NEUREGULIN BASED METHODS FOR TREATING HEART FAILURE

Abstract

The present invention features methods of treating patients with chronic heart failure by administering a neuregulin polypeptide within a dosage range which is both effective and safe.

Claims

1. A method for preventing, treating or delaying heart failure in mammals in need thereof, which method comprises administering to the mammal in need thereof a neuregulin 1 protein in an amount: a) from about 0.3 μg to about 2.4 μg/kg/day; b) from about 6 EU to about 48 EU/kg/day; or c) from about 0.04 nmol to about 0.34 nmol/kg/day.

2. The method of claim 1, wherein the neuregulin 1 protein is neuregulin 1 β2 isoform.

3. The method of claim 1, wherein the neuregulin 1 protein comprises the amino acid sequence set forth in SEQ ID NO:1.

4. The method of claim 1, wherein the neuregulin 1 protein consists of the amino acid sequence set forth in SEQ ID NO:1.

5. The method of claim 1, wherein the neuregulin 1 protein is administered to the mammal intravenously.

6. The method of claim 1, wherein the neuregulin 1 protein is administered to the mammal for one day or multiple days.

7. The method of claim 6, wherein the neuregulin 1 protein is administered to the mammal for at least 10 days.

8. The method of claim 7, wherein the neuregulin 1 protein is administered to the mammal for from 10 minutes to 12 hours per day.

9. The method of claim 8, wherein the neuregulin 1 protein is administered to the mammal 10 hours per day for 10 days.

10. The method of claim 1, wherein the mammal has or is suspected of having heart failure.

11. The method of claim 10, wherein the heart failure is caused by one or more ischaemic, congenital, rheumatic, idiopathic, viral or toxic factors.

12. The method of claim 1, where in the mammal is human.

Description

B. EXAMPLES

[0037] The invention is illustrated by the following examples which are not intended to be limiting in any way.

Example 1: A Phase I, Open-Label, Non-Comparative, Single-Dose Study to Evaluate the Tolerance of Recombinant Human Neuregulin-1 for Injection in Healthy Volunteers

[0038] To determine the safety and tolerance of single dose of recombinant human Neuregulin-1 for injection in healthy subjects, the phase I clinical trial (Protocol ID: KW-70112) was carried out in Peking University First Hospital in China, 28 healthy volunteers were enrolled and were randomized into six dosage groups according to their enrolment order, among which the gender ratio was about 1:1.

[0039] Investigational Product:

[0040] Specification: Neucardin™, a 61-amino acid polypeptide comprising the EGF-like domain of Neuregulin-1 β2 isoform and having the amino acid sequence set forth in SEQ ID NO:1, with the molecular weight of 7054 Dal (1 μg=0.14 nmol). 250 μg (5000 EU)/vial (1 μg=20 EU).

[0041] Preparation: For Injection.

[0042] Mode of administration: Slow bolus (20 ml/10 min, infusion pump control).

[0043] Storage: in safe place, with limited access and protected from light, at 3-8° C.

[0044] Dosage Groups:

[0045] Twenty eight volunteers were divided into six groups with escalating doses equivalent to 0.2 μg, 0.4 μg, 0.8 μg, 1.2 μg, 1.6 μg and 2.4 μg (refer to Table 1 for details). Started from 0.2 μg/kg, under precondition of confirmation of the safety and tolerability of the former dosage group, the study escalated to next dose.

TABLE-US-00001 TABLE 1 Dosage groups and subjects in each group Group Dosage Samples A 0.2 μg/kg (4 EU/kg) 4 B 0.4 μg/kg (8 EU/kg) 4 C 0.8 μg/kg (16 EU/kg) 4 D 1.2 μg/kg (24 EU/kg) 5 E 1.6 μg/kg (32 EU/kg) 6 F 2.4 μg/kg (48 EU/kg) 5

[0046] Study Procedures

[0047] 1) Screening period: [0048] Obtain the written informed consent form; [0049] Verify the inclusion/exclusion criteria; [0050] Demographics [0051] Collect the medical history; [0052] Physical examination; [0053] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0054] Haematology, urinalysis; [0055] Blood biochemistry; [0056] Serum cardiac markers; [0057] ECG; [0058] Ultrasonic cardiogram; [0059] Blood coagulation test: APTT, PT; [0060] Serology test (including HBsAg, Anti-HCV, Anti-HIV assay); [0061] Chest X-ray; [0062] Urine HCG test (female subject, if necessary); [0063] Record adverse event and serious adverse event; [0064] Record concomitant medications.

[0065] 2) Baseline: The time from the night before scheduled study drug administration to initiation of study drug administration. [0066] Physical examination; [0067] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0068] Specific antibody assay; [0069] Re-verify the inclusion/exclusion criteria; [0070] Record adverse event and serious adverse event; [0071] Record concomitant medication.

[0072] 3) The day study drug was administrated [0073] Study drug administration; [0074] ECG; [0075] Echocardiography; [0076] Record adverse event and serious adverse event; [0077] Record concomitant medication (each day).

[0078] 4) The 1st day and 7th day after cessation of drug administration [0079] Physical examination; [0080] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0081] Specific antibody assay; [0082] Haematology, urinalysis; [0083] Blood biochemistry; [0084] Serum cardiac markers; [0085] ECG; [0086] Ultrasonic cardiogram (7th day after cessation of drug administration); [0087] Blood coagulation test: APTT, PT; [0088] Urine HCG test (female subject, if necessary); [0089] Record adverse events and serious adverse events; [0090] Record concomitant medications.

[0091] 5) The 10th day after cessation of drug administration

[0092] As to any abnormal item in the 7th day test results after cessation of drug administration, this item was re-tested on the 10th days after dose. After that, it was re-tested with a certain interval until the result became normal.

[0093] Results and Analysis

[0094] Totally 28 healthy subjects were enrolled into 6 dosage groups of 0.2, 0.4, 0.8, 1.2, 1.6, 2.4 μg/kg, the number of subjects who completed study in each dosage groups was 4, 4, 4, 5, 6, 5 respectively (refer to Table 1).

[0095] Adverse events (AE) were monitored from the time informed consent was signed until the end-of-treatment visit. Sixteen subjects (57.1%) experienced at least one adverse event during the course of the study, all adverse events were mild. None of subject experienced moderate or severe adverse events during the study.

[0096] During the study, the most frequently reported adverse events were listed in Table 2, adverse events by severity were listed in Table 3.

TABLE-US-00002 TABLE 2 Listing of adverse events Group Subject AE Severity Outcome 0.2 μg/kg 402 Abnormal ECG Mild Recovery 0.8 μg/kg 101 Nausea Mild Recovery 102 Nausea; Lower limb fatigue Mild Recovery 103 Nausea; Fatigue; Loose stolls; Mild Recovery Light headness 104 Nausea Mild Recovery 1.2 μg/kg 601 Abnormal ECG Mild Recovery 603 Hematology abnormality Mild Recovery 605 Abnormal ECG Mild Recovery 1.6 μg/kg 201 Poor appetite; Hematology Mild Recovery abnormality 202 ECG and Urinary abnormality Mild Recovery 204 Nausea; Abnormal ECG Mild Recovery 206 Nausea; Poor appetite Mild Recovery 2.4 μg/kg 302 Abnormal ECG Mild Recovery 303 Common cold Mild Recovery 304 Abnormal ECG Mild Recovery 305 Nausea; Light headness; Mild Recovery Abnormal ECG

TABLE-US-00003 TABLE 3 Summary of adverse events by severity 0.2 μg/kg 0.4 μg/kg 0.8 μg/kg 1.2 μg/kg 1.6 μg/kg 2.4 μg/kg Total (N = 4) (N = 4) (N = 4) (N = 5) (N = 6) (N = 5) (N = 28) n (%) n (%) n (%) n (%) n (%) n (%) n (%) Subjects experienced at least one 1 (25.0) 0 (0.0) 4 (100.0) 3 (60.0) 4 (66.7) 4 (80.0) 16 (57.1) adverse events Subjects classified by worst adverse events Mild 1 (25.0) 0 (0.0) 4 (100.0) 3 (60.0) 4 (66.7) 4 (80.0) 16 (57.1) Moderate 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  0 (0.0) Severe 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  0 (0.0) Subjects experienced at least one SAE 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  0 (0.0) Subjects experienced at least one adverse 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  0 (0.0) events leading to premature termination Death 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)  0 (0.0)

[0097] The study drug related adverse events were ECG abnormality (8 subjects, 28.6%) and gastrointestinal disorders (7 subjects, 25.0%). ECG abnormalities included slight ST-T segment depression, T-wave flattening or minimal T-wave inversion. During study drug administration period and observation period, none of the subjects experienced precordial discomfort, choking sensation, chest pain, dyspnea; blood pressure was normal at 2 hour and 24 hour after dosing; no abnormality was found in blood biochemistry, electrolytes, serum cardiac markers and echocardiography.

[0098] All subjects of each dosage were not changed in physical examination parameters from baseline. Serum antibody assay showed negative results in all subjects (refer to Table 4).

TABLE-US-00004 TABLE 4 Serum anti-NRG-1 antibody of each group First sampling Second sampling Third sampling Group 1:50 1:100 1:50 1:100 1:50 1:100 0.2 μg/kg 0.18 ± 0.05 0.12 ± 0.02 0.18 ± 0.06 0.13 ± 0.02 0.17 ± 0.06 0.14 ± 0.02 0.4 μg/kg 0.12 ± 0.01 0.10 ± 0.01 0.12 ± 0.06 0.10 ± 0.02 0.11 ± 0.01 0.10 ± 0.01 0.8 μg/kg 0.16 ± 0.05 0.15 ± 0.02 0.18 ± 0.03 0.15 ± 0.04 0.18 ± 0.05 0.16 ± 0.05 1.2 μg/kg 0.12 ± 0.02 0.10 ± 0.02 0.12 ± 0.03 0.10 ± 0.02 0.11 ± 0.02 0.10 ± 0.02 1.6 μg/kg 0.21 ± 0.12 0.17 ± 0.08 0.22 ± 0.10 0.17 ± 0.07 0.22 ± 0.10 0.18 ± 0.06 2.4 μg/kg 0.15 ± 0.04 0.15 ± 0.03 0.17 ± 0.04 0.13 ± 0.03 0.14 ± 0.03 0.14 ± 0.03

[0099] The results of the phase I clinical trial showed that in the dosage of not exceeding 2.4 μg/kg/day, the drug-related adverse events are mainly mild ECG abnormality and gastrointestinal disorders, no moderate or severe adverse events were observed. So, dosage not exceeding 2.4 μg/kg/day when administered with a single dose is tolerable.

Example 2: A Phase I, Open-Label, Non-Comparative, Multi-Dose Study to Evaluate the Tolerance of Recombinant Human Neuregulin-1 for Injection in Healthy Volunteers

[0100] To evaluate the safety and tolerance of multi-dose of recombinant human Neuregulin-1 for injection in healthy subjects for 5 consecutive days, the phase I clinical trial (Protocol ID: KW-70112) was carried out in Peking University First Hospital in China, 32 healthy volunteers were enrolled and were randomized into 4 dosage groups.

[0101] Investigational Product:

[0102] Specification: Neucardin™, a 61-amino acid polypeptide comprising the EGF-like domain of Neuregulin-1 β2 isoform and having the amino acid sequence set forth in SEQ ID NO:1, with the molecular weight of 7054 Dal (1 μg=0.14 nmol). 250 μg (5000 EU)/vial (1 μg=20 EU).

[0103] Preparation: For injection.

[0104] Mode of administration: Slow bolus (20 ml/10 min, infusion pump control).

[0105] Storage: in safe place, with limited access and protected from light, at 3-8° C.

[0106] Dosage Groups:

[0107] Thirty two volunteers were divided into four groups with escalating doses equivalent to 0.2 μg, 0.4 μg, 0.8 μg and 1.2 μg (refer to Table 5 for details). Started from 0.2 μg/kg, under precondition of confirmation of the safety and tolerability of the former dosage group, the study escalated to next dose.

TABLE-US-00005 TABLE 5 Dosage groups and subjects in each group Group Dosage Samples I (5 days) 0.2 μg/kg (4 EU) 8 II (5 days) 0.4 μg/kg (8 EU) 8 III (5 days) 0.8 μg/kg (16 EU) 8 IV (5 days) 1.2 μg/kg (24 EU) 8

[0108] Study Procedures

[0109] 1) Screening period: [0110] Obtain the written informed consent form; [0111] Verify the inclusion/exclusion criteria; [0112] Demographics; [0113] Collect the medical history; [0114] Physical examination; [0115] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0116] Haematology, urinalysis; [0117] Blood biochemistry; [0118] Serum cardiac markers; [0119] ECG; [0120] Ultrasonic cardiogram; [0121] Blood coagulation test: APTT, PT; [0122] Serology test (including HBsAg, Anti-HCV, Anti-HIV assay); [0123] Chest X-ray; [0124] Urine HCG test (female subject, if necessary); [0125] Record adverse event and serious adverse event; [0126] Record concomitant medications.

[0127] 2) Baseline: The time from the night before scheduled study drug administration to initiation of study drug administration. [0128] Physical examination; [0129] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0130] Re-verify the inclusion/exclusion criteria; [0131] Record adverse event and serious adverse event; [0132] Record concomitant medication.

[0133] 3) 1.sup.st day to 5.sup.th day of drug administration [0134] Study drug administration; [0135] ECG; [0136] Record adverse event and serious adverse event (each day); [0137] Record concomitant medication (each day).

[0138] 4) The 1st day and 7th day after cessation of drug administration [0139] Physical examination; [0140] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0141] Haematology, urinalysis; [0142] Blood biochemistry; [0143] Serum cardiac markers; [0144] ECG; [0145] Ultrasonic cardiogram (7th day after cessation of drug administration); [0146] Blood coagulation test: APTT, PT; [0147] Urine HCG test (female subject, if necessary); [0148] Record adverse events and serious adverse events; [0149] Record concomitant medications.

[0150] 5) The 14th day after cessation of drug administration

[0151] As to any abnormal item in the 7th day test results after cessation of drug administration, this item was re-tested on the 10th days after dose. After that, it was re-tested with a certain interval until the result became normal.

[0152] Results and Analysis

[0153] A total of 32 healthy subjects were enrolled and randomized into 4 different dosage groups of 0.2, 0.4, 0.8 and 1.2 μg/kg, with 8 subjects in each group and the gender ratio of 1:1. One subject in 1.2 μg/kg dosage group discontinued the study because of adverse event, all other subjects completed the study.

[0154] Adverse events were monitored from the time informed consent was signed until the end-of-treatment visit. Twenty-three subjects (71.9%) experienced at least one adverse event during the course of the study, all adverse events were mild. None of subject experienced severe or serious adverse events during the study (refer to Table 6).

TABLE-US-00006 TABLE 6 Adverse events by severity 0.2 μg/kg 0.4 μg/kg 0.8 μg/kg 1.2 μg/kg Total (N = 8) (N = 8) (N = 8) (N = 8) (N = 32) n(%) n(%) n(%) n(%) n(%) Subjects experienced at least one 4 (50.0) 5 (62.5) 7 (87.5) 7 (87.5) 23 (71.9) adverse events Subjects classified by worst adverse events Mild 4 (50.0) 5 (62.5) 7 (87.5) 7 (87.5) 23 (71.9) Moderate 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Severe 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) Subjects experienced at least one 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) SAE Subjects experienced at least one 0 (0.0) 0 (0.0) 0 (0.0) 1 (12.5) 1 (3.1) adverse events leading to premature termination Death 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0)

[0155] The frequently reported study drug related adverse events included ECG abnormalities (14 cases, 40.63%) and gastrointestinal disorders (9 cases, 28.13%).

[0156] ECG abnormalities included slight ST-T segment depression, T-wave flattening or minimal T-wave inversion. During study drug administration period and observation period, none of the subjects with abnormal ECG experienced precordial discomfort, choking sensation, chest pain, and dyspnea. No treatment was taken in the whole process of study, almost all ECG abnormalities return to normal within 48 hours after 5.sup.th study drug administration.

[0157] Gastrointestinal disorders related to study drug were stomach discomfort, nausea, vomit and loose stools (refer to Table 7).

TABLE-US-00007 TABLE 7 Frequently reported study drug related adverse events 0.2 μg/kg 0.4 μg/kg 0.8 μg/kg 1.2 μg/kg Total MedDRA system organ class/ (N = 8) (N = 8) (N = 8) (N = 8) (N = 32) MedDRA preferred term n(%) n(%) n(%) n(%) n(%) Subjects experienced at least one 2 (25.0) 4 (50.0) 7 (87.5) 7 (87.5) 20 (62.5) adverse event Skin and hypodermis disorders 0 (0.0) 0 (0.0) 2 (25.0) 0 (0.0) 2 (6.3) Rash 0 (0.0) 0 (0.0) 2 (25.0) 0 (0.0) 2 (6.3) Nervous system disorder 0 (0.0) 0 (0.0) 1 (12.5) 0 (0.0) 1 (3.1) Headache 0 (0.0) 0 (0.0) 1 (12.5) 0 (0.0) 1 (3.1) Gastrointestinal disorder 0 (0.0) 1 (12.5) 4 (50.0) 4 (50.0) 9 (28.1) Nausea 0 (0.0) 0 (0.0) 4 (50.0) 2 (25.0) 6 (18.8) Vomit 0 (0.0) 0 (0.0) 0 (0.0) 2 (25.0) 2 (6.3) Stomach discomfort 0 (0.0) 1 (12.5) 0 (0.0) 0 (0.0) 1 (3.1) Loose stool 0 (0.0) 0 (0.0) 0 (0.0) 1 (12.5) 1 (3.1) Cardiac disorder 2 (25.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (6.3) Ventricular arrhythmia 2 (25.0) 0 (0.0) 0 (0.0) 0 (0.0) 2 (6.3) ECG abnormalities 0 (0.0) 3 (37.5) 5 (62.5) 6 (75.0) 14 (43.8) T wave inversion 0 (0.0) 2 (25.0) 2 (25.0) 3 (37.5) 7 (21.9) T wave depression 0 (0.0) 2 (25.0) 5 (62.5) 4 (50.0) 11 (34.4) T wave abnormalities 0 (0.0) 0 (0.0) 0 (0.0) 1 (12.5) 1 (3.1)

[0158] The results of the phase I clinical trial showed that in the dosage of not exceeding 1.2 μg/kg/day, the drug-related adverse events are mainly mild ECG abnormality and gastrointestinal disorders, no moderate or severe adverse events were observed. The AEs experienced in this multi-dose trial were similar with that of single-dose trial, supporting that the incidence and intensity of AEs will not increase when the study drug was administered for multiple days.

Example 3: An Open-Label, Single-Center, Parallel Group Study to Evaluate the Efficacy and Safety of Recombinant Human Neuregulin-1 for Injection in Heart Failure Patients on Standard Therapy

[0159] To evaluate the efficacy and safety of recombinant human neuregulin-1 for injection on chronic heart failure and to investigate the effective dosage range of recombinant human neuregulin-1 for injection for treating chronic heart failure, the phase II, open-label, single-center, parallel group, standard treatment based study (Protocol ID: HREC 06/035) was carried out in St Vincent's Hospital in Australia. Fifteen patients with chronic heart failure were enrolled and randomized into 3 groups.

[0160] Investigational Product:

[0161] Specification: Neucardin™, a 61-amino acid polypeptide comprising the EGF-like domain of Neuregulin-1 β2 isoform and having the amino acid sequence set forth in SEQ ID NO:1, with the molecular weight of 7054 Dal (1 μg=0.14 nmol). 250 μg (5000 EU)/vial (1 μg=20 EU).

[0162] Preparation: For injection.

[0163] Mode of administration: Intravenously drip.

[0164] Storage: in safe place, with limited access and protected from light, at 3-8° C.

[0165] Dosage Groups:

[0166] Fifteen patients with chronic heart failure (LVEF≤40%, NYHA class II/III, established on ACEI/ARA and beta blocker with stable doses of these medications for the preceding 3 months) were enrolled and randomized into 3 dosage groups with 5 patients in each group (refer to Table 8).

TABLE-US-00008 TABLE 8 Dosage regimen of phase II trial in Australia Dosage group Sample Dosage (1.sup.st day) Dosage (2.sup.nd-11.sup.th day) A 5 1.2 μg/kg/6 hrs 2.4 μg/kg/12 hrs B 5 1.2 μg/kg/6 hrs 1.2 μg/kg/12 hrs C 5 1.2 μg/kg/6 hrs 0.6 μg/kg/12 hrs

[0167] Study Procedures

[0168] 1) Screening and baseline: [0169] Obtain the written informed consent form; [0170] Collect the medical history; [0171] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0172] Physical examination; [0173] Verify the inclusion/exclusion criteria; [0174] ECG; [0175] FBC; [0176] Neurohormonal, immunological marker (NT-proBNP, PIIINP. NA, Aldos, Endothelin, hsCRP, TNF-α, IL-6); [0177] Ultrasonic cardiogram; [0178] MRI.

[0179] 2) 1.sup.st day of drug administration [0180] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0181] Study drug administration; [0182] ECG; [0183] RH Catheter; [0184] NT-proBNP.

[0185] 3) 2.sup.nd to 11.sup.th day of drug administration [0186] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0187] Study drug administration; [0188] ECG

[0189] 4) 12.sup.th day of the study [0190] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0191] ECG; [0192] RH Catheter; [0193] FBC; [0194] Neurohormonal, immunological marker (NT-proBNP, PIIINP. NA, Aldos, Endothelin, hsCRP, TNF-α, IL-6); [0195] Ultrasonic cardiogram; [0196] MRI.

[0197] 5) 30.sup.th day of the study [0198] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0199] ECG; [0200] FBC; [0201] Neurohormonal, immunological marker (NT-proBNP, PIIINP, NA, Aldos, Endothelin, hsCRP, TNF-α, IL-6); [0202] Ultrasonic cardiogram; [0203] MRI.

[0204] 6) 60.sup.th day of the study [0205] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0206] ECG; [0207] FBC; [0208] Neurohormonal, immunological marker (NT-proBNP, PIIINP, NA, Aldos, Endothelin, hsCRP. TNF-a, IL-6);

[0209] 7) 90.sup.th day of the study [0210] Collect the medical history; [0211] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0212] Physical examination; [0213] ECG; [0214] FBC; [0215] RH Catheter; [0216] Neurohormonal, immunological marker (NT-proBNP, PIIINP, NA, Aldos, Endothelin, hsCRP, TNF-a, IL-6); [0217] Ultrasonic cardiogram; [0218] MRI.

[0219] Results and Analysis

[0220] The primary efficacy endpoint is evaluating the left ventricular function and remodeling (as determined by change in LVEF, LVEDV and LVESV measured by MRI). Hemodynamics parameters and neurohormonal and immunological markers act as secondary efficacy endpoints.

[0221] As a total count, 13 subjects (13/15, 86.7%) received the test drug by IV infusion for 11 consecutive days, one subject (1/15, 6.7%) received the test drug by IV infusion for 10 days, one subject (1/15, 6.7%) received the test drug by IV infusion for 7 days. No subject withdrew.

[0222] In Group A (1.2 μg/kg/day*1 day+2.4 μg/kg/day*10 days), 4 subjects (4/5, 80.0%) received the test drug by IV infusion for 11 consecutive days, one subject (1/5, 20.0%) received the test drug by IV infusion for 7 days. In Group B (1.2 μg/kg/day*1 day+1.2 μg/kg/day*10 days), 5 subjects (5/5, 100.0%) received the test drug by IV infusion for 11 consecutive days. In Group C (1.2 μg/kg/day*1 day+0.6 μg/kg/day*10 days), 4 subjects (4/5, 80.0%) received the test drug by IV infusion for 11 consecutive days, one subject (1/5, 20%) received the test drug by IV infusion for 10 days.

[0223] In group A, after the administration of Neucardin™, at Day 12, the LVEF (%) was improved by 5.4% compared to baseline; the percentage increase was 16.0%; at 1 Month, the LVEF (%) was improved by 3.3% compared to baseline, the percentage of increase was 8.8%; at 3 Months, the LVEF (%) was still improved by 2.6% compared to baseline, the percentage of increase was 6.1%.

[0224] In group B, at Day 12, the LVEF (%) was improved by 3.8% compared to baseline; the percentage increase was 11.8%; at 1 Month, the LVEF (%) was improved by 3.4% compared to baseline, the percentage of increase was 9.7%; at 3 Months, the improvement of LVEF (%) was increased to 4.8% compared to baseline, the percentage of increase was 15.7%.

[0225] In group C, at Day 12, the LVEF (%) was improved by 2.2% compared to baseline; the percentage increase was 8.4%; at 1 Month, the LVEF (%) was improved by 4.6% compared to baseline, the percentage of increase was 15.4%; and the improvement of LVEF (%) sustained to 3 Months by 4.4% compared to baseline, the percentage of increase was 15.5%.

[0226] And for all 15 subjects who completed the study (combined), the improvement of LVEF (%) was 3.8%, 3.8% and 3.9% at day 12, 1 month, 3 month, compared to baseline, respectively (refer to Table 9).

[0227] For all 15 subjects who completed the study (combined) and for each individual dosage group, there was no significant change compared to baseline in LVEDV (ml) at any time point.

[0228] Concerning LVESV (ml), in group A, at Day 12, the LVESV (ml) was decreased by 8.4 ml compared to baseline, the percentage decrease was 4.9%; At 1 Month, the LVESV (ml) was decreased by 14.3 ml compared to baseline, the percentage decrease was 8.8%; At 3 month, the LVESV (ml) was decreased by 12.0 ml compared to baseline, the percentage decrease was 8.3%.

TABLE-US-00009 TABLE 9 Measured values (Mean ± SD) of LVEF (%) Percent change Measured Absolute of LVEF LVEF change of compared to Dosage group Time points value (%) LVEF (%) baseline (%) Group A Baseline (n = 5) 33.2 ± 9.7 (1.2 mcg/kg/day*1 day + Day 12 (n = 5)  38.6 ± 11.6 5.4 ± 2.9 16.0 ± 8.2  2.4 mcg/kg/day*10 days) 1 Month (n = 4)  37.5 ± 13.1 3.3 ± 2.8 8.8 ± 6.3 3 Months (n = 5)  35.8 ± 13.1 2.6 ± 4.0  6.1 ± 11.0 Group B Baseline (n = 5) 30.8 ± 8.0 (1.2 mcg/kg/day*1 day + Day 12 (n = 5)  34.6 ± 10.0 3.8 ± 2.6 11.8 ± 5.9  1.2 mcg/kg/day*10 days) 1 Month (n = 5)  34.2 ± 10.9 3.4 ± 4.1  9.7 ± 12.4 3 Months (n = 5) 35.6 ± 9.4 4.8 ± 3.4 15.7 ± 11.2 Group C Baseline (n = 5) 32.6 ± 7.3 (1.2 mcg/kg/day*1 day + Day 12 (n = 5) 34.8 ± 5.4 2.2 ± 3.7  8.4 ± 11.9 0.6 mcg/kg/day*10 days) 1 Month (n = 5) 37.2 ± 6.8 4.6 ± 3.4 15.4 ± 11.5 3 Months (n = 5) 37.0 ± 5.2 4.4 ± 3.0 15.5 ± 12.9 All subjects Baseline (n = 15) 32.2 ± 7.9 Day 12 (n = 15) 36.0 ± 8.9 3.8 ± 3.2 12.1 ± 8.9  1 Month (n = 14) 36.2 ± 9.6 3.8 ± 3.3 11.5 ± 10.3 3 Months (n = 15) 36.1 ± 9.1 3.9 ± 3.4 12.4 ± 11.8

[0229] In group B, at Day 12, the LVESV (ml) was decreased by 1.0 ml compared to baseline, the percentage decrease was 2.5%; At 1 Month, the LVESV (ml) was decreased by 3.8 ml compared to baseline, the percentage decrease was 2.5%; At 3 month, the LVESV (ml) was decreased by 12.6 ml compared to baseline, the percentage decrease was 6.3%.

[0230] In group C, at Day 12, the LVESV (ml) was decreased by 11.4 ml compared to baseline, the percentage decrease was 8.6%; At 1 Month, the LVESV (ml) was decreased by 8.0 ml compared to baseline, the percentage decrease was 5.1%; At 3 month, the LVESV (ml) was decreased by 10.8 ml compared to baseline, the percentage decrease was 7.1%.

[0231] And for all 15 subjects who completed the study (combined), the decrease of LVESV (ml) was 6.9, 8.3 and 11.8 ml at day 12, 1 month, 3 month, compared to baseline, respectively (refer to Table 10).

TABLE-US-00010 TABLE 10 Measured values (Mean ± SD) of LVESV (ml) Percent change Measured Absolutely of LVESV value of change of compared to Dosage groups Time points LVESV (ml) LVESV (ml) baseline (%) Group A Baseline (n = 5) 194.2 ± 74.0 (1.2 mcg/kg/day*1 day + Day 12 (n = 5) 185.8 ± 73.2  −8.4 ± 10.4 −4.9 ± 6.3 2.4 mcg/kg/day*10 days) 1 Month (n = 4) 172.8 ± 81.5 −14.3 ± 14.9 −8.8 ± 9.8 3 Months (n = 5) 182.2 ± 83.4 −12.0 ± 17.6  −8.3 ± 11.4 Group B Baseline (n = 5) 179.6 ± 34.7 (1.2 mcg/kg/day*1 day + Day 12 (n = 5) 178.6 ± 60.5  −1.0 ± 29.0  −2.5 ± 16.6 1.2 mcg/kg/day*10 days) 1 Month (n = 5) 175.8 ± 40.1  −3.8 ± 14.5 −2.5 ± 8.3 3 Months (n = 5) 167.0 ± 26.0 −12.6 ± 15.6 −6.3 ± 8.3 Group C Baseline (n = 5) 182.4 ± 70.5 (1.2 mcg/kg/day*1 day + Day 12 (n = 5) 171.0 ± 81.6 −11.4 ± 22.2  −8.6 ± 14.2 0.6 mcg/kg/day*10 days) 1 Month (n = 5) 174.4 ± 73.4 −8.0 ± 9.2 −5.1 ± 6.9 3 Months (n = 5) 171.6 ± 74.4 −10.8 ± 8.9  −7.1 ± 6.6 All subjects Baseline (n = 15) 185.4 ± 58.1 Day 12 (n = 15) 178.5 ± 67.2  −6.9 ± 20.8  −5.3 ± 12.4 1 Month (n = 14) 174.4 ± 60.7  −8.3 ± 12.7 −5.2 ± 8.1 3 Months (n = 15) 173.6 ± 61.7 −11.8 ± 13.5 −7.2 ± 8.4

[0232] A 6-hour infusion of Neucardin™ acutely and significantly influenced several hemodynamic parameters when monitored over 24 hours. As shown in Table 11, the acute influence of Neucardin™ on hemodynamics was quite positive.

TABLE-US-00011 TABLE 11 Summary of significant changes in hemodynamics parameters during & after a 6-hour infusion of Neucardin ™ Time points when Time points when the parameters the parameters significantly significantly Hemodynamics increased compared decreased compared parameters to baseline to baseline Heart rate (HR, 1 Hour, 2 Hours, beat/min) 4 Hours & 6 Hours Systolic Blood 6 Hours Pressure (SBP, mmHg) Diastolic Blood 24 Hours Pressure (DBP, mmHg) Mean Blood Pressure 24 Hours (MBP, mmHg) Right Atrial Pressure 1 Hour, 2 Hours, (RA, mmHg) 4 Hours, 6 Hours, 12 Hours & 24 Hours Pulmonary Artery 30 minutes, 1 Hour Systolic Pressure (PASP, mmHg) Pulmonary Artery 2 Hours, 12 Hours & Diastolic Pressure 24 Hours (PADP, mmHg) Mean Pulmonary 2 Hours, 12 Hours Arterial Pressure & 24 Hours (MPAP, mmHg) Pulmonary Arterial 30 minutes, 1 Hour, Wedge Pressure 2 Hours, 4 Hours, (PAWP, mmHg) 6 Hours, 12 Hours & 24 Hours Transpulmonary 30 minutes, 1 Hour, Pressure Gradient 2 Hours, 4 Hours, (TPG, mmHg) 6 Hours & 24 Hours Pulmonary Vascular Resistance (PVR, dynes .Math. sec .Math. cm.sup.−5) Systemic Vascular 30 minutes, 2 Hours, Resistance (SVR, 4 Hours, 6 Hours & dynes .Math. sec .Math. cm.sup.−5) 24 Hours Cardiac Output 30 minutes, 1 Hour, (CO, L/min) 2 Hours, 4 Hours, 6 Hours & 24 Hours Cardiac Index 30 minutes, 1 Hour, (CI, L/min/m.sup.2) 2 Hours, 4 Hours, 6 Hours & 24 Hours Stroke Volume 30 minutes, 2 Hours, (SV, ml) 4 Hours, 12 Hours & 24 Hours Stroke Volume 30 minutes, 2 Hours, Index (SVI, ml/m.sup.2) 4 Hours, 12 Hours & 24 Hours

[0233] The long term significant changes in hemodynamics for the study subjects are summarized in the following table, and details are listed below (Table 12).

TABLE-US-00012 TABLE 12 Summary of significant changes in long-term hemodynamic parameters Time points when the Time points when the parameters significantly parameters significantly Hemodynamics increased compared to decreased compared to parameters baseline baseline SBP (mmHg) Day 12, 3 Months DBP (mmHg) Day 12, 3 Months MBP (mmHg) Day 12, 3 Months TPG (mmHg) Day 12, 3 Months PAWP (mmHg) 3 Months

[0234] Concerning the neurohormonal and immunological biomarkers, the NT-proBNP was increased after the 6-hour infusion of Neucardin™ on the first day, and did not return to baseline at 24 hours from the start of the administration, and after the end of medication, it decreased and returned to baseline at the end of the study.

[0235] No significant change was observed in hs-CRP.

[0236] Regarding the safety evaluation, 10 subjects (10/15, 66.7%) experienced at least one adverse event (AE). The most commonly reported individual AEs were nausea (20%), lethargy (20%) and chest pain (20%), without a clear causal relationship to dose. Approximately 67% of subjects had ECG abnormalities during or following study drug dosing, including: T wave inversions or flattening, ST-T segment changes, premature ventricular contractions, non-sustained runs of supraventricular or ventricular tachycardia or atrial flutter/fibrillation. As ECG abnormalities are common in CHF subjects and there was no placebo group for comparison, it is difficult to assess causality or relationship to drug in this study.

[0237] One subject in Group A (2.4 μg/kg/day) and one subject in Group C (0.6 μg/kg/day) experienced serious adverse event(s) (SAEs).

[0238] There were no clinical significant abnormalities identified concerning to vital signs, physical examination, laboratory parameters.

[0239] According to the results of the phase II trial, recombinant human neuregulin-1 is believed to be effective and safe to treat patients with chronic heart failure in the dosage range between 0.6 to 2.4 μg/kg/day (12-48 EU/kg/day or 0.08-0.34 nmol/kg/day).

Example 4: A Randomized, Double-Blinded, Multi-Center, Placebo Controlled Study to Evaluate the Efficacy and Safety of Recombinant Human Neuregulin 1 in Patients with Chronic Heart Failure Based on Standard Treatment

[0240] To evaluate the efficacy and safety of recombinant human neuregulin-1 for injection on chronic congestive heart failure and to investigate the effective dosage range of recombinant human neuregulin-1 for injection for treating chronic congestive heart failure, the phase II, double-blinded, multi-center, placebo controlled, standard treatment based study (Protocol ID: ZS-01-206) was carried out in multiple clinical centers in China. Sixty four patients diagnosed as chronic heart failure originating from one or more factors including dilated cardiomyopathy, hypertension, viral myocarditis, myocardial infarction, and alcoholic cardiomyopathy, were enrolled and randomized into 4 groups.

[0241] Investigational Product:

[0242] Specification: Neucardin™, a 61-amino acid polypeptide comprising the EGF-like domain of Neuregulin-1 β2 isoform and having the amino acid sequence set forth in SEQ ID NO:1, with the molecular weight of 7054 Dal (1 μg=0.14 nmol). 250 μg (5000 EU)/vial (1 μg=20 EU).

[0243] Preparation: For injection.

[0244] Mode of administration: Intravenously drip.

[0245] Storage: in safe place, with limited access and protected from light, at 3-8° C.

[0246] Placebo:

[0247] Specification: Excipient for Neucardin™. 250 μg/vial and without active recombinant human neuregulin-1 protein.

[0248] Dosage Groups:

[0249] Sixty four patients with chronic heart failure (LVEF≤40%, NYHA class established on ACEI/ARA, beta blocker, diuretic or digoxin with stable doses of these medications for 1 month) were designed to be enrolled and randomized into 4 dosage groups with 16 patients in each group (refer to Table 13).

TABLE-US-00013 TABLE 13 Dosage groups and regimen Dosage 0 μg/kg/day 0.3 μg/kg/day 0.6 μg/kg/day 1.2 μg/kg/day Administration Intravenous infusion Volume 50 ml Course 10 hours per day, for consecutive 10 days

[0250] Study Procedures

[0251] 1) Screening and baseline: [0252] Obtain the written informed consent form; [0253] Demography; [0254] Collect the medical history; [0255] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0256] Physical examination; [0257] Blood/Urine routine, blood biochemistry; [0258] Blood coagulation test: APTT, PT; [0259] NT-proBNP; [0260] ECG; [0261] NYHA classification; [0262] Ultrasonic cardiogram; [0263] Chest radiography; [0264] MRI; [0265] Tissue Doppler (breast, liver, gallbladder, kidney, adrenal gland, pancrease, spleen, ovary, uterus, prostate); [0266] Urine pregnancy test (women of child-bearing age); [0267] Dyspnea evaluation at rest stage; [0268] Quality of life; [0269] Six minute walking test; [0270] Verify the inclusion/exclusion criteria; [0271] Urine volume for 24 hours; [0272] Record adverse events and serious adverse events; [0273] Record concomitant medications.

[0274] 2) 1.sup.st to 10.sup.th day of drug administration [0275] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0276] ECG; [0277] Study drug administration; [0278] Urine volume for 24 hours; [0279] Record adverse events and serious adverse events; [0280] Record concomitant medications.

[0281] 3) 11.sup.th-13.sup.th day of the study [0282] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0283] Physical examination; [0284] ECG; [0285] Blood/Urine routine, blood biochemistry; [0286] Blood coagulation test: APTT, PT; [0287] NT-proBNP; [0288] Urine pregnancy test (women of child-bearing age); [0289] Quality of life; [0290] Six minute walking test; [0291] MRI; [0292] NYHA classification; [0293] Dyspnea evaluation at rest stage; [0294] Urine volume for 24 hours; [0295] Record adverse events and serious adverse events; [0296] Record concomitant medications.

[0297] 4) 30.sup.th and 90.sup.th day of the study [0298] Vital signs (supine blood pressure, pulse, respiratory rate, temperature); [0299] Physical examination; [0300] Blood/Urine routine, blood biochemistry; [0301] Blood coagulation test: APTT, PT; [0302] NT-proBNP; [0303] ECG; [0304] Chest radiography; [0305] Six minute walking test; [0306] MRI; [0307] NYHA classification; [0308] Dyspnea evaluation at rest stage; [0309] Quality of life; [0310] Record adverse events and serious adverse events; [0311] Record concomitant medications.

[0312] Results and Analysis

[0313] Forty patients completed the trial. The primary efficacy endpoint is heart function parameters (LVEF, LVEDV and LVESV) at day 30. Concerning LVEF, the mean value of LVEF is decreased from 21.54% at baseline to 20.93% at day 30 in the group of placebo. While in the group of 0.3 μg/kg/day, the mean value of LVEF is slightly improved from 25.08% at baseline to 26.61%, the percentage increase is 4.88%. And in the group of 0.6 μg/kg/day, the mean value of LVEF is significantly increased from 23.03% at baseline to 28.01% at day 30, the percentage increase is 27.11% (refer to Table 14).

[0314] Concerning LVEDV, the mean value of LVEDV is increased from 392.16 ml at baseline to 413.35 ml at day 30 in the group of placebo, with the percentage increase of 5.93%. While in the group of 0.3 μg/kg/day, the mean value of LVEDV slightly decreased from 333.62 ml at baseline to 323.22 ml at day 30, with the percentage decrease 3.28%. In the group of 0.6 μg/kg/day and 1.2n/kg/day, the mean value of LVEDV significantly decreased from 408.51 ml and 397.04 ml at baseline to 386.89 ml and 374.46 ml at day 30, respectively, both with the percentage decrease more than 5% (refer to Table 15).

TABLE-US-00014 TABLE 14 LVEF value before and after treatment Group A Group B Group C Group D (Placebo) 0.3 μg/kg/day 0.6 μg/kg/day 1.2 μg/kg/day n = 10 n = 11 n = 11 n = 8 Baseline (%) 21.54 ± 4.73 25.08 ± 7.64 23.03 ± 10.23 22.64 ± 4.24 Day 30 (%) 20.93 ± 8.53 26.61 ± 9.68 28.01 ± 11.27 22.01 ± 7.21 ΔLVEF (%) −0.61 ± 8.22  1.53 ± 3.81 4.98 ± 5.37 −0.63 ± 4.34 Changed percentage (%)  −1.39 ± 33.34  4.88 ± 17.07 27.11 ± 31.12  −4.01 ± 19.42

TABLE-US-00015 TABLE 15 LVEDV value before and after treatment Group A Group B Group C Group D (Placebo) 0.3 μg/kg/day 0.6 μg/kg/day 1.2 μg/kg/day n = 10 n = 11 n = 11 n = 8 Baseline (ml) 392.16 ± 107.38 333.62 ± 106.14 408.51 ± 142.85 397.04 ± 86.94 Day 30 (ml) 413.35 ± 143.98 323.22 ± 111.86 386.89 ± 150.14 374.46 ± 84.47 ΔLVEDV (ml)  21.20 ± 108.13 −10.40 ± 33.19  −21.63 ± 42.72  −22.58 ± 37.72 Changed percentage (%)  5.93 ± 31.48 −3.28 ± 11.34 −5.64 ± 10.03 −5.41 ± 8.53

[0315] The result of LVESV is similar with that of LVEDV, in the group of placebo, the mean value of LVESV increased from 310.54 ml at baseline to 335.78 ml at day 30, with the percentage increase 9.45%. While in each group of recombinant neuregulin treatment, the mean value of LVESV decreased. For the group of 0.6 μg/kg/day as an example, the mean value of LVESV decreased from 325.02 ml at baseline to 291.71 ml at day 30, with the percentage decrease 11.58% (refer to Table 16).

TABLE-US-00016 TABLE 16 LVESV value before and after treatment Group A Group B Group C Group D (Placebo ) 0.3 μg/kg/day 0.6 μg/kg/day 1.2 μg/kg/day n = 10 n = 11 n = 11 n = 8 Baseline (ml) 310.54 ± 97.54  254.85 ± 100.86 325.02 ± 142.70 308.46 ± 74.37 Day 30 (ml) 335.78 ± 145.37  244.01 ± 110.30 291.71 ± 147.66 292.22 ± 69.20 ΔLVESV (ml) 25.24 ± 119.19 −10.84 ± 25.10  −33.31 ± 44.54  −16.24 ± 20.89 Changed percentage (%) 9.45 ± 48.04 −5.33 ± 12.89 −11.58 ± 12.74  −5.01 ± 6.77

[0316] NT-proBNP is an important independent prognostic factor for survival rate of patients with chronic heart failure. From the result, the value of NT-proBNP in the group of placebo and 1.2 μg/kg/day at day 30 and day 90 increased significantly compared to that of baseline. While in the group of 0.3 μg/kg/day and 0.6 μg/kg/day, it shows a trend of decrease (refer to Table 17), suggesting a better prognosis.

TABLE-US-00017 TABLE 17 Change of NT-proBNP at day 30 and day 90 Group A Group B Group C Group D (Placebo) 0.3 μg/kg/day 0.6 μg/kg/day 1.2 μg/kg/day n = 10 n = 11 n = 11 n = 8 Baseline (fmol/ml) 1013 ± 634.5  1732 ± 1638.4 1403 ± 1755.2 859 ± 460.7 Day 30 (fmol/ml)  811 ± 436.6 1631 ± 1222.1 1021 ± 1045.9 822 ± 349.4 Changed Percentage (%) 6.54 ± 74.29 25.68 ± 72.09.sup.  −4.27 ± 41.37.sup.  49.42 ± 118.19  Day 90 (fmol/ml)  869 ± 440.9 1663 ± 1664.1 1112 ± 1418.5 847 ± 316.2 Changed Percentage (%) 26.79 ± 102.61 −3.5 ± 60.07  8.66 ± 74.83  45.99 ± 97.45.sup. 

[0317] There were adverse events in each group, particularly, in the group of 1.2 μg/kg/day, 100% patients in the group experienced at least on adverse event. The most commonly reported individual AEs were nausea, poor appetite and headache. There was no serious adverse events reported (refer to Table 18). Most reported adverse events relieved without treatment and the rest relieved after the cease of study drug administration or by symptomatic treatment and without sequelae.

[0318] There were no clinical significant abnormalities identified concerning to vital signs and physical examination.

[0319] From the results of efficacy and safety, we can find that in the dosage of 0.3-1.2 μg/kg/day (6-24 EU/kg/day or 0.04-0.17 nmol/kg/day), recombinant human neuregulin-1 is effective and safe for the treatment of chronic heart failure.

TABLE-US-00018 TABLE 18 Incidence of AE and SAE in each group Dosage group With Without Incidence (%) AE Group A (n = 11) 6 5 54.5 Group B (n = 11) 7 4 63.64 Group C (n = 12) 8 4 66.67 Group D (n = 10) 10 0 100 SAE Group A (n = 11) 0 11 0 Group B (n = 11) 0 11 0 Group C (n = 12) 0 12 0 Group D (n = 10) 0 10 0

[0320] From the results of these clinical trials, we can make a conclusion that in the dosage of 0.3-2.4 μg/kg/day (6-48 EU/kg/day or 0.04-0.34 nmol/kg/day), recombinant human neuregulin-1 is tolerant and effective for the treatment of chronic heart failure.