COMPOSITIONS AND METHODS FOR TREATING HEART FAILURE IN DIABETIC PATIENTS

Abstract

The present invention provides the use of neuregulin protein for the preparation of medication for preventing, treating or delaying heart failure in humans and methods for preventing, treating or delaying heart failure in humans using said medication.

Claims

1.-10. (canceled)

11. A composition comprising neuregulin, wherein the composition is suitable for use in preventing, treating or delaying cardiac complications in a diabetic patient.

12. The composition of claim 11, wherein the neuregulin is neuregulin-1.

13. The composition of claim 11, wherein the neuregulin protein comprises of EGF-like domain of neuregulin-1β.

14. The composition of claim 11, wherein the neuregulin comprises of SEQ ID NO: 1.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0049] FIG. 1: The results of NT-proBNP in diabetic patients with rhNRG-1 and placebo in day 11, day 30 and 90.

EXAMPLES

Example 1: The Effect of Neucardin™ Administration by Different Routes on the Survival Rate of Rats with CHF

[0050] Introduction:

[0051] In this study, we used a coronary artery ligation (CAL) model mimicking the myocardial infarction in the diabetic patients to investigate whether administration of Neucardin™ by IV drip using a by micro-injection pump or by subcutaneous (SC) bolus had any effects on survival rate and cardiac hemodynamics, 120 days after the initiation of administration of Neucardin™ 4 weeks after CAL. Echocardiography and cardiac remodeling were also used to determine cardiac function and recovery from CAL.

[0052] 2. Methods:

[0053] 2.1. Test Animals:

[0054] Strain, Origin: Wistar rats, Shanghai SLAC Laboratory Animal CO. LTD; Weight, 200±10 g, male;

[0055] 2.2 Test Article:

[0056] 2.2.1 Neucardin™

[0057] Identification: Recombinant human neuregulin-1 for injection (rhNRG-1, Neucardin™)

[0058] Lot Number: 200607009

[0059] Manufacturer: Zensun (Shanghai) Sci & Tech Co., Ltd

[0060] Dose form: Lyophilized powder

[0061] Appearance: White or off-white cake

[0062] Labeled Content of rhNRG-1: 250 μg/vial

[0063] Specific activity: 4897 U/vial

[0064] Storage conditions: 2-8° C.

[0065] 2.2.2 Vehicle:

[0066] Identification: Placebo for recombinant human neuregulin-1

[0067] Dose form: Lyophilized powder

[0068] Appearance: White or off-white cake

[0069] Composition: Human serum albumin, mannitol, phosphate, NaCl

[0070] Storage conditions: 2-8° C.

[0071] 2.3 Procedure:

[0072] 2.3.1 to Establish the Rat CHF Model:

[0073] The LAD of the rats was ligated. Briefly, the rats were anesthetized with ketamine hydrochloride (100 mg/kg, IP) and their chest was shaved and sterilized. The rats were endotracheally intubated and mechanically ventilated with room air (respiratory rate 60 breaths/min, tidal volume 20 ml). A left thoracotomy was then performed at the 4th and 5th intercostal space and then the skin was incised along the left sternal border. The fourth rib was then cut proximal to the sternum. The pericardial sac was perforated and the heart was exposed. The LAD was ligated approximately 2 mm from its origin using a 6-0 silk suture. Subsequently, the air within the thorax was removed and the chest was closed in three layers (ribs, muscles and then skin). The rats were then allowed to resume spontaneous respiration, recover from the anesthesia and were then returned to their cages. Rats were maintained during a period of 4 weeks, then echocardiography evaluated, included in the formal study if they were shown an EF % value of 30-45%. Animals from all Groups were housed 5 per cage, fed ad libitum with standard diet and had free access to pure water. Room temperature was maintained at 21±1° C. and in a12 h light/dark cycle.

[0074] 2.3.2 IV Drip Via Microinjection Pump:

[0075] The method of IV drip of vehicle or Neucardin™ was through the tail vein. For this procedure, an appropriate rat restrainer was used according to the weight of the animal. The rat was placed near the restrainer and was gently placed into the apparatus. Normally the rats entered the restrainer without aid. Subsequently the tail of the rat was swabbed with a gauze dampened with alcohol to increase blood flow to the tail vein and to the intenerate skin corneum. The two lateral (on the side) tail veins were located and with the bevel of the needle facing upward with the needle almost parallel to the vein, the needle was inserted 2 mm into the tail vein 2-3 cm from the end of the tail. To confirm that the needle was successfully inserted into the tail vein, blood was extracted into the hub of the needle. The needle was fixed into the tail using medical tape. The infusion of drug or vehicle at the appropriate rate (0.2-0.4 ml/h) by microinjection pump or bolus injection was initiated.

[0076] 2.3.3 SC Bolus

[0077] The SC bolus of vehicle or Neucardin™ was from the back of the rat. For this procedure, an appropriate rat restrainer was used according to the weight of the animal. The back of the rat was swabbed with gauze dampened with alcohol to sterilize the skin. With the bevel of the needle facing upward with the needle almost parallel to the skin, the needle was subcutaneously inserted 3-4 cm into the back of the rat. The needle was fixed onto the back using medical tape and connected to the perfusion tube. Then, the rat was placed near the restrainer and was gently placed into the apparatus. Normally the rats entered the restrainer with no aid. After fasten the restrainer, the bolus injection was initiated.

[0078] 2.3.4 Experiment Groups and Drug Infusion:

[0079] MI rats were randomized by EF % value into four Groups as follows:

[0080] Group A (Negative control) for both IV and SC bolus. n=58 rats: IV drip of vehicle for 10-days by micro-injection pump at a speed of 0.2 ml/h for 8 h each day for the first 10 days, SC bolus of vehicle (same volume as Neucardin™), every 5 days until Day 120;

[0081] Group B (SC bolus Neucardin™), n=58: IV drip of vehicle by micro-injection pump at a speed of 0.2 ml/h for 8 h each day in the first 10 days, SC bolus Neucardin™ (10 μg/day), every 5 days until Day 120;

[0082] Group C (IV drip Neucardin™), n=57: IV drip of Neucardin™ (0.625 m/kg/h) by micro-injection pump at a speed of 0.2 ml/h for 8 h each day for the first 10 days, SC bolus of vehicle (same volume as Neucardin™), every 5 days until Day 120.

[0083] Group D (IV drip and SC bolus Neucardin™), n=57: IV drip of Neucardin™ (0.625 μg/kg/h) by micro-injection pump at a rate of 0.2 ml/h for 8 h per day for the first 10 days, SC bolus of vehicle (same volume as Neucardin™) at 1st, 6th, 11th day, and then SC bolus Neucardin™ (10 μg/kg), every 5 days from 16th day to the end.

[0084] 2.3.5 Data Acquisition

[0085] Survival rate; Echocardiography parameters; Hemodynamics parameters;

[0086] 3. Results

[0087] 3.1 Survival Rate:

[0088] Table 1 illustrates the survival rates between each Group. The survival rates were 48.3%, 62.1%, 64.9% and 82.5% in the IV drip & SC bolus of vehicle Group A, SC bolus of Neucardin™ Group B, IV drip of Neucardin™ Group C and IV drip & SC bolus of Neucardin™ Group D, respectively. All the survival rate or mean survival time of mortalities in Group B, C and D were improved or prolonged compared to Group A with Group D had best efficacy.

TABLE-US-00001 TABLE 1 Mortality, Survival rate and Mean survival time in the four Groups Mean survival time of Start rat Survival Survival mortalities Group Treatment number Deaths rat number rate (%) in days ± S.E. A Vehicle 58 30 28 48.3% 83.8 ± 5.9 B SC bolus Neucardin ™ 58 22 36 62.1% 91.4 ± 5.5 C IV drip Neucardin ™ 57 20 37 64.9% 97.5 ± 5.1 D SC bolus & IV drip 57 10 47 82.5% 107.5 ± 4.1  Neucardin ™

[0089] 3.2 Echocardiography Parameters:

[0090] Echocardiography parameters were shown in Table 2. Four-weeks after coronary artery ligation and before administration of the test article, the CHF rats were randomized into four Groups according to their EF % values. As shown in Table 2, there were no significant differences between the four Groups before treatment (BT). 120 days after the start of administration, the EF % values were 30.7±3.1, 32.9±4.1, 33.5±3.4, 36.2±4.8% in the vehicle, Neucardin™ via SC bolus, Neucardin™ via IV drip and Neucardin™ via IV drip plus SC bolus Groups, respectively. After treatment, EF % and FS % of Group B, C and D were all higher than that of Group A.

TABLE-US-00002 TABLE 2 Echocardiography parameters in the four Groups BT LVEDd LVEDs EF % FS % Group AT N (cm) (cm) (%) (%) A. Negative BT 58 0.987 ± 0.829 ± 38.0 ± 16.2 ± control 0.083 0.088 5.5 2.7 AT 25 1.100 ± 0.961 ± 30.7 ± 12.7 ± 0.089 0.090 3.1 1.4 B. SC bolus BT 58 0.992 ± 0.831 ± 38.2 ± 16.3 ± Neucardin ™ 0.066 0.066 4.0 2.0 AT 33 1.104 ± 0.952 ± 33.1 ± 13.9 ± 0.063 0.070 4.1 1.9 C. IV drip BT 57 0.985 ± 0.824 ± 38.5 ± 16.3 ± Neucardin ™ 0.061 0.068 4.4 2.1 AT 36 1.080 ± 0.929 ± 33.4 ± 14.0 ± 0.072 0.073 3.4 1.6 D. SC bolus & BT 57 0.979 ± 0.818 ± 38.7 ± 16.5 ± IV drip 0.065 0.066 4.3 2.1 Neucardin ™ AT 44 1.052 ± 0.893 ± 36.2 ± 15.3 ± 0.087 0.092 4.8 2.4 BT: Before treatment; AT: After treatment;

[0091] 3.3 Hemodynamic Parameters:

[0092] Table 3 shows the MAP, HR, ±dp/dt, LVEDP and LVSP values as measured in the four Groups of anesthetized animals on day 121. When Neucardin™ was administered by SC bolus or by IV drip alone (Group B and C), Neucardin™ significantly increased dp/dt and −dp/dt by 19.6% and 27.1%, 22.5% and 29.8% compared to Group A. When Neucardin™ was administered by both IV drip and SC bolus routes (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/s), −dp/dt (−4353.2±847.6 mmHg/s) compared to vehicle were obtained. Interestingly, these values of MAP, LVSP, +dp/dt and −dp/dt were 10.6%, 9.2%, 38.5% and 37.8% higher than vehicle treated rats, respectively. The results showed that Group B, C and D were all better than Group A in hemodynamic parameters with Group D had best efficacy.

TABLE-US-00003 TABLE 3 Hemodynamics parameters in the four Groups SBP DBP MAP LVSP Group Treatment N (mmHg) (mmHg) (mmHg) (mmHg) A Vehicle 14 118.7 ± 11.5  94.1 ± 12.3  102.3 ± 11.7 128.5 ± 14.7 B SC bolus Neucardin ™ 27 123.8 ± 11.5 95.3 ± 8.9 104.9 ± 9.5 129.5 ± 13.6 C IV drip Neucardin ™ 25 122.5 ± 10.5 95.0 ± 7.5 104.4 ± 8.2 131.7 ± 10.0 D SC bolus & IV drip 35 132.6 ± 7.1  102.1 ± 5.3  112.3 ± 5.5 139.4 ± 9.8  Neucardin ™ LVEDP dp/dt (-dp/dt) Heart rate Group Treatment N (mmHg) (mmHg/s) (mmHg/s) (Beat/min) A Vehicle 14 5.8 ± 3.5 4995.6 ± 532.2 3087.5 ± 715.7 297.2 ± 16.0 B SC bolus Neucardin ™ 27 4.5 ± 2.8  6050.9 ± 1231.3 4013.8 ± 838.3 292.6 ± 23.0 C IV drip Neucardin ™ 25 4.0 ± 3.2 6199.9 ± 709.5 4098.9 ± 823.5 296.3 ± 13.5 D SC bolus & IV drip 35 3.9 ± 2.5 7012.1 ± 903.0 4353.2 ± 847.6 292.5 ± 19.1 Neucardin ™

[0093] 4. Conclusion

[0094] A combined administration of Neucardin™ by IV drip & SC bolus or administration of the peptide given by each route alone all increased the survival rate of rats with CHF induced by CAL and improved cardiac functional parameters compared to rats treated with vehicle.

Example 2: 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

[0095] To evaluate the efficacy of recombinant human neuregulin-1 for injection on chronic heart failure including those diabetic patients with chronic heart failure, a phase II, double-blinded, multi-center, placebo controlled, standard treatment based study was carried out in multiple clinical centers in China. A total of 195 patients with NYHA Class II or III stable chronic heart failure including 21 diabetic patients were enrolled and randomized into three groups: placebo, or 0.6 μg/kg and 1.2 μg/kg of rhNRG-1. There were 13 patients with diabetic in Neucardin group (5 in 0.6 μg/kg/day arm and 8 in 1.2 μg/kg/day arm), and 9 patients in placebo group. There were no significant variations in demographics or background therapies among groups. According to the schedule, patients were administered the drug for 10 consecutive days in the hospital first, after finishing the day 11 follow up, they were discharged from the hospital. Another two on site follow up were at day 30 and day 90. A telephone interview was conducted one year after the last patient enrolled.

[0096] Investigational Product:

[0097] Specification: Neucardin™, 61 amino acid polypeptide comprises the EGF-like domain of Neuregulin-1 P2 isoform, with the molecular weight of 7054 Dal (1 μg=0.14 nmol). 250 μg (5000 EU)/vial (lpg=20 EU).

[0098] Preparation: For injection.

[0099] Mode of administration: Intravenously drip.

[0100] Storage: in safe place, with limited access and protected from light, at 3-8° C.

[0101] Placebo:

[0102] Specification: Excipient for Neucardin™. 250 μg/vial and without active recombinant human neuregulin-1 protein.

[0103] Dosage groups:

TABLE-US-00004 Dosage 0 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

[0104] Study Procedure

[0105] Patients were randomly assigned to three groups, treated with placebo or rhNRG-1 (0.6 or 1.2 μg/kg/day) for 10 consecutive days, after finishing the day 11 follow up, they were discharged from the hospital. Another two on site follow up were at day 30 and day 90. Blood samples of each patient were collected before treatment and at day 11, 30 and 90. Plasma NT-proBNP was tested in the core lab with NT-proBNP assays (kit from Biomedica).

[0106] With regards to the diabetic patients (5 in 0.6 μg/kg/day arm and 8 in 1.2 μg/kg/day arm, and 9 in placebo group), as shown in FIG. 1, decreased NT-proBNP levels in Neucardin™ treated patients in day 30 and 90 were observed, indicating improved long-term prognosis. For the increased level in day 11, it was caused by direct release of BNP from cardiomyocytes by Neucardin™ stimulation, which has been confirmed in preclinical studies.

[0107] These results showed that rhNRG-1 treatment can reduce the plasma level of NT-proBNP, which may indicate rhNRG-1 can provide long-term benefits to diabetic patients with chronic heart failure.

Example 3: A Randomized, Double-Blinded, Multi-Center, Placebo Controlled Survival Study of Recombinant Human Neuregulin 1 in Patients with Chronic Heart Failure Based on Standard Treatment

[0108] To evaluate the efficacy of recombinant human neuregulin-1 for injection on chronic heart failure including those diabetic patients with chronic heart failure, a phase II, double-blinded, multi-center, placebo controlled, standard treatment based study was carried out in multiple clinical centers in China. A total of 351 patients with NYHA Class III or IV stable chronic heart failure were enrolled and randomized into placebo group or rhNRG-1 group (0.6 μg/kg). 68 of 351 patients were diabetic patients, with 35 cases in placebo group, and 33 cases in Neucardin™ group. There were no significant variations in demographics or background therapies among groups. According to the schedule, patients were administered with the drug for 10 consecutive days in the hospital, after finishing the day 11 follow up, they were discharged from the hospital, and were administered with the drug once weekly from the 3.sup.rd week till the 25.sup.th week as out-patient. Blood sampals of each patient were collected before treatment (baseline) and at each follow up. The survival information was collected at 52th week of the study.

[0109] Investigational Product:

[0110] Specification: Neucardin™, 61 amino acid polypeptide comprises the EGF-like domain of Neuregulin-1 β2 isoform, with the molecular weight of 7054 Dal (1 μg=0.14 nmol). 250 μg (5000 EU)/vial (1 μg=20 EU).

[0111] Preparation: For injection.

[0112] Mode of administration: Intravenously drip or infusion.

[0113] Storage: in safe place, with limited access and protected from light, at 3-8° C.

[0114] Placebo:

[0115] Specification: Excipient for Neucardin™. 250 μg/vial and without active recombinant human neuregulin-1 protein.

[0116] Dosage and Regimens:

TABLE-US-00005 Day 1-10 Week 3-25 Dose 0.6 μg/kg/day rhNRG-1 or 0.8 μg/kg/day rhNRG-1or placebo placebo Route Intravenous drip Intravenous infusion regimen 10 hours per day for 10 days 10 minures infusion weekly

[0117] Regarding the diabetic patients, the all-cause mortality of the placebo group at 52 week is 20%, with 7 death in 35 patients, while the number is 9.1% in rhNRG-1 group, with 3 death in 33 patients completed the trial. So from the results we can find around 55% decrease of the mortality of rhNRG-1 administration compared with placebo group, even the placebo group were still maintain their previous standard treatment for chronic heart failure.

[0118] Results of this study showed rhNRG-1 could significantly lower the mortality of diabetic patients with cardiac complications especially HF.