Recombinant Human Neuregulin Derivatives and Use Thereof

Abstract

Disclosed is a use of recombinant human neuregulin derivatives in preparing a medicine for preventing, treating, or reducing the progression of cardiovascular diseases in mammals. In particular, the present invention relates to a novel recombinant human NRG-FC protein and a use thereof in the treatment of cardiovascular diseases. The protein has a prolonged half-life and enhanced biological activity.

Claims

1. An NRG fusion polypeptide, comprising an amino acid sequence of an EGF-like domain of NRG and an amino acid sequence of an IgG Fc or Fc analogue.

2. The NRG fusion polypeptide according to claim 1, wherein the amino acid sequence of the EGF-like domain of NRG is shown in the amino acid sequence of SEQ ID NO: 1 or an analogue thereof.

3. The NRG fusion polypeptide according to claim 1, wherein the IgG Fc has the amino acid sequence of IgG1 or IgG4 subtype Fc or an analogue thereof.

4. The NRG fusion polypeptide according to claim 3, further comprising a linker peptide sequence, and the EGF-like domain of NRG is linked to the IgG Fc through the linker peptide.

5. The NRG fusion polypeptide according to claim 3, wherein the N-terminus of the polypeptide further comprises an amino acid sequence of an IL-2 signal peptide, and the amino acid sequence of the IL-2 signal peptide is cleaved during extracellular secretion of the recombinantly prepared NRG fusion polypeptide.

6. The NRG fusion polypeptide according to claim 1, comprising an amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

7. Use of an NRG fusion polypeptide in the manufacture of a medicament for preventing, treating or relieving cardiovascular diseases in mammals.

8. The use according to claim 7, wherein the NRG fusion polypeptide comprises an amino acid sequence of an EGF-like domain of NRG, and an amino acid sequence of an IgG Fc or Fc analogue.

9. A pharmaceutical formulation, comprising an NRG fusion polypeptide and a pharmaceutically acceptable carrier, excipient or diluent, wherein the NRG fusion polypeptide comprises an amino acid sequence of an EGF-like domain of NRG, and an amino acid sequence of an IgG Fc or Fc analogue.

10. A method of treating cardiovascular diseases, comprising administering an effective dose of an NRG fusion polypeptide in an individual in need thereof, wherein the NRG fusion polypeptide comprises an amino acid sequence of an EGF-like domain of NRG, and an amino acid sequence of an IgG Fc or Fc analogue.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0074] FIG. 1 is a schematic diagram of the expression vector

[0075] FIG. 2 is a schematic diagram of NRG-IgG1/IgG4-Fc fusion protein

[0076] FIG. 3 shows the SDS-PAGE/Western blot results of expression in IFN/IL2-eGFP 293F

[0077] FIG. 4 shows the SDS-PAGE/Western blot results of expression in 1L2-NRG-IgG1-Fc 293F

[0078] FIG. 5 shows the SDS-PAGE results of purified NRG-IgG1-Fc fusion polypeptide (in reduced state)

[0079] FIG. 6 shows the echocardiographic results of all rats with heart failure before and after medication administration

EMBODIMENTS

Embodiment 1 Construction of a Vector for Expressing Fusion Polypeptide

[0080] A full-length DNA sequence containing human NRG (SEQ ID NO: 1), linkers and human immunoglobulin (IgG1 or IgG4) Fc fragments was subcloned to the pcDNA3.1 (+) vector. A sequence containing the restriction enzyme Hind III site, Kozak sequence and melittin signal peptide was introduced at the 5′ end, while a sequence containing the EcoRI site was introduced at the 3′ end. After correct sequencing, it was transferred into E. coli through CaCl.sub.2) for amplification, with the plasmid preserved.

[0081] A DNA sequence containing IL-2-eGFP and IFN-eGFP was subcloned into the pcDNA3.1 (+) vector. A sequence containing the restriction enzyme Hind III site and Kozak sequence was introduced at the 5′ end, while a sequence containing the EcoRI site was introduced at the 3′ end. After correct sequencing, it was transferred into E. coli through CaCl2 for amplification, with the plasmid preserved.

[0082] A full-length DNA sequence containing human NRG (SEQ ID NO: 1), linkers and human immunoglobulin (IgG1 or IgG4) Fc fragments was subcloned to the pcDNA3.1 (+) vector. A sequence containing the restriction enzyme Hind III site, Kozak sequence and IL-2 signal peptide or IFN signal peptide sequence was introduced at the 5′ end, while a sequence containing the EcoRI site was introduced at the 3′ end. After correct sequencing, it was transferred into E. coli through CaCl2 for amplification, with the plasmid preserved.

[0083] For example, the above full-length DNA sequence containing NRG, linker peptide and human immunoglobulin (IgG1 or IgG4) Fc fragments has the corresponding amino acid sequence of SEQ ID NO: 2 and SEQ ID NO: 3. FIG. 1 shows vector constructed. FIG. 2 is a schematic diagram of NRG-IgG1/IgG4-Fc fusion protein.

Embodiment 2 Protein Expression and Detection

[0084] The correctly sequenced plasmid was transiently transfected into 293F cells. Preparation before transfection: 293F cells at the logarithmic growth phase, with activity>95%, were inoculated into a fresh SMM 293-TII culture medium mixed with 1% penicillin/streptomycin mixture, and the density was adjusted to 1.2-1.5*10.sup.6 cells/ml, cultured for 24 h. On the day of transfection, the cell viability should be >90%, the cell density should be adjusted to 2.0-2.5×10.sup.6 cells/mL, and the volume should be 20 mL.

[0085] Melittin-NRG-IgG1/IgG4-Fc and IFN-eGFP/IL-2-eGFP vector transfection: 30 ug of plasmid was taken and transfected according to the transfection reagent and method of Sinofection® (100 uL, Sino Biological). Feed was supplemented 24 h after transfection and every 48 h thereafter. At 37° C., 8% CO.sub.2 orbital shake culture was performed at 120 rpm, with samples collected every 24 h. After sample collection, the expression of the target protein in the supernatant and cell lysis buffer was detected by SGS-PAGE and Western blot. The target protein was not detected in the cell lysis buffer or supernatant in the melittin-NRG-IgG1/IgG4-Fc expression system, indicating that NRG-IgG1/IgG4-Fc fusion polypeptide was not expressed in 293F cells when melittin was used as a signal peptide.

[0086] In contrast, when IFN/IL-2 was used as a signal peptide, the eGFP fusion polypeptide was highly expressed in 293F cells, and the IL2 signal peptide was significantly better than IFN in terms of the extracellular secretion efficiency of eGFP (see FIG. 3).

[0087] IL-2-NRG-IgG1/IgG4-Fc vector transfection: Plasmid DNA (30 ug and 45 ug) was mixed with 150 mM NaCl solution to 0.5 ml, and then let it stand for 5-10 min. The PEI solution with molecular weight of 40K was mixed with 150 mM NaCl solution to 0.5 mL, and then let it stand for 5-10 min. Then, PEI was mixed with DNA and incubated at room temperature for 20-30 min to form a DNA-PEI complex. The transfection solution was added dropwise to the cell culture solution while the culture flask was shaken gently. Then, it was put back into the shaker for further culture. Feed was supplemented 24 h after transfection and every 48 h thereafter. At 37° C., 8% CO.sub.2 orbital shake culture was performed, and samples were taken every 24 h to detect the cell transfection efficiency. According to the expression characteristics of different proteins, samples could be collected as long as 6-10 d after transfection. The supernatant was collected by centrifugation and stored at −20° C.

[0088] Samples with different DNA/PEI mass ratios were taken at different time points to detect the expression by SDS-PAGE and Western-blot analysis (see FIG. 4 for the results) to determine optimum conditions for transfection and sample collection.

Embodiment 3 Purification of Fusion Protein

[0089] An equal volume of binding buffer (0.02M of disodium hydrogen phosphate, pH 7.0) was added to the supernatant, with the pH value adjusted to 7.0. Samples could also be pretreated in a dialysis system or desalting column. Then, the supernatant was first filtered through a 1 um filter head, then through a 0.45 um filter membrane, and finally through an affinity column. Samples at peak value of OD280 were collected and tested by SDS-PAGE (see FIG. 5 for the results).

Embodiment 4 Binding of a Receptor to NRG Polypeptide

[0090] MCF-7 cells were collected, counted, centrifuged and re-suspended in DMEM (10% serum, 9 μg/ml of insulin), at a cell density of 5×10.sup.4/ml. A 96-well plate was spread. 100 μl of suspension was put in each well and left standing overnight at 37° C. The cells were washed with PBS three times the next day, and cultured in serum-free DMEM for 24 h.

[0091] The ErbB2 antibody H4 was diluted with a coating buffer (50 mM Na.sub.2CO.sub.3—NaHCO.sub.3, pH9.6) to 4 μg/ml, and put in a 96-well plate, with 50 μl in each well, and then left standing overnight at 4° C. so that the antibody can bind to the plate.

[0092] The DMEM medium in MCF-7 cells was sucked out. Then, NRG and NRG-IgG1-Fc were diluted with DMEM serially and then put in the wells, with 100 μl in each well. Here, NRG was a recombinant NRG polypeptide with an amino acid sequence of SEQ ID NO: 1, and NRG-IgG1-Fc was a recombinant NRG fusion protein with an amino acid sequence of SEQ ID NO: 2. Only DMEM was added to the blank control. After being incubated at 37° C. for 20 min, the cells were washed with PBS buffer. Then, 100 μl/well of lysis buffer (50 mM Hepes, pH8.0, 150 mM NaCl, 2 mM sodium orthovanadate, 0.01% thimerosal, 1% Triton X-100 and 25 ml of protease inhibitor cocktail) was added to each well, lysed at 4° C. for 30 min. Then, the plate was shaken gently to remove the cells from the plate, with it centrifuged at 15,000 rpm for 15 min.

[0093] The antibody-coated plate was washed with washing liquid (10 mM PBS, pH7.4, 0.05% Tween 20) for 5 times. 200 μl of washing liquid containing 5% skimmed milk was added to each well, incubated at 37° C. for 2 h, and then washed with washing liquid for 3 times.

[0094] The lysed cell sap was added to the coated plate, with 90 μl in each well, incubated at 37° C. for 1 hour, and then washed with washing liquid for 5 times. 100 μl of horseradish peroxidase (HRP) with proper concentration was added to it, incubated at 37° C. for 1 hour. After washing 5 times, a fresh prepared HRP substrate solution ((50 mM citric acid, 100 mM Na.sub.2HPO.sub.4, pH 5.0, 0.2 mg/ml of tetramethylbenzidine (TMB, 0.003% H.sub.2O.sub.2) was added, incubated at 37° C. for 10 min. Finally, 50 μl 1M H.sub.2SO.sub.4 was added to each well to destroy HRP activity to terminate the reaction. The OD value of each well was measured at 450 nm on a microplate reader. EC50 was the concentration of NRG or fusion protein reaching the midpoint of maximum absorbance. The lower the EC50 value was, the higher the affinity of the receptor to NRG or fusion protein was.

[0095] Table 1 and 2 show the EC50 values of NRG NRG-IgG1-Fc and NRG-IgG4-Fc. The EC50 values of NRG-IgG1-Fc and NRG-IgG4-Fc are slightly higher than that of NRG

TABLE-US-00001 TABLE 1 EC50 Values of NRG and NRG-IgG1-Fc Samples EC.sub.50(nM) NRG 0.9245 NRG-IgG1-Fc 2.483

TABLE-US-00002 TABLE 2 EC50 Values of NRG and NRG-IgG4-Fc Samples EC.sub.50(nM) NRG 1.651 NRG-IgG4-Fc 3.659

Embodiment 5 ELISA Method Used to Detect the Half-Life Period of Intravenously or Subcutaneously Injected NRG-IgG1-F and NRG-IgG4-Fc Fusion Peptides in Rats

[0096] Rats were injected with 250 ug/kg of NRG-IgG1-Fc through the tail vein or 500 ug/kg of NRG-IgG1-Fc subcutaneously or 250 ug/kg of NRG-IgG4-Fc through the tail vein. Then, blood was collected from the jugular vein at different time points after administration. Let it stand at room temperature for at least 30 min. It was centrifuged after blood coagulation to collect the supernatant. The rat serum containing NRG-IgG1-Fc was diluted with dilution buffer in a ratio of 1:1 for later use.

[0097] Standard NRG-IgG1-Fc samples were prepared with rat serum in a concentration range of 5,000 ng/ml, 2,500 ng/ml, 1,000 ng/ml, 200 ng/ml, 40 ng/ml, 8 ng/ml, 1.6 ng/ml, 0.32 ng/ml, 0.064 ng/ml and 0 ng/ml, and then diluted with dilution buffer in a ratio of 1:1.

[0098] Plate coating and blocking: Human NRG1/HRG1-β1 EGF domain antibody was diluted with a coating buffer, and coated overnight at 4° C. after adding 50 uL to each reaction well in a 96-well plate. The coating buffer was discarded the next day, while the plate was washed, and a block buffer was added to it for blocking at room temperature. After the plate was dried on a piece of absorbent paper, 50 uL of corresponding standard sample or sample to be tested was added to each reaction well, and incubated at room temperature for 2 h. The plate was washed. Then, Anti-Human IgG1 Fc (HRP) antibody was added to it, and incubated at room temperature for 1 hour. The plate was washed. Then, temporarily prepared TMB substrate solution was added to each reaction well. After 20 min of photophobic reaction at 37° C., 50 uL 1M of sulfuric acid was added to terminate the reaction. The absorbance of each reaction well at 450 nM was measured, and the content of NRG and protein in the sample was calculated according to the standard curve. The data were analyzed using GraphPad Prism 5.0.

[0099] The results are shown in Tables 3, 4 and 5, respectively:

TABLE-US-00003 TABLE 3 Half-life results of intravenously injected NRG-IgG1-Fc Parameters Unit Mean AUC.sub.(0-t) ng/ml*h 104777.97 T.sub.(1/2) h 4.66 CL.sub.z/F L/h/kg 0.00 C.sub.max ng/ml 34148.32

TABLE-US-00004 TABLE 4 Half-life results of subcutaneously injected NRG-IgG1-Fc Parameters Unit Mean AUC.sub.(0-t) ng/ml*h 6330.97 T.sub.(1/2) h 13.45 CL.sub.z/F L/h/kg 0.08 C.sub.max ng/ml 282.33

TABLE-US-00005 TABLE 5 Half-life results of intravenously injected NRG-IgG4-Fc Parameters Unit Mean AUC.sub.(0-t) ng/ml*h 38034.83 T.sub.(1/2) h 8.48 CL.sub.z/F L/h/kg 0.01 C.sub.max ng/ml 9816.47

[0100] The experimental data showed that compared with the 10 min half-life period of intravenously injected NRG and 1.5 h half-life period of subcutaneously injected NRG, the fusion peptides NRG-IgG1-Fc and NRG-IgG4-Fc could significantly prolong the half-life of NRG fragments injected intravenously and subcutaneously into rats.

Embodiment 6 Pharmacodynamic Experiment of Intravenously Injected NRG-IgG1-Fc Fusion Polypeptide in Treating Rats with Heart Failure

6.1 Experimental Objectives

[0101] In the rat model of heart failure induced by left coronary artery ligation, rats were injected intravenously with NRG-IgG1-Fc fusion protein once a day and injected intravenously with recombinant human NRG (rhNRG) in a sustained way through a Medtronic insulin injection pump to compare the therapeutic effects of four administration methods on the rat model of heart failure.

6.2 Experimental Materials

6.2.1 Experimental Animals

[0102] 6.2.1.1 Strain & source: Wistar rat, provided by Shanghai Sippe-Bk Lab Animal Co., Ltd.
6.2.1.2 Gender, weight and number: male, 200-250 g, 150

6.2.2 Reagents

[0103] 6.2.2.1 Excipient: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd. Dosage form: freeze-dried powder; specification: 2 mg Alb/bottle
6.2.2.2 Recombinant human NRG: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd. Dosage form: freeze-dried powder; specification: 250 μg/piece
6.2.2.3 Recombinant human NRG-IgG1-Fc fusion protein: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd. Dosage form: injection

6.2.2.4 Isoflurane: RWD Life Technology Co., Ltd.

6.3 Experimental Equipment

[0104] 6.3.1 Anesthesia apparatus (isoflurane evaporator): MSS INTERNATIONAL LTD
6.3.2 Ultrasonic heart detector: Vivid E95; probe type: 12S-D
6.3.3 Insulin pump: Medtronic; model: MMT-712EWS, MMT-722NAS/L
6.3.4 PowerLab multi-channel physiological recorder: ADInstruments (Shanghai) Co., Ltd.; model: ML-845

6.4 Experimental Methods

6.4.1 Establishment of a Rat Model of Heart Failure Induced by Coronary Artery Ligation

[0105] Rats were anesthetized with 4% isoflurane in a gas anesthesia apparatus, and then fixed in supine position and sterilized with 75% alcohol after chest hair removal. After the left anterior chest skin was cut open, the chest muscles were bluntly dissected, with the 4.sup.th and 5.sup.th ribs exposed. The muscles between the 4.sup.th and 5.sup.th ribs were bluntly dissected with hemostatic forceps, squeezed with both hands, so that the heart was squeezed out of the thoracic cavity and fully exposed. Lung inflation and heartbeat were put under observation. The left atrial appendage and pulmonary arterious cone were fully exposed, and between them, the left anterior descending coronary artery was ligated with 6-0 sutures. Then, the chest was squeezed hard to discharge air, and then the chest muscles and skin were sutured. After surgery, the rats were put back into the cage and placed under close observation. In case of acute arrhythmia, the heart was massaged urgently for 3-5 min. After the operation, 80,000 U of penicillin sodium was injected intramuscularly into each rat's muscles for 2 d.

6.4.2 Grouping and Drug Administration

6.4.2.1 Grouping

[0106] The grouping results are shown in Table 6. At week 2 and 4 after surgery, the rats' cardiac function was detected by B-ultrasound Vivid E95. After cardiac ultrasound at week 4, rats with EF value of 28%-45% were selected for the next experiment.

TABLE-US-00006 TABLE 6 Experimental animal grouping and administration arrangement Dosing Dose Route of Cycle of Group Dosage Volume Concentration Administration Administration Model — 2 ml/kg each — Intravenous Qd × 10 d Group time injection NRG-IgG1- 10 ug/kg 2 ml/kg each 5 ug/ml Intravenous Qd × 10 d Fc Group time injection NRG-IgG1- 30 ug/kg 2 ml/kg each 15 ug/ml Intravenous Qd × 10 d Fc Group time injection NRG Group  6 ug/kg 5 ml/kg 1.2 ug/ml Tail vein 8 h/d × 10 d infusion Sham — — — — — Operation Group

[0107] The rats were randomly divided into 4 groups according to the results of cardiac ultrasound. The rats that received an intravenous injection were divided into the excipient group, the NRG-IgG1-Fc 30 μg/kg qid group, and the NRG-IgG1-Fc 6 μg/kg qid group. According to Table 1, intravenous injection was performed every day for 10 d, and the dose volume was set to 2 ml/kg/time, while the dose concentration was set to 5 μg/ml and 15 μg/ml respectively.

[0108] The NRG tail vein group was treated with an insulin pump for 8 h a day and for 10 d; the dosing volume was 5 ml/kg, the dosage of NRG was 6 μg/kg and the concentration was 1.2 μg/ml.

[0109] The sham operation group underwent threading only rather than coronary artery ligation and medication.

6.4.2.2 Dispensing Methods

[0110] 1) Excipient: 2 mg Alb/bottle, with 1 ml of normal saline added to each bottle to prepare mother liquor, and 0.24 ml of mother liquor was diluted with 49.76 ml of normal saline into 9.6 μg/ml of Alb solution.
2) NRG-IgG1-Fc: 0.4 mg-0.8 mg/ml of NRG-IgG1-Fc mother liquor was diluted with normal saline into NRG-IgG1-Fc with a certain working concentration.
3) NRG: 250 μg NRG/bottle, with 1 ml of normal saline added to each bottle to prepare mother liquor, and 0.24 ml of mother liquor was diluted with 49.76 ml normal saline into 1.2 μg/ml of NRG solution.

6.4.3 Observation Indexes

6.4.3.1 Cardiac Function Test

[0111] After being anesthetized with 4% isoflurane in the gas anaesthesia apparatus, the rats were fixed to a surgical stent in left recumbent position. The rat head was fixed in the breathing mask of the gas anaesthesia apparatus, with the concentration of isoflurane maintained at 2%. After chest hair removal and sterilization with 75% alcohol, the rats were smeared with a couplant, and then a rat cardiac ultrasound probe was used to test the left ventricular echo signal. In “B-mode”, the cardiac ultrasound probe was placed near the left side of the sternum with the probe pointing to 2-3 o'clock. The sound beam cut the heart in the direction perpendicular to the long axis of the heart. The probe was adjusted until it became horizontally parallel to both papillary muscles to obtain a horizontal left ventricular short axis view of the papillary muscles to collect a dynamic image of the papillary muscle of left ventricle and save it. In “M-mode”, the probe was kept on the left ventricular short axis section, and the M-mode sampling line was adjusted so that it could pass through the weakest point of heartbeat on the anterior wall. The focal length was adjusted, and an M-shaped curve (the left ventricular cavity and the anterior and posterior walls of the left ventricle should be clearly displayed) was used to measure the left ventricular end diastolic diameter (D) and left ventricular end systolic diameter (D). The left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated by Teichholtz's formula V=7/(2.4+D)*D 3. Also, the ejection fraction (EF) was calculated as follows: EF=(EDV−ESV)/EDV*100%.

6.4.3.2 Hemodynamic Test of Heart

[0112] A physiological recorder was used to record hemodynamic indexes such as carotid arterial pressure, intraventricular pressure, +dp/dt and −dp/dt. Main steps: The rats were anesthetized by intraperitoneal injection of 20% urethane, with an injection volume of 6 ml/kg. The right common carotid artery was separated, and its distal end was ligated. Its proximal end was blocked with an artery clamp, and a small opening was cut between both ends. Then, the PE50 catheter connected with the probe was inserted into the common carotid artery through the opening. The waveform displayed by the Powerlab physiological recorder was observed. After stabilization, the carotid artery pressure was recorded, and then the catheter was further inserted into the left ventricle for 10-min indwelling. After stabilization, the indexes such as LVSP, LVEDP, +dp/dt and −dp/dt were recorded, and LabChart7 was used for analysis.

6.4.4 Data Processing

[0113] All experimental data were expressed in x±SD. GraphPad Prism 6 was used for one-way ANOVA analysis. P<0.05 indicated that there was a significant difference between the groups, while P<0.01 indicated that there was an extremely significant difference between the groups.

6.5 Experimental Results

[0114] The results of cardiac ultrasound are shown in Table 7 and FIG. 6. The experimental results showed that after random grouping of heart failure rats, there was no significant difference in cardiac ultrasound data between the groups before administration. After administration, there were obvious differences between the groups. 10 d of injection of NRG-IgG1-Fc (30 ug/kg a day) could significantly increase the EF and FS values (P<0.001) of the rat heart, and significantly decrease the values of LVESV (P<0.001), LVEDV (P<0.05), LVDs (P<0.001) and LVDd (P<0.05), suggesting that it could significantly improve the cardiac function of rats with heart failure. After continuous infusion for 8 h*10 d, the EF, LVESV, FS and LVDs values of the NRG group were improved. The results showed that an equimolar amount (30 ug/kg) of NRG-IgG1-Fc produced a better therapeutic effect on heart failure in rats in the treatment group than the NRG group. Moreover, NRG-IgG1-Fc was easier to use than NRG.

TABLE-US-00007 TABLE 7 Results of cardiac ultrasound in rats before and after treatment of each group Model Group NRG (6 ug/kg/8 h) NRG-IgG1-Fc (10 ug/kg) NRG-IgG1-Fc (30 ug/kg) Pre- Post- Pre- Post- Pre- Post- Pre- Post- Administration Administration Administration Administration Administration Administration Administration Administration LVDd 0.969 ± 0.035  0.99 ± 0.058  0.963 ± 0.049 0.97 ± 0.046  0.96 ± 0.044 0.977 ± 0.058 0.959 ± 0.055 0.94 ± 0.086*  (cm) LVDs 0.814 ± 0.042 0.855 ± 0.066  0.81 ± 0.048 0.801 ± 0.047* 0.806 ± 0.045 0.819 ± 0.067 0.806 ± 0.051 0.744 ± 0.097*** (cm) LVEDV 1.896 ± 0.186  2.02 ± 0.308 1.868 ± 0.25 1.905 ± 0.242  1.85 ± 0.22 1.934 ± 0.317  1.85 ± 0.276 1.768 ± 0.414*  (cm.sup.3) LVESV 1.183 ± 0.168 1.359 ± 0.277  1.166 ± 0.175 1.133 ± 0.18**  1.15 ± 0.175  1.21 ± 0.268 1.154 ± 0.19   0.95 ± 0.106*** (cm.sup.3) FS %  16 ± 2.1 13.8 ± 3.1  15.9 ± 1.9 17.4 ± 3.5** 16.1 ± 2.0  16.3 ± 2.8*  16 ± 2.0 21.1 ± 1.9***  EF % 37.8 ± 4.4  33.0 ± 6.5  37.7 ± 3.9  40.4 ± 6.8***  38 ± 4.2 38.3 ± 5.7* 37.7 ± 4.1  47.4 ± 7.0****

[0115] The scope of the present invention is not limited to the embodiments. As is clear to those skilled in the art, the present invention can be modified and changed in many manners without departing from its spirit and scope. The embodiments described herein are provided in the form of embodiments only, and the present invention is subject only to the appended claims and their equivalents in their entirety.