METHOD FOR PREVENTING, TREATING OR DELAYING MYOCARDIAL DAMAGE USING NEUREGULIN AND COMPOSITION
20220096599 · 2022-03-31
Assignee
Inventors
Cpc classification
C07K14/4756
CHEMISTRY; METALLURGY
A61K38/1883
HUMAN NECESSITIES
International classification
Abstract
A method for preventing, treating or delaying myocardial damage in a mammal using neuregulin and a composition. An administration method for, an administration frequency of and an administration dosage of a pharmaceutical formulation or composition for reducing myocardial damage. It can be proved in a rat myocardial damage model that neuregulin can improve the cardiac function after myocardial infarction, suggesting that neuregulin can be used for preventing, treating, or delaying myocardial infarction damage.
Claims
1. Use of neuregulin (NRG) or a functional fragment thereof in the manufacturing of a medicament for preventing, treating, or relieving myocardial injury in a mammal.
2. The use according to claim 1, wherein the NRG is NRG-1, NRG-2, NRG-3 or NRG-4.
3. The use according to claim 1, wherein the NRG is NRG-1.
4. The use according to claim 1, comprising the use of other drugs or therapies that can be used to prevent, treat or relieve myocardial infarction injury in a mammal.
5. The use according to claim 1, wherein the NRG enhances the cardiac function of mammals and reduces cardiac remodeling.
6. The use according to claim 1, wherein the mammal is human.
7. A composition for preventing, treating, or relieving myocardial injury in a mammal, wherein the composition comprises an effective amount of NRG or a functional fragment thereof.
8. A method for preventing, treating, or relieving myocardial injury in a mammal, comprising subcutaneously administering neuregulin (NRG) with at a dose of 2.5 μg/kg/day to 50 μg/kg/day.
9. A method for preventing, treating, or relieving myocardial injury in a mammal, comprising administering neuregulin (NRG) multiple times a day for consecutive days.
10. The method according to claim 9, comprising administering the NRG multiple times a day for consecutive days and then slowly withdrawing the NRG.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0036]
[0037]
[0038]
[0039]
EXAMPLES
Example 1: Therapeutic Effect of rhNRG on Acute Myocardial Infarction in Rats Through Long-Term Subcutaneous Administration of Different Doses—a Study on the Dose-Effect Relationship of NRG
1. Objective
[0040] To investigate the dose-effect relationship of NRG for treatment of acute myocardial infarction in rats by observing the therapeutic effect of different doses of rhNRG on acute myocardial infarction in rats in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0041] 2.1 Excipient: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
[0042] 2.2 rhNRG: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0043] 3.1 Strain and Source: Wistar Rats, Provided by Shanghai Sippe-Bk Lab Animal Co., Ltd.
[0044] 3.2 Gender, Body Weight and Certificate: Male, 200-270 g
4. Experimental Materials and Equipment
[0045] An anesthesia apparatus, an isoflurane evaporator, from MSS INTERNATIONAL LTD.
[0046] Isoflurane 100 ml/bottle, from RWD Life Technologies Co., Ltd.
[0047] A cardiac ultrasonic detector Vivid E95 Ningbo Lingqiao suture needle with threads, from Ningbo Medical Needle Co.,
[0048] Ltd.
5. Experimental Methods
[0049] 5.1 Establishment of a Rat Model of Heart Failure Caused by Coronary Artery Ligation
[0050] The rats were anesthetized with isoflurane through a gaseous anesthesia apparatus. Then, the rats were fixed in a supine position. After chest hair removal, the skin was disinfected with 75% alcohol. After the incision of the left anterior chest skin, the thoracic muscles were bluntly separated, with the 4.sup.th and 5.sup.th ribs exposed. Hemostatic forceps were used to bluntly cut off the muscle between the 4.sup.th and 5.sup.th ribs. Both hands were used to squeeze the heart out of the thoracic cavity so that the heart should be fully exposed for observation of lung inflation and heartbeats. The left atrial appendage and pulmonary conus were fully exposed to ligate the left anterior descending coronary artery (LADCA) with surgical suture between them. The heart was quickly restored upon after ligation. Then, the thoracic muscles and skin were sutured. After surgery, the rats were put back into the cages for feeding and close observations.
[0051] 5.2 Grouping and Administration
TABLE-US-00003 TABLE 1 Experimental animal grouping and administration schedule Frequency and Administered Route of Cycle of Group Dose Administration Administration Control Group — Subcutaneous 3 times a day × injection 60 days High-dose NRG 15 μg/kg/D Subcutaneous 3 times a day × injection 60 days Middle-dose NRG 7.5 μg/kg/D Subcutaneous 3 times a day × injection 60 days Low-dose NRG 3.75 μg/kg/D Subcutaneous 3 times a day × injection 60 days
[0052] Administration began on the day following the animal model establishment of myocardial infarction.
[0053] 5.3 Observation Indexes
[0054] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating board in the left lateral recumbent position. The head of the rats was fixed in the breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol and coated with a coupling agent. An echocardiography probe was used to detect any echo signal from the left ventricle of the rats. The left ventricular end-diastolic diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. Also, the ejection fraction (EF) value was found, EF=(EDV−ESV)/EDV*100%.
[0055] 5.3.2 Data Processing
[0056] All experimental data were expressed in ±SD.
6. Experimental Results
[0057] 6.1 Echocardiographic Results
[0058] Echocardiography was performed after 60 days of continuous NRG administration. The results showed that the LVEDd, LVEDs and EF value of the excipient group were 0.971±0.07 cm, 0.832±0.08 cm and 34.6±7.00%, respectively; the LVEDd, LVEDs and EF value of the 15 μg/kg-NRG group were 0.975±0.07 cm, 0.794±0.10 cm and 42.9±11.32%, respectively; the LVEDd, LVEDs and EF value of the 7.5 μg/kg-NRG group were 0.965±0.07 cm, 0.808±0.11 cm and 38.4±12.17%, respectively; the LVEDd, LVEDs and EF value of the 3.75 μg/kg-NRG group were 0.994±0.08 cm, 0.839±0.12 cm and 37.0±12.23%, respectively. According to the data of LVEDd and LVEDs, the LVEDd and LVEDs of the high-dose NRG group could be reduced. According to the data of EF value, the cardiac function of the rats in the high-dose, middle-dose and low-dose groups was all improved after 60 days of continuous administration, and there was dose-effect relationship among the three groups. See Table 2 and
TABLE-US-00004 TABLE 2 Echocardiographic results of the effect of NRG in treating rats with acute myocardial infarction through 60-day subcutaneous administration of different doses (
7. Conclusions
[0059] After 60-day treatment with rhNRG, the EF value of the treatment groups, which were subcutaneously injected with 5 μg/kg, 2.5 μg/kg, 1.25 μg/kg of NRG 3 times a day, was higher than that of the control group, and there was a certain dose-effect relationship for the three doses.
Example 2: Therapeutic Effect of rhNRG on Acute Myocardial Infarction in Rats Through Long-Term Subcutaneous Administration at Different Frequencies
1. Objective
[0060] To investigate the therapeutic effect of a certain dose of rhNRG on cute myocardial infarction in rats through long-term subcutaneous administration at different frequencies in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0061] 2.1 Excipient: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
[0062] 2.2 rhNRG: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0063] 3.1 Strain and Source: Wistar Rats, Provided by Shanghai Sippe-Bk Lab Animal Co., Ltd.
[0064] 3.2 Gender, Body Weight and Certificate: Male, 200-270 g
4. Experimental Materials and Equipment
[0065] The same as “4. Experimental Materials and Equipment” in Example 1.
5. Experimental Methods
[0066] 5.1 Establishment of a Rat Model of Heart Failure Caused by Coronary Artery Ligation
[0067] Established in the same way as the rat model of heart failure caused by coronary artery ligation in 5.1, Embodiment 1
[0068] 5.2 Grouping and Administration
TABLE-US-00005 TABLE 3 Experimental animal grouping and administration schedule Frequency and Administered Route of Cycle of Group Dose Administration Administration Control Group — Subcutaneous 3 times a day × injection 35 days NRG/30 μg/kg/ 30 μg/kg/ Subcutaneous 3 times a day × Day Day injection 35 days NRG/30 μg/kg/ 30 μg/kg/ Subcutaneous 3 times a day, BIW Day injection 2 days a week × 5 weeks Nrg/30 μG/KG/ 30 μg/kg/ Subcutaneous 3 times a day × Day*7 + QW Day + injection 7 days 30 μg/kg/ once a day, 7 days 1 day/week × 4 weeks
[0069] All the experimental animals were randomly divided into groups upon coronary artery ligation. According to post-ligation survival, the rats were randomly divided into 4 groups by body weight: the excipient group (control group), the NRG30 μg/kgDay group, the NRG30 μg/kg/BlW group and the NRG30 μgkgDay*7+QW group. Administration began on the day following the animal model establishment of myocardial infarction. For the first three groups and for the fourth group on the first 7 days, the rats were given subcutaneous injection 3 times a day and weighed once a day. They took medicine by weight and the dose was 30 μg/kg/day. For the fourth group in the last four weeks, the rats were injected with NRG on one day per week, and the daily dose was 30 μg/kg.
[0070] 5.3 Observation Indexes
[0071] 5.3.1 Cardiac Function Test
[0072] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating board in the left lateral recumbent position. The head of the rats was fixed in the breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol and coated with a coupling agent. An echocardiography probe was used to detect any echo signal from the left ventricle of the rats. The left ventricular end-diastolic diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. Also, the ejection fraction (EF) value was found, EF−(EDV−ESV)/EDV*100%. The cardiac function of the rats was performed by echocardiography in the 1.sup.st, 2.sup.nd, 3.sup.rd and 5.sup.th weeks after the occurrence of myocardial infarction.
[0073] 5.3.2 Data Processing
[0074] All experimental data were expressed in ±SD.
6. Experimental Results
[0075] 6.1 Echocardiographic Results
[0076] Echocardiography was performed after 35 consecutive days of NRG administration. The LVEDd, LVEDs, EF value of the control group were 0.925±0.084 cm, 0.756±0.107 cm and 42.5±10.174%, respectively; the LVEDd, LVEDs, EF value of the NRG/30 μg/kg/Day group were 0.879±0.058 cm, 0.694±0.077 cm and 47.9±8.342%, respectively; the LVEDd, LVEDs, EF value of the NRG/30 μg/kg/BIW group were 0.928±0.084 cm, 0.746+0.110 cm and 45.2+10.248%, respectively; the LVEDd, LVEDs, EF value of the NRG/30 μg/kg/Day*7+QW group were 0.931±0.070 cm, 0.760±0.097 cm and 42.7±9.892%, respectively.
[0077] After 35 days of continuous administration, as shown by the data of LVEDd and LVEDs, NRG/30 μg/kg/Day could significantly reduce the LVEDd and LVEDs; according to the data of EF value, it was significantly higher in the NRG/30 μg/kg/Day group than in the control group; it showed a rising trend in the NRG/30 μg/kg/BIW group compared with the control group; compared with the control group, the cardiac function of the rats in the NRG/30 μg/kg/Day*7+QW group was improved to some extent on the first 7 days of continuous administration, i.e., it showed a rising trend. Then, an injection was given every 7 days to maintain the efficacy. See Table 4 and
TABLE-US-00006 TABLE 4 Echocardiographic results of the therapeutic effect of NRG on myocardial infarction in rats through long-term subcutaneous administration at different frequencies (
Example 3: Therapeutic Effect of rhNRG on Acute Myocardial Infarction in Rats Through Long-Term Subcutaneous Administration Followed by Withdrawal Through Frequency Reduction
1. Objective
[0078] To observe the therapeutic effect of rhNRG on acute myocardial infarction in rats through long-term administration followed by withdrawal through frequency reduction in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0079] 2.1 Excipient: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
[0080] 2.2 rhNRG: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0081] 3.1 Strain and Source: Wistar Rats, Provided by Shanghai Sippe-Bk Lab Animal Co., Ltd.
[0082] 3.2 Gender, Body Weight and Certificate: Male, 200-270 g
4. Experimental Materials and Equipment
[0083] The same as “4. Experimental Materials and Equipment” in Embodiment 1
5. Experimental Methods
[0084] 5.1 Establishment of a Rat Model of Heart Failure Caused by Coronary Artery Ligation
[0085] Established in the same way as the rat model of heart failure caused by coronary artery ligation in 5.1, Embodiment 1
[0086] 5.2 Grouping and Administration
[0087] The rats were randomly divided into groups and medicated upon coronary artery ligation. According to post-ligation survival, the rats were randomly divided into two groups, including the excipient group and the NRG30 μg/kg group. There were 19 rats in the excipient group and there were 18 rats in the NRG group. Continuous administration of medicine started on the day following the modeling, with subcutaneous administration for 3 times a day, at a dose of 10 μg/kg. Echocardiography was performed on day 14 after modeling. All the animals were medicated continuously until day 38, and the animals in the NRG group were examined by echocardiography. The animals in the NRG group were averagely divided into two subgroups, with the animals in one subgroup continuing to be medicated while the animals in the other subgroup stopped taking medicine at an early stage. The excipient group continued to be medicated. For the continuous NRG administration subgroup, a 3-week withdrawal plan was implemented on day 49: medicated every other day in the first week; medicated every three days in the second week; subcutaneously injected with NRG every four days in the third week. In terms of administration method, the rats were subcutaneously injected with NRG 3 times a day, exactly the same as above. For the NRG withdrawal subgroup, the clinical symptoms of the rats were observed. All the animals underwent echocardiography every week for monitoring of the changes in the cardiac function.
[0088] 5.3 Observation Indexes
[0089] 5.3.1 Cardiac Function Test
[0090] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating board in the left lateral recumbent position. The head of the rats was fixed in the breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol and coated with a coupling agent. An echocardiography probe was used to detect any echo signal from the left ventricle of the rats. The left ventricular end-diastolic diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. Also, the ejection fraction (EF) value was found, EF=(EDV−ESV)/EDV*100%.
[0091] 5.3.2 Data Processing
[0092] All experimental data were expressed in ±SD. GraphPad Prism6 was used for one-way ANOVA analysis. P<0.05 indicates a significant inter-group difference; P<0.01 indicates an extremely significant inter-group difference.
6. Experimental Results
[0093] 6.1 Echocardiographic Results
[0094] Echocardiography was performed after 35 consecutive days of NRG administration. The LVEDd, LVEDs and EF value of the excipient group were 0.988±0.08 cm, 0.850±0.10 cm and 33.6±11.36%, respectively; the LVEDd, LVEDs and EF value of the NRG group were 0.953±0.05 cm, 0.767±0.06 cm and 44.9±6.09%, respectively; the results showed that NRG could significantly reduce the LVEDd and LVEDs and strengthen the cardiac systolic function, thereby reversing left ventricular remodeling; after 49 days of continuous administration, the LVEDd, LVEDs and EF value of the excipient group were 1.020±0.10 cm, 0.881±0.15 cm and 33.1±14.55%, respectively; the LVEDd, LVEDs and EF value of the NRG withdrawal subgroup were 0.987±0.05 cm, 0.807±0.06 cm and 42.2±5.48%, respectively, while the LVEDd, LVEDs and EF value of the continuous NRG administration subgroup were 0.973±0.07 cm, 0.783±0.08 cm and 45.0±5.51%, respectively; the results showed that the sudden withdrawal of NRG exerted some effect on the cardiac function of the rats. A gradual withdrawal plan was implemented in the continuous NRG administration subgroup, and echocardiography was performed in the second week after drug withdrawal. The LVEDd, LVEDs and EF value of the early NRG withdrawal subgroup were 1.043±0.06 cm, 0.887±0.06 and 35.4±6.78%, respectively; the LVEDd, LVEDs and EF value of the gradual NRG withdrawal subgroup were 0.989±0.07 cm, 0.814±0.08 and 41.3±4.92%, respectively. There was a significant difference from the excipient group. Echocardiography was performed in the third week after withdrawal. The LVEDd, LVEDs and EF value of the early NRG withdrawal subgroup were 1.010±0.06 cm, 0.842+0.06 cm and 38.9±5.04%, respectively; the LVEDd. LVEDs and EF value of the gradual NRG withdrawal subgroup were 0.976±0.06 cm, 0.805±0.07 cm, 40.8±4.67%, respectively. Compared with the excipient group, the effect of gradual withdrawal of NRG on the cardiac function of the rats was allayed. See Tables 5 & 6 and
TABLE-US-00007 TABLE 5 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction through 35-day subcutaneous administration (
TABLE-US-00008 TABLE 6 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction in the two subgroups after 38-day subcutaneous administration (
7. Conclusions
[0095] Through long-term subcutaneous administration followed by withdrawal through frequency reduction, rhNRG can improve the cardiac function of the rats with myocardial infarction and reduce cardiac remodeling.
Example 4: Therapeutic Effect of rhNRG on Acute Myocardial Infarction in Rats Through Long-Term Subcutaneous Administration Followed by Withdrawal Through Dose Reduction
1. Objective
[0096] To observe the therapeutic effect of rhNRG on acute myocardial infarction in rats through long-term administration followed by withdrawal through frequency reduction in a rat model of myocardial infarction caused by left coronary artery ligation.
2. Experimental Drugs
[0097] 2.1 Excipient: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
[0098] 2.2 rhNRG: Developed by Zensun (Shanghai) Sci & Tech Co., Ltd.
3. Experimental Animals
[0099] 3.1 Strain and Source: Wistar Rats, Provided by Shanghai Sippe-Bk Lab Animal Co., Ltd.
[0100] 3.2 Gender, Body Weight and Certificate: Male, 200-270 g
4. Experimental Materials and Equipment
[0101] The same as “4. Experimental Materials and Equipment” in Embodiment 1
5. Experimental Methods
[0102] 5.1 Establishment of a Rat Model of Heart Failure Caused by Coronary Artery Ligation
[0103] Established in the same way as the rat model of heart failure caused by coronary artery ligation in 5.1, Embodiment 1
[0104] 5.2 Grouping and Administration
[0105] 5.3 Observation Indexes
[0106] The rats were randomly divided into groups and medicated upon coronary artery ligation. According to post-ligation survival, the rats were randomly divided into 2 groups by body weight. Subcutaneous injection was given 3 times a day, and the animals were weighed once a day. The animals were medicated by weight. Echocardiography was performed on day 10 after modeling. All the animals underwent echocardiography every 10 days, underwent echocardiography every week after dose reduction, and underwent echocardiography every 2 weeks after complete withdrawal. All the animals were medicated continuously till day 60, and then a three-week dose-reduction withdrawal plan was implemented: The administered dose was reduced to 15 μg/kg, 7.5 μg/kg and 3.75 μg/kg in the first, second and third week, respectively. The drug was completely withdrawn after three-week dose reduction to observe the clinical symptoms.
[0107] 5.3.1 Cardiac Function Test
[0108] After being anesthetized with 4% isoflurane, the rats were fixed onto the operating board in the left lateral recumbent position. The head of the rats was fixed in the breathing mask of the gaseous anesthesia machine, with isoflurane used for maintenance of anesthesia. After chest hair removal, the skin was disinfected with 75% alcohol and coated with a coupling agent. An echocardiography probe was used to detect any echo signal from the left ventricle of the rats. The left ventricular end-diastolic diameter and left ventricular end-systolic diameter (D) were measured. The left ventricular end-diastolic volume (EDV) and end-systolic volume (ESV) were calculated. The ejection fraction (EF) value was found, EP=(EDV−ESV)/EDV*100%.
[0109] 5.3.2 Data Processing
[0110] All experimental data were expressed in ±SD. GraphPad Prism6 was used for one-way ANOVA analysis. P<0.05 indicates a significant inter-group difference; P<0.01 indicates an extremely significant inter-group difference.
6. Experimental Results
[0111] 6.1 Echocardiographic Results
[0112] Echocardiography was performed after 60 consecutive days of NRG administration. The LVEDd, LVEDs and EF of the excipient group were 1.048±0.07 cm, 0.910±0.09 cm and 32.1±6.6%, respectively; the LVEDd, LVEDs and EF of the NRG30 μg/kg/Day group were 0.981±0.08 cm, 0.794±0.08 cm and 43.8±8.0%, respectively. According to the data of LVEDd and LVEDs, the LVEDd and LVEDs of the everyday NRG administration group were significantly reduced, showing an extremely significant difference from the control group (p<0.001). According to the data of EF value, the EF value of the NRG group increased significantly after 60 days of continuous administration, showing an extremely significant difference from the control group (p<0.001). Frequency-unchanged and dose-reduction treatment was given 60 days later. Echocardiography was performed in the third week. The LVEDd, LVEDs and EF value of the control group were 1.038±0.07 cm, 0.899±0.10 cm and 32.4+9.5%, respectively; the LVEDd, LVEDs and EF value of the NRG/30 μg/kg/Day group were 0.981±0.08 cm, 0.799±0.08 and 42.3±11.2%. The drug was completely withdrawn after three-week dose reduction for an observation. The LVEDd, LVEDs and EF value of the control group were 1.065±0.07 cm, 0.942±0.10 cm and 28.3±9.4%, respectively; the LVEDd, LVEDs and EF value of the NRG/30 μg/kg/Day group were 0.994±0.08 cm, 0.826±0.10 cm and 39.3±12.7%, respectively. Echocardiography was performed in the ninth week after withdrawal. The LVEDd, LVEDs and EF value of the control group were 1.137±0.08 cm, 1.006±0.08 cm and 28.0+5.7%, respectively; the LVEDd, LVEDs and EF value of the NRG/30 μg/kg/Day group were NRG/30 μg/kg/day group was 1.104±0.08 cm, 0.950±0.09 cm, and 33.4±7.6%, respectively. Nine weeks after withdrawal, there remained a significant difference in LVEDd and LVEDs between the everyday NRG administration group and the control group; the EF value still showed a rising trend compared with the control group. See Table 7, 8, 9 and
7. Conclusions
[0113] Given a certain dose and different frequencies of administration, rhNRG exerted some therapeutic effect on myocardial infarction in rats during continuous administration, enhancing the cardiac function of the rats with acute myocardial infarction and improving ventricular remodeling, and deferring aging caused by myocardial infarction. It still has a significant improving effect on the cardiac function of the rats with myocardial infarction a long time after withdrawal.
TABLE-US-00009 TABLE 7 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction through 60-day subcutaneous administration (
TABLE-US-00010 TABLE 8 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction three weeks after dose reduction following 60-day subcutaneous administration (
TABLE-US-00011 TABLE 9 Echocardiographic results of the effect of NRG in treating rats with myocardial infarction after withdrawal on day 81 of subcutaneous administration (