药方案：
Figure PCTCN2021076772-appb-000003
临床试验的入组标准包括年龄为18-80岁、左心室射血分数(LVEF)≤40％的慢性心衰患者(NYHA心功能分级III或IV级)，临床症状稳定(包括临床症状、体征和接受心衰标准基础治疗已达目标剂量或最大耐受量1个月以上)。主要的排除标准包括急性心肌梗死，肥厚型心肌病，缩窄性心包炎，明显的瓣膜病变或先天性心脏病，重度肺动脉高压，收缩压<90mmHg或>160mmHg，严重室性心律失常，前6个月内有心脏外科治疗或脑血管意外者，幽闭恐惧症或怀孕女性。所用患者均需理解并签署知情同意书。
实施例3：多中心、随机、双盲、标准治疗基础上的安慰剂平行对照评价重组人纽兰格林对慢性心力衰竭患者的生存研究(305试验)
为了评价注射用重组人纽兰格林(rhNRG-1，
Figure PCTCN2021076772-appb-000004
)对慢性心力衰竭的疗效，在中国的多个临床中心进行了双盲、多中心、标准治疗基础上的安慰剂并行对照的III期临床试验。共有679位心功能为NYHA II或III级的慢性心力衰竭入组，并被随机分为安慰组或rhNRG-1组(0.6μg/kg)。各组之间在统计学和背景治疗方面无显著性差异。按照计划，病人在医院连续10天施用药物，第11天允许出院。从第3周到第25周做为门诊病人每周用药一次。采集病人治疗前，和每次用药后的血样。血浆NT-proBNP水平在核心实验室里检测(试剂盒来自Biomedica)。在该研究的第52周采集生存信息。
受试药物：
规格：rhNRG-1，61个氨基酸构成Neuregulin-1β2异构体的EGF类似功能域，分子量为7054Dal(1μg＝0.14nmol)。
安慰剂：
规格：rhNRG-1的赋形剂(250μg/支，不含重组人纽兰格林蛋白活性成分)
给药方案：
  1-10天 3-25周
剂量 0.6μg/kg/day rhNRG-1或安慰剂 0.8μg/kg/day rhNRG-1或安慰剂
给药途径 静脉滴注 静脉输注
疗程 每天10小时，连续10天 每周输注10分钟
临床试验的入组标准包括年龄为18-75岁、左心室射血分数(LVEF)≤40％的慢性心衰患者(NYHA心功能分级II或III级)，临床症状稳定(包括临床症状、体征和接受心衰标准基础治疗已达目标剂量或 最大耐受量1个月以上)。主要的排除标准包括急性心肌梗死，肥厚型心肌病，缩窄性心包炎，明显的瓣膜病变或先天性心脏病，重度肺动脉高压，收缩压<90mmHg或>160mmHg，严重室性心律失常，前6个月内有心脏外科治疗或脑血管意外者，幽闭恐惧症或怀孕女性。所用患者均需理解并签署知情同意书。
申请人对三项独立临床试验的数据进行了统计分析。包含全部女性受试者(包括纽约心功能分级NYHA II-IV级的轻、中度与重度患者)的三个独立试验合并分析数据表明：与标准治疗基础上的安慰剂对照组相比，女性用药组受试者获得了统计学显著的生存率获益(P＝0.016)。在研究周期内，受试者相对全因死亡率降低达50％以上。在每个独立试验中，全部女性用药组受试者都显示出生存率获益，与三个试验合并分析的趋势一致。
合并分析显示在研究周期内，整体而言受试者基线期NT-proBNP水平越低，生存获益(全因死亡率相对降低幅度)越大；在基线期NT-proBNP水平不超过3000fmol/mL的女性受试者中，用药组女性受试者死亡率为0％，而标准治疗基础上的安慰剂对照组全因死亡率为近10％，受试者生存获益统计学极其显著(P<0.001)。各独立试验也展现出与合并分析一致的女性受试者生存获益趋势。
临床结果表明，rhNRG-1对轻中度心衰患者更有效的发现与经主动脉缩窄诱发的心衰大鼠模型结果一致。表明当心衰进入偏心肥大和泵血功能衰竭的更严重阶段，ErbB2和ErbB4受体表达明显下调。因此，在心衰的早期阶段，也即ErbB2/ErbB4受体表达下调之前，重组人纽兰格林能为患者带来更大受益。
表1.全因死亡率-亚组分析总结：(对209/301/305研究进行数据汇总分析)
Figure PCTCN2021076772-appb-000005
缩写：LVEF＝左心射血分数；N＝分析总量；n＝特定类别数量；NYHA＝美国纽约心脏病学会心力衰竭分级
分析发现基线NT-proBNP≤1600fmol/ml时，无论男性还是女性受试者，rhNRG-1均显示出统计学上显著降低死亡率(表1)，但这一结论并不适用于具有较高基线NT-proBNP的两性合并数据分析。进一步 分析发现，rhNRG-1在所有接受测试的女性受试者中均显示出统计学上显著降低死亡率(表1)。在单项研究209、301和305中也发现了女性受试者具有相同趋势，如表2所示。
表2.全因死亡率-亚组分析总结：单项临床试验(209/301/305分析)
Figure PCTCN2021076772-appb-000006
缩写：LVEF＝左心射血分数；N＝分析总量；n＝特定类别数量；NYHA＝美国纽约心脏病学会心力衰竭分级
由于女性受试者在汇总研究和单项研究中均显示出更好的死亡率改善，因此基于女性受试者的基线期NT-proBNP百分位数进行了进一步的亚组分析。亚组包括NT-proBNP≤1600fmol/ml(～50％)，NT-proBNP≤3000fmol/ml(～75％)和所有女性患者，对这些亚组进行汇总和单项分析，如表3所示。
在三项临床研究209、301和305的汇总分析中发现，在女性rhNRG-1治疗组患者的三个亚组NT-proBNP≤1600，NT-proBNP≤3000fmol/ml和所有女性用药患者的死亡率分别为0％、0％和7.1％。这三个亚组相对应的安慰剂组的死亡率分别为8.4％，10.4％和16.1％，表明rhNRG-1能显著降低死亡率,尤其是在NT-proBNP≤3000fmol/ml的亚组中，约占注册女性受试者的75％，如表3所示。
在209、301和305单项试验研究分析中发现，女性rhNRG-1治疗组NT-proBNP≤3000fmol/ml亚组的死亡率分别为0％，0％和0％，而相对应的安慰剂组死亡率分别为21.4％，8.3％和6.3％，如表3所示。
对于男性患者，在NT-proBNP≤4000fmol/ml或NT-proBNP≤3000fmol/ml的亚组中，部分临床试验显示，rhNRG-1治疗组的死亡率优于安慰剂组。但更优选的是，对于NT-proBNP≤1600fmol/ml的男性受试者，在三项临床研究209、301和305的汇总分析中，发现rhNRG-1治疗组与安慰剂组相比，治疗组的死亡率大幅降低(2.6％vs 5.6％)，与NT-proBNP≤1600fmol/ml的所有性别受试者的综合分析一致。
表3.全因死亡率-女性亚组分析总结：(209/301/305研究数据汇总分析&单项分析)
Figure PCTCN2021076772-appb-000007
基于以上分析，将三项临床试验中的NT-proBNP≤3000fmol/ml的女性受试者和NT-proBNP≤1600fmol/ml的男性受试者，进行单项和汇总分析，如表4所示。在这些男性和女性组合的受试者中rhNRG-1治疗组的死亡率结果均明显优于安慰剂。
表4.全因死亡率-亚组分析总结：(209/301/305研究数据汇总分析&单项分析)
Figure PCTCN2021076772-appb-000008
上述列举的实施例不会限制本发明的保护范围。在没有背离本发明的宗旨和范围的情况下，本技术领域内的技术人员可以对本发明进行调整和改变。因此，本发明的保护范围应当根据权利要求来定义，而不是通过具体的实施例来限定。

Claims (10)

  1. NRG用于制备治疗心力衰竭的药物的用途，其中心力衰竭患者是治疗前NT-proBNP的血浆水平不超过3000fmol/ml的女性心衰患者。
  2. 根据权利要求1所述的用途，其中所述NRG是NRG-1。
  3. 根据权利要求1所述的用途，其中所述NRG包含NRG-1的EGF样功能域。
  4. 根据权利要求1所述的用途，其中所述NRG包含SEQ ID NO:1的氨基酸序列。
  5. 一种筛选适用于纽兰格林治疗的心衰患者的方法，包含在治疗之前进行诊断测试，根据测试结果决定是否适合用纽兰格林进行治疗。
  6. 根据权利要求5所述的方法，其中所述诊断测试是检测NT-proBNP或BNP的血浆水平。
  7. 根据权利要求6所述的方法，当女性心衰患者治疗前NT-proBNP的血浆水平不超过3000fmol/ml时，测试结果表明适合用纽兰格林治疗。
  8. 一种诊断试剂盒用于筛选适合用纽兰格林治疗的心衰病人，其中诊断试剂盒包含免疫分析试剂用来检测心衰患者的NT-proBNP血浆水平。
  9. 根据权利要求8所述的诊断试剂盒，当女性心衰患者治疗前NT-proBNP的血浆水平不超过3000fmol/ml时，测试结果表明适合用纽兰格林治疗物。
  10. 一种用于治疗心力衰竭的药盒，其中含有如权利要求8所述的诊断试剂盒以及有效剂量的NRG。
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