Compositions and diagnostic kits for the treatment of heart failure

Abstract

To provide a method of treating a patient with chronic heart failure using a medicament comprising neuregulin.SOLUTION: A companion diagnostic test is first performed on each patient prior to treatment, and the appropriate treatment is provided to each patient according to the companion diagnostic test results. If the test results are within a preferred therapeutic range for treatment, the patient is suitable for treatment of heart failure by administration of an effective amount of neuregulin.SELECTED DRAWING: None

Description

Detailed Description of the Invention

[0001] [1. Technical Field] The present invention relates to the use of neuregulin proteins for the preparation of medicaments for the prevention, treatment, or delay of heart failure in humans, and to methods for the prevention, treatment, or delay of heart failure in humans using said medicaments. In particular, the present invention provides methods for the prevention, treatment, or delay of heart failure in specific populations of patients with chronic heart failure using said medicaments comprising neuregulin proteins.

[0002] [2. Background technology] Approximately 5 million Americans suffer from heart failure, with more than 550,000 new cases diagnosed each year. Current drug treatments for heart failure primarily involve angiotensin-converting enzyme (ACE) inhibitors, which are vasodilators that dilate blood vessels, lower blood pressure, and reduce the heart's workload. While mortality rates have declined significantly, the actual mortality reduction with ACE inhibitor use averages only 3–4%, and there are several potential side effects. Additional limitations exist with other options for preventing or treating heart failure. For example, heart transplantation is significantly more expensive and invasive than drug therapy and is further limited by the availability of donor hearts. The use of mechanical devices, such as biventricular pacemakers, is similarly invasive and expensive. Therefore, there is a need for new therapies that take into account the shortcomings of current treatments.

[0003] One promising novel therapeutic approach involves administering neuregulin (NRG) to patients with or at risk for heart failure. NRG is a member of the EGF-like growth factor family, which includes a family of structurally related growth and differentiation factors, including NRG1, NRG2, NRG3, and NRG4, and their isoforms. These factors are involved in a range of biological responses, including stimulating breast cancer cell differentiation and milk protein secretion, inducing neural crest cell differentiation into Schwann cells, stimulating skeletal muscle cell synthesis of acetylcholine receptors, and promoting cardiac cell survival and DNA synthesis. In vivo studies of neuregulin gene-targeted homozygous mouse embryos, which exhibit severe defects in ventricular trabecular formation and dorsal root ganglion development, have demonstrated that neuregulin is essential for cardiac and neural development.

[0004] NRGs bind to the EGF receptor family, which includes EGFR, ErbB2, ErbB3, and ErbB4, each of which plays an important role in multiple cellular functions, such as cell proliferation, differentiation, and survival. These are protein tyrosine kinase receptors, consisting of an extracellular ligand-binding domain, a transmembrane kinase domain, and a cytoplasmic tyrosine kinase domain. After binding to the extracellular domain of ErbB3 or ErbB4, NRGs induce a conformational change, resulting in heterodimerization between ErbB3, ErbB4, and ErbB2, or homodimerization of ErbB4 itself. This leads to phosphorylation of the receptor's C-terminal domain within the plasma membrane. The phosphorylated intracellular domain then binds to additional signaling proteins within the cell, activating the corresponding downstream AKT or ERK signaling pathways, triggering a series of cellular responses, such as stimulation or inhibition of cell proliferation, differentiation, apoptosis, migration, or adhesion. Among these receptors, ErbB2 and ErbB4 are mainly expressed in the heart.

[0005] The EGF-like domain of NRG-1 (50 to 64 amino acids in size) has been shown to be sufficient to bind and activate these receptors. Previous studies have shown that neuregulin-1β (NRG-1β) can directly bind to ErbB3 and ErbB4 with high affinity. The orphan receptor ErbB2 can form heterodimers with ErbB3 and ErbB4 with higher affinity than either ErbB3 or ErbB4 homodimers. Studies in neural development have shown that an intact NRG-1β, ErbB2, and ErbB3 signaling pathway is required for the formation of the sympathetic nervous system. Targeted disruption of NRG-1β or ErbB2 or ErbB4 results in embryonic lethality due to defects in cardiac development. Recent studies have also highlighted the role of NRG-1β, ErbB2, and ErbB4 in cardiovascular development, as well as in maintaining normal cardiac function in adults. NRG-1β has been shown to enhance sarcomere organization in adult myocardium. In clinical trials and in different animal models of heart failure, administration of recombinant NRG-1β EGF-like domain significantly improved or prevented myocardial deterioration. These results highlight the promise of NRG-1 as a potential treatment for heart failure. However, more evidence is still needed about whether treatment with NRG-1 can provide long-term benefits to patients with heart failure and whether these benefits can be provided to all patients with chronic heart failure or to a subset of patients.

[0006] 3. Summary of the Invention In human clinical trials of neuregulin for the treatment of heart failure, applicants discovered that by assessing the New York Heart Association (NYHA) cardiac performance class or by measuring the patient's plasma levels of NT-proBNP or BNP, they could select heart failure patients who would receive significant therapeutic benefit from neuregulin, including a significant reduction in mortality.

[0007] Applicant has discovered that NRG enhances cardiomyocyte differentiation, sarcomere and cytoskeletal organization, and cell adhesion. Applicant has also discovered that NRG significantly improves or significantly protects against the deterioration of myocardial function in clinical trials and in different animal models of heart failure. Neuregulin, neuregulin polypeptides, neuregulin derivatives, or compounds mimicking the activity of neuregulin are within the scope of the present invention.

[0008] Thus, in a first aspect of the present invention, a pharmaceutical composition comprising an effective amount of neuregulin is administered to treat a patient with chronic heart failure, and the patient derives a significant benefit from the pharmaceutical composition. In one embodiment, the benefit is a significant reduction in mortality. In one embodiment, the benefit is a significant reduction in rehospitalization. In one embodiment, the benefit is an improvement in biomarker levels indicating improvement in chronic heart failure. In one embodiment, the pharmaceutical composition is administered to the patient in an induction regimen. In one optimized embodiment, the induction regimen comprises administering the pharmaceutical composition for at least 3, 5, 7, or 10 consecutive days. In one optimized embodiment, the pharmaceutical composition is administered to the patient in a maintenance regimen for at least 3, 6, or 12 months after the induction regimen. In one optimized embodiment, the maintenance regimen comprises administering the pharmaceutical composition every 3, 5, 7, or 10 days.

[0009] In a second aspect, the present invention provides a method for improving survival or reducing mortality in a patient with chronic heart failure, comprising administering to the patient a pharmaceutical composition comprising an effective amount of neuregulin. In one embodiment, the pharmaceutical composition is administered to the patient in an induction regimen. In one optimized embodiment, the induction regimen comprises administering the pharmaceutical composition for at least 3, 5, 7, or 10 consecutive days. In one optimized embodiment, the pharmaceutical composition is administered to the patient in a maintenance regimen for at least 3, 6, or 12 months following the induction regimen. In one optimized embodiment, the maintenance regimen comprises administering the pharmaceutical composition every 3, 5, 7, or 10 days.

[0010] In a third aspect of the present invention, a pharmaceutically effective amount of neuregulin is used to treat a patient with chronic heart failure whose plasma level of NT-proBNP is within a preferred therapeutic range before treatment with neuregulin. In one embodiment, the preferred therapeutic range is 4000 fmol / ml or less. In another embodiment, the preferred therapeutic range is between 1600 fmol / ml and 4000 fmol / ml. In yet another embodiment, the preferred therapeutic range is 1600 fmol / ml or less. In another preferred embodiment, the plasma level is measured by immunoassay.

[0011] In a fourth aspect of the present invention, a therapeutically effective amount of neuregulin is used to treat a patient with chronic heart failure who has been classified into a specific cardiac function class according to the NYHA functional classification. In one embodiment, the specific cardiac function class is NYHA class II. In one embodiment, the specific cardiac function class is NYHA class III.

[0012] In a fifth aspect, the present invention provides a method for selecting a patient with heart failure for treatment with neuregulin. The method comprises measuring the patient's plasma level of NT-proBNP. In one embodiment, a level of 4000 fmol / ml or less indicates a patient's suitability for neuregulin treatment for heart failure. In another embodiment, a level between 1600 fmol / ml and 4000 fmol / ml indicates a patient's suitability for neuregulin treatment for heart failure. In yet another embodiment, a level of 1600 fmol / ml or less indicates a patient's suitability for neuregulin treatment for heart failure.

[0013] In a sixth aspect, the present invention provides a method for selecting a patient with heart failure for treatment with neuregulin. The method includes assessing the patient's cardiac function class according to the NYHA functional classification. In one embodiment, NYHA class II indicates that the patient is suitable for treatment with neuregulin for heart failure. In another embodiment, NYHA class III indicates that the patient is suitable for treatment with neuregulin for heart failure.

[0014] In a seventh aspect, the present invention provides a diagnostic kit for selecting heart failure patients for neuregulin therapy. In one embodiment, the diagnostic kit includes an immunoassay reagent for measuring plasma levels of NT-proBNP in heart failure patients, with a level of 4000 fmol / ml or less indicating a patient's suitability for neuregulin therapy for heart failure. In another embodiment, a level between 1600 fmol / ml and 4000 fmol / ml indicates a patient's suitability for neuregulin therapy for heart failure. In yet another embodiment, a level of 1600 fmol / ml or less indicates a patient's suitability for neuregulin therapy for heart failure.

[0015] In an eighth aspect of the present invention, there is provided a use of a neuregulin protein for preparing a medicament. The medicament can be administered to a patient with chronic heart failure to obtain a long-term benefit. In one embodiment, the long-term benefit is improved survival. In one embodiment, the long-term benefit is reduced rehospitalization. In another embodiment, the long-term benefit is an improvement in a biomarker indicative of long-term prognosis of chronic heart failure. In one embodiment, the medicament is administered to the patient in an induction regimen. In one optimized embodiment, the induction regimen comprises administering the medicament for at least 3, 5, 7, or 10 consecutive days. In one optimized embodiment, the medicament is administered to the patient in a maintenance regimen for at least 3, 6, or 12 months after the induction regimen. In one optimized embodiment, the maintenance regimen comprises administering the medicament every 3, 5, 7, or 10 days.

[0016] In a ninth aspect of the present invention, a companion diagnostic test for treating chronic heart failure with neuregulin protein is provided. N-terminal pro-brain natriuretic peptide (NT-proBNP) is used as a biomarker in the companion diagnostic test. In one embodiment, a level of 4000 fmol / ml or less indicates a patient suitable for neuregulin-based heart failure treatment. In another embodiment, a level between 1600 fmol / ml and 4000 fmol / ml indicates a patient suitable for neuregulin-based heart failure treatment. In yet another embodiment, a level of 1600 fmol / ml or less indicates a patient suitable for neuregulin-based heart failure treatment.

[0017] A tenth aspect of the present invention provides a method for treating chronic heart failure using neuregulin. The method includes a pre-treatment evaluation procedure, and determines whether each patient is suitable for neuregulin treatment according to the evaluation results. In one embodiment, the evaluation procedure includes the NYHA functional classification of the chronic heart failure patient. In another embodiment, the evaluation procedure includes testing plasma NT-proBNP or plasma BNP in each chronic heart failure patient.

[0018] In an eleventh aspect of the present invention, there is provided a companion diagnostic kit for determining whether a patient with chronic heart failure is suitable for neuregulin protein therapy, which comprises a plasma NT-proBNP or plasma BNP test kit and instructions on how to use the kit and how to determine whether a patient is suitable for neuregulin protein therapy according to the test results.

[0019] 4. Detailed Description of the Invention For clarity of disclosure, and not by way of limitation, the following detailed description of the invention is divided into the following sections: All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0020] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, applications, published applications, and other publications mentioned herein are incorporated herein by reference in their entirety. To the extent that a definition set forth in this section contradicts or is inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth in this section shall take precedence over the definition incorporated herein by reference.

[0021] As used herein, the singular forms "a," "an," and "the" mean "at least one" or "one or more," unless the context clearly dictates otherwise.

[0022] As used herein, "neuregulin" or "NRG" refers to a protein or peptide capable of binding to and activating ErbB2, ErbB3, ErbB4, or a combination thereof, and includes, but is not limited to, all neuregulin isoforms, the neuregulin EGF-like domain alone, polypeptides containing the neuregulin EGF-like domain, neuregulin variants or derivatives, and any type of neuregulin-like gene product that similarly activates the above-mentioned receptors (described in detail below). Neuregulin also encompasses NRG-1, NRG-2, NRG-3, and NRG-4 proteins, peptides, fragments, and compounds that mimic neuregulin activity. Neuregulins used in the present invention can activate the above-mentioned ErbB receptors and regulate their biological responses. For example, they stimulate the synthesis of acetylcholine receptors in skeletal muscle cells and / or improve cardiac cell differentiation, survival, and DNA synthesis. Neuregulins also encompass variants thereof with conservative amino acid substitutions that do not substantially alter their biological activity. Suitable conservative substitutions of amino acids are known to those skilled in the art and can be made without generally altering the biological activity of the resulting molecule. Those skilled in the art recognize that single amino acid substitutions in non-essential regions of a polypeptide generally do not substantially alter biological activity (see, for example, Watson et al., Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin / Cummings Pub. Co., p. 224). In a preferred embodiment, the neuregulin used in the present invention binds to and activates ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimers. Examples of peptides include, but are not limited to, peptides comprising residues 177-237 of the NRG-1β2 isoform, including the amino acid sequence: SHLVKCAEKEKTFCVNGGECFMVKDLSNPSRYLCKCPNEFTGDRCQNYVASFYKAEELYQ (SEQ ID NO: 1).The peptide, comprising residues 177-237 of the NRG-1β2 isoform, contains the EGF-like domain that has been shown to be sufficient to bind to and activate the receptor.

[0023] As used herein, "epidermal growth factor-like domain" or "EGF-like domain" refers to a polypeptide motif encoded by the neuregulin gene that binds to and activates ErbB2, ErbB3, ErbB4, or a combination thereof, and has structural similarity to the EGF receptor binding domain disclosed in 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. Oncol., 13:1061-1067 (1998); Chang et al., Nature, 387:509-512 (1997); Carraway et al., Nature, 387:512-516 (1997); Higashiyama et al., J. Biochem., 122:675-680 (1997); and WO 97 / 09425, the entire contents of which are incorporated herein by reference. In certain embodiments, the EGF-like domain binds to and activates ErbB2 / ErbB4 or ErbB2 / ErbB3 heterodimers. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor-binding domain of NRG-1. In certain embodiments, the EGF-like domain comprises the amino acid sequence corresponding to amino acid residues 177-226, 177-237, or 177-240 of NRG-1. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor-binding domain of NRG-2. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor-binding domain of NRG-3. In certain embodiments, the EGF-like domain comprises the amino acid sequence of the receptor-binding domain of NRG-4. In one embodiment, the EGF-like domain comprises the amino acid sequence Ala Glu Lys Glu Lys Thr Phe Cys Val Asn Gly Gly Glu Cys Phe Met Val Lys Asp Leu Ser Asn Pro, as described in U.S. Patent No. 5,834,229.

[0024] The neuregulin protein is preferably in the form of a pharmaceutical composition, the composition, dosage, and route of administration of which can be determined according to methods known in the art (e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor), Mack Publishing Company, April 1997; Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Banga, 1999; and 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 and Rojanasakul (Ed.), Humana Press, 1999; Therapeutic Angiogenesis: From Basic Science to the Clinic, Vol. 28, Dole et al. (Ed.), Springer-Verlag New York, 1999).

[0025] The neuregulin protein can be formulated for oral, rectal, topical, inhalation, buccal (e.g., sublingual), parenteral (e.g., subcutaneous, intramuscular, intradermal, or intravenous), transdermal, or other suitable administration route. The optimal route in any particular case will depend on the nature and severity of the condition being treated and the properties of the particular neuregulin protein used. The neuregulin protein can be administered alone. Alternatively, the neuregulin protein is preferably administered with a pharmaceutically acceptable carrier or excipient. Any suitable pharmaceutically acceptable carrier or excipient can be used in this manner (see, e.g., Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor), Mack Publishing Company, April 1997).

[0026] According to the present invention, the neuregulin protein, alone or with other drugs, carriers, or additives, may be formulated for any suitable route of administration, such as intracavernous, subcutaneous, intravenous, intramuscular, intradermal, oral, or topical administration. This method may utilize a unit-dose injection formulation in an ampule or multi-dose container containing a preservative. The formulation may take the form of a suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulating agents such as suspending agents, stabilizers, and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, sterile pyrogen-free water, or other solution before use. Topical administration in the present invention may utilize foams, gels, creams, ointments, skin patches, or pastes.

[0027] Pharmaceutically acceptable compositions that can be used in the present invention, and methods of administration thereof, include, but are not limited to, those described in U.S. Patents 5,736,154; 6,197,801B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374B1; and 5,686,102.

[0028] The therapeutic or prophylactic dosage will vary according to the severity of the condition to be treated and the route of administration. The dosage, and perhaps the frequency of administration, will also vary according to the age, weight, condition, and response of the individual patient.

[0029] Additionally, the attending physician will know when and how to stop, discontinue, or adjust treatment to a lower dose due to toxic or adverse effects. Conversely, the attending physician will also know when and how to adjust treatment to a higher dose if the clinical response is inadequate (excluding toxic side effects).

[0030] Any suitable route of administration may be used. Dosage forms include tablets, lozenges, cachets, dispersions, suspensions, solutions, capsules, salves, and the like. See Remington's Pharmaceutical Sciences. In practical use, the neuregulin, alone or in combination with other drugs, may be combined with pharmaceutical carriers or additives, such as β-cyclodextrin and 2-hydroxypropyl-β-cyclodextrin, as the active ingredient in a mixture, according to conventional drug compounding techniques. The carrier may take a wide variety of formulations, such as those desired for topical or parenteral administration. Compositions for parenteral administration, such as intravenous injection or infusion, may be prepared using water, glycols, oils, buffers, sugars, preservatives, liposomes, and other similar pharmaceutical vehicles known to those skilled in the art. Examples of such parenteral compositions include, but are not limited to, 5% w / v dextrose, normal saline, or other solutions. The total dose of the neuregulin protein to be administered, alone or in combination with other agents, may be administered in a vial of intravenous fluid ranging from about 1 to 2000 ml, with the amount of diluent varying depending on the total dose.

[0031] The present invention also provides kits for carrying out the therapeutic regimens of the present invention. Such kits include one or more containers of a therapeutically effective amount of the neuregulin protein in a pharmaceutically acceptable form, alone or in combination with other agents. A preferred dosage form may be a combination with sterile saline, dextrose solution, buffer, or other pharmaceutically acceptable sterile fluid. The composition may also be lyophilized or desiccated, in which case the kit may further include a pharmaceutically acceptable solution, preferably sterile, in the container for reconstituting the compound to form an injectable solution. Examples of pharmaceutically acceptable solutions include saline and dextrose solution.

[0032] In another embodiment, the kit further comprises a needle or syringe, preferably packaged in sterile form, for injecting the composition, and / or a packaged alcohol pad, and may include instructions for administering the composition by a physician or patient.

[0033] As used herein, "treat," "treatment," and "treating" refer to any manner in which the symptoms of a disease, disorder, or condition are ameliorated or otherwise altered for the better. The effect of treatment may be prophylactic, in that the condition or its symptoms are prevented, either completely or partially, and / or therapeutic, in that the condition and / or harmful effects resulting from said condition are cured, either partially or completely. Treatment also includes any pharmaceutical use of the compositions herein.

[0034] As used herein, "heart failure" refers to an abnormality in cardiac function in which the heart does not pump blood at a rate required by metabolic tissues. Heart failure encompasses a wide range of disease states, including congestive heart failure, myocardial infarction, tachyarrhythmia, familial hypertrophic cardiomyopathy, ischemic heart disease, idiopathic dilated cardiomyopathy, and myocarditis. Heart failure can result from many causes, including, but not limited to, ischemic, congenital, rheumatic, viral, toxic, or idiopathic forms. Chronic cardiac hypertrophy is a serious disease state that is a precursor to congestive heart failure and cardiac arrest.

[0035] As used herein, "protein" is synonymous with "polypeptide" or "peptide," unless the context clearly dictates otherwise.

[0036] As used herein, "plasma" is synonymous with "serum" unless the context clearly dictates otherwise.

[0037] As used herein, "long-term benefit" refers to a benefit resulting from a treatment or intervention that is not seen in the short term after the treatment or intervention. For patients with chronic heart failure, a long-term benefit may be improved survival, reduced rehospitalization, or an improvement in a biomarker indicative of long-term prognosis. In one embodiment, the period of observation for the benefit is about six months. In one embodiment, the period of observation for the benefit is about one year. In one embodiment, the period of observation for the benefit is about two years. In other embodiments, the period of observation for the benefit is about three, five, ten, or more years.

[0038] As used herein, "survival" refers to the length of time or probability that a patient is alive and may be expressed as survival time or survival rate. Survival time is the time from diagnosis or treatment to death. Survival rate refers to the proportion of people who are alive for a certain period of time after diagnosis or treatment. For an individual patient, an increase in survival time due to a treatment or intervention can be considered a benefit. For a patient population or a large population, an increase in average survival time or an increase in survival rate can be considered a benefit.

[0039] As used herein, "readmission" refers to the number or frequency of hospitalizations of a patient admitted to a hospital within a given period of time. The hospital admissions may be for any disease, or may be solely for the same disease for which treatment is being given. For an individual patient, a reduction in the number of readmissions within a given period of time can be considered a benefit. For a patient population or large population, a reduction in the total or average number of readmissions can be considered a benefit.

[0040] As used herein, "N-terminal brain natriuretic peptide" or "NT-proBNP" refers to the inactive residue N-terminal proBNP, which is a prohormone of the hormonally active natriuretic peptide BNP, released primarily from cardiac myocytes in the left heart wall. In response to the stretch and tension of the myocardial wall, the prohormone proBNP is split by proteolytic cleavage into BNP and the hormonally inactive residue NT-proBNP.

[0041] BNP and NT-proBNP plasma levels are promising items in the daily management of suspected or established heart failure. Many studies on the clinical use of BNP and NT-proBNP have addressed their diagnostic properties, and more and more evidence is available to support the prognostic value of BNP and NT-proBNP. NT-proBNP has a half-life in the blood that is approximately six times longer than BNP, making it more widely used as a diagnostic or prognostic marker for heart failure. Plasma NT-proBNP levels can be analyzed using commercially available kits. For illustrative purposes, but not limited to, commercially available kits from Roche or Biomedica are mentioned. In the present example, NT-proBNP levels were detected using a kit from Biomedica (Austria).

[0042] Because the values ​​of both markers are generally elevated in patients with poor prognosis, blood levels of BNP and NT-proBNP are used for screening and diagnosing heart failure, and are also useful for determining the prognosis of heart failure. In the present invention, it has been discovered that plasma levels of BNP or NT-proBNP indicate patients suitable for neuregulin-based heart failure treatment. In fact, any diagnostic or prognostic marker for heart failure can be used to determine whether a patient is suitable for neuregulin-based heart failure treatment. The NT-proBNP plasma levels identified in the present invention should be used as a guideline, rather than a limitation, for selecting heart failure patients who will benefit significantly from neuregulin treatment. For example, using a plasma level of 5000 fmol / ml, it is still possible to select heart failure patients who will benefit from neuregulin treatment, although some of these patients will benefit less from treatment.

[0043] As used herein, the "New York Heart Association" or "NYHA" cardiac functional classification is a simplified method for classifying the severity of heart failure. This classification categorizes patients into four categories based on the degree of limitation during physical activity (limitations / symptoms related to the degree of variation between normal breathing and shortness of breath and / or severe pain): I, no symptoms or limitations during ordinary physical activity (e.g., shortness of breath when walking or climbing stairs); II, mild symptoms (mild shortness of breath and / or pain) and mild limitations during ordinary activity; III, symptoms significantly limiting activity even during less than ordinary activity (e.g., walking short distances (20-100 meters)) and only stable at rest; IV, patients frequently bedridden and severely limited by symptoms even at rest).

[0044] As used herein, "activity unit," "EU," or "U" refers to the amount of a standard that can induce 50% of the maximum response. That is, to determine the activity unit for a given active substance, the EC50 should be measured. For example, if the EC50 for a batch of product was 0.1 μg, this would be 1 unit. Furthermore, if 1 μg of the product was used, 10 EU (1 / 0.1) would be used. The EC50 can be determined by any method known in the art, including the method used by the inventors. This determination of the activity unit is important in the quality control of genetically engineered products and clinically used pharmaceuticals. This determination also allows products from different formulations and / or different batch numbers to be quantified using the same standard.

[0045] The following is an exemplary, rapid, sensitive, flexible, and quantitative method for determining the biological activity of NRG-1 through binding of NRG to cell surface ErbB3 / ErbB4 molecules and indirect mediation of ErbB2 phosphorylation (see, e.g., Michael D. Sadick et al., 1996, Analytical Biochemistry, 235:207-214 and WO03 / 099300).

[0046] Briefly, the assay, termed the kinase receptor activity enzyme-linked immunosorbent assay (KIRA-ELISA), involves two separate microtiter plates: one for cell culture, ligand stimulation, and cell lysis / receptor solubilization; and the other for receptor capture and phosphotyrosine ELISA. The assay was performed to analyze NRG-induced ErbB2 activity in the adherent breast cancer cell line, MCF-7, utilizing intact receptor stimulation. Membrane proteins were solubilized via Triton X-100 lysis, and the receptor was captured in ELISA wells coated with an ErbB2-specific antibody that does not cross-react with ErbB3 or ErbB4. The degree of receptor phosphorylation was quantified by anti-phosphotyrosine ELISA. A reproducible standard curve was generated for heregulin β1(177-244) with an EC50 of approximately 360 pM. When an ideal HRGβ1(177-244) sample is analyzed by KIRA-ELISA and quantitative anti-phosphotyrosine Western blot analysis, the results show a high correlation with each other. The assay described in this report can clearly quantify the tyrosine phosphorylation of ErbB2 upon the interaction of HRG with ErbB3 and / or ErbB4.

[0047] Because most genetically engineered drugs are proteins and polypeptides, their activity can be evaluated by their amino acid sequence or the active center formed by their three-dimensional structure. Because the activity potency of proteins and polypeptides does not correspond to their absolute quantity, activity cannot be evaluated by weight units, as with chemical drugs. However, the biological activity of genetically engineered drugs generally corresponds to their pharmacodynamic properties, and potency units can be determined using established potency determination systems based on a given biological activity. Therefore, evaluation of biological activity can be part of the potency measurement process for biologically active substances and is an important element of the quality control of genetically engineered drugs. Determining biological activity standards is important for the quality control of genetically engineered products and clinically used drugs.

[0048] The amount of standard that can induce 50% of the maximum response is defined as an activity unit (1 EU). Thus, products from different preparations and different batch numbers can be quantified by the same standard.

[0049] B. Working Example Example 1: Effect of different routes of Neucardin™ administration on survival in rats with CHF introduction In this study, we used a coronary artery ligation (CAL)-induced CHF model to examine whether Neucardin™ administered by IV infusion using a microinjection pump or subcutaneous (SC) bolus had any effect on survival and cardiac hemodynamics 120 days after Neucardin™ administration, which began 4 weeks after CAL. Echocardiography and cardiac remodeling were also used to assess cardiac function and recovery from CAL.

[0050] 2. Method 2.1. Test animals: Strain, origin: Wistar rat, Shanghai SLAC Laboratory Animal CO. LTD; body weight 200±10g, male; 2.2 Test Subjects 2.2.1 Neucardin(TM) ID: Injectable recombinant human neuregulin-1 (rhNRG-1, Neucardin™) Lot number: 200607009 Manufacturer:Zensun (Shanghai) Sci & Tech Co., Ltd Dosage form: Lyophilized powder Appearance: White or off-white cake Labeled rhNRG-1 dosage: 250 μg / vial Specific activity: 4897U / vial Storage conditions: 2~8℃ 2.2.2 Excipients: ID: placebo of recombinant human neuregulin-1 Dosage form: Lyophilized powder Appearance: White or off-white cake Composition: Human serum albumin, mannitol, phosphate, sodium chloride Storage conditions: 2~8℃ 2.3 Steps: 2.3.1 Method for creating a rat CHF model The LAD of rats was ligated. Briefly, rats were anesthetized with ketamine hydrochloride (100 mg / kg, IP), and the chest was shaved and sterilized. The rats were intubated and mechanically ventilated with room air (respiratory rate: 60 breaths / min, tidal volume: 20 ml). A left thoracotomy was then performed between the fourth and fifth intercostal spaces, and a skin incision was made along the left sternal line. The fourth rib was resected toward the sternum. The pericardial sac was punctured to expose the heart. The LAD was ligated approximately 2 mm from its origin using silk suture (6-0). The chest cavity was then evacuated, and the chest was closed in three layers (rib, muscle, and skin). After the rats were allowed to resume spontaneous breathing and recovered from anesthesia, they were returned to their cages. Rats were maintained for 4 weeks, then evaluated by echocardiography. Those showing EF values ​​of 30-45% were included in the formal study. Rats in all groups were housed in five cages and provided with free access to standard chow and pure water. The room temperature was maintained at 21±1°C, with a 12-hour light / dark cycle.

[0051] 2.3.2 IV infusion with a microinjection pump Vehicle or Neucardin™ was administered intravenously via the tail vein. To perform this procedure, a rat restrainer appropriate for the rat's weight was used. The rat was placed near the restrainer and gently placed into the apparatus. Typically, the rat entered the restrainer unassisted. The rat's tail was then wiped with alcohol-moistened gauze to increase blood flow in the tail vein and soften the stratum corneum of the skin. Two lateral tail veins were identified, and the needle was inserted 2 mm into the tail vein, 2–3 cm from the end of the tail, with the bevel facing upward and approximately parallel to the vein. Blood was withdrawn into the needle hub to confirm secure entry into the tail vein. The needle was then secured to the tail with medical tape. Drug or vehicle infusion was initiated at an appropriate rate (0.2–0.4 ml / h) using a microinjection pump or bolus injection.

[0052] 2.3.3 SC bolus A subcutaneous bolus of vehicle or Neucardin™ was administered through the rat's back. To perform this procedure, a rat restrainer appropriate for the rat's weight was used. The rat's back was wiped with alcohol-moistened gauze to disinfect the skin. The needle was inserted subcutaneously into the rat's back 3–4 cm, with the bevel facing upward and approximately parallel to the skin. The needle was secured to the back with medical tape and connected to the perfusion tubing. The rat was then placed near the restrainer and gently placed into the apparatus. The rat typically entered the restrainer without assistance. After the restrainer was closed, the bolus injection was initiated.

[0053] 2.3.4 Experimental groups and drug infusions The MI rats were randomly divided into the following four groups according to their EF values:

[0054] Group A (control study of IV and SC bolus): n=58 rats, IV infusion of vehicle by microinjection pump at a rate of 0.2 ml / hour for 8 hours daily for the first 10 days, and SC bolus of vehicle (same amount as Neucardin™) every 5 days until day 120.

[0055] Group B (Neucardin™ SC bolus): n=58, vehicle was administered IV via microinjection pump at a rate of 0.2 ml / hour for 8 hours daily for the first 10 days, and Neucardin™ SC bolus (10 μg / day) was administered every 5 days until day 120.

[0056] Group C (Neucardin™ IV infusion): n=57, Neucardin™ IV infusion (0.625 μg / kg / hr) via microinjection pump at a rate of 0.2 ml / hr for 8 hours daily for the first 10 days, with an SC bolus of vehicle (same volume as Neucardin™) administered every 5 days until day 120.

[0057] Group D (Neucardin™ IV infusion and SC bolus): n=57, Neucardin™ IV infusion (0.625 μg / kg / hr) via microinjection pump at a rate of 0.2 ml / hr for 8 hours daily for the first 10 days, SC bolus of vehicle (same amount as Neucardin™) on days 1, 6, and 11, then SC bolus of Neucardin™ (10 μg / day) every 5 days from day 16 to the last day.

[0058] 2.3.5 Acquired Data Survival rate; echocardiography parameters; hemodynamic parameters; 3. Results 3.1 Survival rate Table 1 shows the survival rates among each group. The survival rates were 48.3% for Group A (vehicle IV infusion and SC bolus), 62.1% for Group B (Neucardin™ SC bolus), 64.9% for Group C (Neucardin™ IV infusion), and 82.5% for Group D (Neucardin™ IV infusion and SC bolus). The mean survival times of surviving or dead rats in Groups B, C, and D were improved or prolonged compared to Group A, with Group D showing the greatest effect.

[0059] [Table 1]

[0060] 3.2 Echocardiography parameters Echocardiography parameters are shown in Table 2. Four weeks after coronary artery ligation and before administration of the test substances, CHF rats were randomly divided into four groups based on EF values. As shown in Table 2, there were no significant differences among the four groups before treatment (BT). At 120 days after the start of treatment, the EF values ​​were 30.7 ± 3.1 in the vehicle group, 32.9 ± 4.1 in the SC bolus Neucardin™ group, 33.5 ± 3.4 in the IV infusion Neucardin™ group, and 36.2 ± 4.8 in the IV infusion and SC bolus Neucardin™ group, respectively. After treatment, the EF and FS values ​​of Groups B, C, and D were higher than those of Group A.

[0061] [Table 2]

[0062] 3.3 Hemodynamic parameters Table 3 shows measurements of MAP, HR, ±dp / dt, LVEDP, and LVSP in the four groups of anesthetized animals on day 121. When Neucardin™ was administered by either SC bolus or IV infusion (Groups B and C), Neucardin™ significantly increased dp / dt by 19.6% and 27.1%, respectively, and −dp / dt by 22.5% and 29.8%, respectively, compared to Group A. When Neucardin™ was administered both by IV infusion and SC bolus (Group D), significant increases in mean arterial pressure (MAP, 112.3 ± 5.5 mmHg), left ventricular systolic pressure (LVSP, 139.4 ± 9.8 mmHg), +dp / dt (7012.1 ± 903.0 mmHg / sec), and -dp / dt (-4353.2 ± 847.6 mmHg / sec) were observed compared with vehicle. Interestingly, these values ​​for MAP, LVSP, +dp / dt, and -dp / dt were 10.6%, 9.2%, 38.5%, and 37.5%, respectively, higher than those in rats administered with vehicle. These results indicated that Groups B, C, and D performed better than Group A with regard to hemodynamic parameters, with Group D showing the greatest effect.

[0063] [Table 3]

[0064] [Table 4]

[0065] 4. Conclusion Both the combined administration of Neucardin™ by IV infusion and SC bolus, and administration of the peptide by either route alone, increased survival and improved cardiac performance parameters in rats with CAL-induced CHF compared to vehicle-treated rats.

[0066] Example 2: A randomized, double-blind, multicenter, placebo-controlled study to evaluate the efficacy and safety of recombinant human neuregulin 1 in patients with chronic heart failure on standard of care To evaluate the efficacy of recombinant human neuregulin-1 infusion in chronic heart failure, a phase II, double-blind, multicenter, placebo-controlled, standard-of-care trial was conducted at multiple clinical sites in China. A total of 195 patients with stable chronic heart failure (NYHA class II or III) were admitted and randomly divided into three groups (placebo, 0.6 μg / kg rhNRG-1, or 1.2 μg / kg rhNRG-1). There were no significant differences in demographics or treatment history between the groups. Patients were first administered the drug for 10 consecutive days in the hospital according to the schedule and were discharged after the 11th follow-up visit. Additional in-home follow-up visits were conducted on days 30 and 90. A telephone interview was conducted one year after the last patient's admission.

[0067] Investigational drug: Specifications: Neucardin™, a 61 amino acid polypeptide containing the EGF-like domain of the neuregulin-1 β2 isoform with a molecular weight of 7054 Da (1 μg = 0.14 nmol). 250 μg (5000 EU) per vial (1 μg = 20 EU).

[0068] Preparation: For injection.

[0069] Administration form: Infusion.

[0070] Storage: 3-8°C, limited use, in a safe place away from light.

[0071] placebo: Specifications: Neucardin™ excipient (250 μg / vial without active recombinant human neuregulin-1 protein).

[0072] [Table 5]

[0073] Test Procedure Inclusion criteria included patients aged 18 to 65 years with CHF (NYHA class II or III), a relatively stable condition (including clinical signs, symptoms, and treatment with acceptable standard of care for CHF at target or maximum tolerated doses for at least 1 month), an LVEF of 40% or less, and a mean age of 18 to 65 years. Key exclusion criteria included acute myocardial infarction, hypertrophic cardiomyopathy, constrictive pericarditis, severe valvular or congenital heart disease, severe pulmonary hypertension, systolic blood pressure <90 mmHg or >160 mmHg, severe arrhythmia, cardiac surgery or cerebrovascular event within the past 6 months, claustrophobia, or pregnant women. All participating patients signed a consent form.

[0074] Patients were randomly assigned to one of three groups, receiving placebo or rhNRG-1 (0.6 or 1.2 μg / kg / day) for 10 consecutive days, and were discharged after the 11th follow-up visit. Two separate in-home follow-up visits were conducted on days 30 and 90. Blood samples were collected from each patient before treatment and on days 11, 30, and 90. NT-proBNP was tested at the core laboratory using the NT-proBNP assay (Biomedica kit). One year after the last patient's hospitalization, a telephone interview was conducted to collect information on readmissions. All telephone interviews were recorded on a special form signed by the investigator.

[0075] Of 48 patients in the placebo group with available readmission information, 12 (25.0%) were readmitted at least once due to worsening heart failure. In the 0.6 μg / kg group, only 4 of 46 patients (8.7%) were readmitted to the hospital (P = 0.05 compared with placebo); the readmission rate in the 1.2 μg / kg group was 22.0% (11 / 50). The mean number of readmissions was 0.458 (22 / 48) per patient in the placebo group, whereas the mean number of readmissions in the 0.6 μg / kg group (8 / 41) and 1.2 μg / kg groups (19 / 50) was reduced by 57.4% and 17.0%, respectively, compared with the placebo group.

[0076] In the placebo group, NT-proBNP levels remained stable throughout the study compared with baseline. On day 11, NT-proBNP levels increased significantly in the rhNRG-1-treated groups (from 1853 ± 1512 to 2399 ± 1841 fmol / ml in the 0.6 μg / kg group, P < 0.01; from 1562 ± 1275 to 2774 ± 1926 fmol / ml in the 1.2 μg / kg group, P < 0.01). However, this increase was transient and not attributable to a deterioration in cardiac function, as cardiac function improved. NT-proBNP levels returned to baseline levels in the 1.2 μg / kg group on days 30 and 90. Furthermore, in the 0.6 μg / kg group, NT-proBNP decreased significantly at 30 days (1323 ± 1124 fmol / ml, P = 0.01) and 90 days (1518 ± 1403 fmol / ml, P = 0.01) compared with baseline.

[0077] These results suggest that treatment with rhNRG-1 can provide long-term benefits to patients with chronic heart failure, as it reduces rehospitalizations and plasma levels of NT-proBNP.

[0078] Example 3: Randomized, double-blind, multicenter, placebo-controlled survival study of recombinant human neuregulin 1 in patients with chronic heart failure on standard of care A phase II, double-blind, multicenter, placebo-controlled study based on standard of care was conducted at multiple clinical sites in China to evaluate the effects of recombinant human neuregulin-1 infusion on chronic heart failure. A total of 351 patients with stable chronic heart failure (NYHA class III or IV) were admitted and randomized to placebo or rhNRG-1 (0.6 μg / kg). There were no significant differences in demographics or treatment history between the groups. Patients received the drug for 10 consecutive days at the hospital according to the schedule. They were discharged after the 11th follow-up visit and continued receiving the drug as outpatients once a week from weeks 3 to 25. Blood samples were collected from each patient before treatment (baseline) and at each follow-up visit. NT-proBNP was tested at the core laboratory using an NT-proBNP assay (Biomedica kit). Survival information was collected at week 52 of the study.

[0079] Investigational drug: Specifications: Neucardin™, a 61 amino acid polypeptide containing the EGF-like domain of the neuregulin-1 β2 isoform with a molecular weight of 7054 Da (1 μg = 0.14 nmol). 250 μg (5000 EU) per vial (1 μg = 20 EU).

[0080] Preparation: For injection.

[0081] Administration: Infusion or intravenous infusion.

[0082] Storage: 3-8°C, limited use, in a safe place away from light.

[0083] placebo: Specifications: Neucardin™ excipient, 250 μg / vial, does not contain active recombinant human neuregulin-1 protein.

[0084] [Table 6]

[0085] Inclusion criteria included patients aged 18 to 80 years with CHF (NYHA class III or IV), a relatively stable condition (including clinical signs, symptoms, and treatment with acceptable standard of care for CHF at target or maximum tolerated doses for at least 1 month), an LVEF of 40% or less, and a history of acute myocardial infarction (acute myocardial infarction, hypertrophic cardiomyopathy, constrictive pericarditis, severe valvular or congenital heart disease, severe pulmonary hypertension, systolic blood pressure <90 mmHg or >160 mmHg, severe arrhythmia, cardiac surgery or cerebrovascular event within the past 6 months, claustrophobia, or pregnant women. All participating patients signed a consent form.

[0086] The all-cause mortality rate at week 52 in the placebo group was 15.91%, with 28 deaths among 176 patients, compared with 9.71% in the rhNRG-1 group, with 16 deaths among 175 patients who completed the study (hazard ratio = 0.425, 95% CI 0.222-0.813, p = 0.0097). Regarding mortality from cardiovascular events, the placebo group had a 14.77% mortality rate at week 52, with 26 deaths among 176 patients, compared with 9.71% in the rhNRG-1 group. These results suggest that even though the placebo group continued with conventional standard treatment for chronic heart failure, the mortality rate for those receiving rhNRG-1 was approximately 40% lower than that for those receiving placebo.

[0087] All-cause mortality was also analyzed based on baseline NT-proBNP stratification. When NT-proBNP levels were stratified into three categories: ≤1600 fmol / ml, >1600 fmol / ml and ≤4000 fmol / ml, or >4000 fmol / ml, the mortality rates in the placebo group compared with those in the rhNRG-1 group were 1.49% vs. 8.49%, 8.96% vs. 23.33%, and 26.67% vs. 28.00%, respectively. When NT-proBNP levels were stratified into ≤4000 fmol / ml and >4000 fmol / ml, the mortality rates in the placebo group compared with those in the rhNRG-1 group were 5.22% vs. 14.89% (P = 0.0092), and 26.67% vs. 28.00%, respectively. The results demonstrate with statistical significance that rhNRG-1 can substantially improve survival in patients with chronic heart failure.

[0088] Additionally, patients were stratified into baseline NYHA cardiac performance class III or IV. The all-cause mortality rate for class III was 6.06% (8 deaths of 132 patients) in the rhNRG-1 group and 15.49% (22 deaths of 142 patients) in the placebo group (p = 0.0189). The all-cause mortality rate for class IV was 20.93% (9 deaths of 43 patients) in the rhNRG-1 group and 17.65% (6 deaths of 34 patients) in the placebo group (p = 0.7789).

Claims

[Claim 1] 10. A pharmaceutical composition substantially as hereinbefore described.

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