USES OF COMPLEX OF ANGIOTENSIN II RECEPTOR ANTAGONIST METABOLITE AND NEP INHIBITOR IN TREATING HEART FAILURE

Abstract

Uses of a complex of an angiotensin II receptor antagonist metabolite and a NEP inhibitor in treating heart failure, specifically related are uses of complex in preparing a medicament for use in heart failure with reduced ejection fraction (HFrEF).

Claims

1. Use of a complex of an angiotensin receptor antagonist metabolite and an NEP inhibitor in preparing a medicament for use in heart failure with reduced ejection fraction, wherein the complex has the structural units as follows:
(aEXP3174.bAHU377).xCa.nA where a:b=1:0.25-4; x is a value between 0.5 and 3; A refers to water, methanol, ethanol, 2-propanol, acetone, ethyl acetate, methyl-tert-butyl ether, acetonitrile, toluene, and dichloromethane; and n is a value between 0 and 3.

2. The drug use according to claim 1, characterized in that a single dose form of the medicament contains 60 mg to 500 mg of the complex.

3. The drug use according to claim 1, characterized in that a single-dose form of the medicament contains 60, 120, 180, 240, 300, 360, 420 and 480 mg of the complex.

4. The drug use according to claim 1, characterized in that the medicament is a solid composition for oral administration, preferably the composition is tablet or capsule.

5. The drug use according to claim 1, characterized in that the value of a:b is selected from 1:0.25, 1:0.5, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5 and 1:4.

6. The drug use according to claim 1, characterized in that the complex has the structural units as follows: Or ##STR00006## where x is a value between 0.5 and 2; and n is a value between 0 and 3.

7. The drug use according to claim 1, characterized in that x is selected from 0.5, 1, 1.5 and 2.

8. The drug use according to claim 1, characterized in that the complex has the structural units as follows:
(EXP3174.AHU377).1.5Ca.nH.sub.2O
Or
(EXP3174.AHU377).2Ca.nH.sub.2O where n is any value between 1 and 3.

9. The drug use according to claim 1, characterized in that n is selected from 0.5, 1, 1.5, 2, 2.5 and 3.

10. The drug use according to claim 1, characterized in that the complex is selected from:
(EX3174.AHU1377).1.5Ca.1H.sub.2O:
(EX3174.AHU1377).1.5Ca.1.5H.sub.2O:
(EX3174.AHU1377).1.5Ca.2H.sub.2O:
(EX3174.AHU1377).1.5Ca.2.5H.sub.2O:
(EX3174.AHU1377).1.5Ca.3H.sub.2O:
(EX3174.AHU1377).2Ca.1H.sub.2O:
(EX3174.AHU1377).2Ca.1.5H.sub.2O:
(EX3174.AHU1377).2Ca.2H.sub.2O:
(EX3174.AHU1377).2Ca.2.5H.sub.2O:
(EX3174.AHU1377).2Ca.2.5H.sub.2O.

Description

DESCRIPTION OF THE DRAWINGS

[0032] FIG. 1 Chinese Guidelines for the Diagnosis and Treatment of Heart Failure 2018-Classification and Diagnostic Criteria of Heart Failure Table.

SPECIFIC EMBODIMENTS

[0033] The invention will now be described in further details with reference to examples and drawings, but the embodiments of the invention are not limited thereto.

[0034] In the following examples:

[0035] An Empyrean X-ray diffractometer was employed for X-ray powder diffraction detection. The detection conditions: Cu target Kα ray, voltage 40 KV, current 40 mA, emission slit 1/32°, anti-scatter slit 1/16°, anti-scatter slit 7.5 mm, 2θ range 3°-60°, step length 0.02°, and residence time per step 40 s.

[0036] DSC204F1 differential scanning calorimeter from NETZSCH, Germany was employed to detect differential scanning calorimetry spectra. Detection conditions: atmosphere: N.sub.2, 20 mL/min; scanning procedure: recording the heating curve by increasing the temperature from room temperature at 10° C./min to 250° C.

[0037] TG209 thermogravimetric analyzer from NETZSCH, Germany was employed to detect the moisture content. Detection conditions: atmosphere: N.sub.2, 20 mL/min; scanning procedure: room temperature to 700° C., heating rate: 10° C./min.

[0038] EXP3174 used in the examples was self-made by the company, with a purity of 98.3%. AHU377 calcium salt used in the examples was self-made by the company, with a purity of 99.4%.

Example 1

[0039] Preparation of AHU377 Free Acid:

[0040] A 250 mL single-necked flask was added with 2.1 g of AHU377 calcium salt and 40 mL of isopropyl acetate, and then added with 4.5 mL of 2 mol/L hydrochloric acid at room temperature and stirred to dissolve. The liquids were separated to collect the organic layer that was washed twice with 20 mL of water; after precipitated under reduced pressure at 35° C., it provided AHU377 free acid.

Example 2

[0041] Preparation of the Complex: (Prepared According to Example 2 of Patent WO2017125031A1)

##STR00004##

[0042] At room temperature, 2.36 g of AHU377 free acid, 2 g of EXP3174 and 40 mL of acetone obtained according to the method in Example 1 were added into a 250 mL three-necked flask, and dissolved; 1.3 equivalents of calcium hydroxide solid to AHU377 and 1 mL of water were added at room temperature, stirred at room temperature for 10 h and added with 40 mL more of acetone to react for another 8 hours. Under nitrogen protection, it was filtered by a Buchner funnel. The solid was rinsed with acetone to provide a white solid that was vacuum dried at 35° C. for 8 h and dried to provide 3.5 g of solid (EXP3174.AHU377).sup.3−.1.5Ca.sup.2+.2.5H.sub.2O, with a purity of 99% as determined by HPLC. The test was repeated to obtain sufficient doses for pharmacodynamic experiments.

Example 3

[0043] Preparation of the Complex: (Prepared According to Example 3 of Patent WO2017125031A1)

##STR00005##

[0044] At room temperature, 2.36 g of AHU377 free acid, 2 g of EXP3174 and 40 mL of acetone obtained according to the method in Example 1 were added into a 250 mL three-necked flask, and dissolved; 1.6 equivalents of calcium hydroxide solid to AHU377 and 0.6 mL of water were added at room temperature, stirred for 6 h at 35° C. and added with 40 mL more of acetone to react for another 8 hours. Under nitrogen protection, it was filtered by a Buchner funnel. The solid was rinsed with acetone to provide a white solid that was vacuum dried at 50° C. for 8 h and dried to provide 3.1 g of solid (EXP3174.AHU377).sup.3−.Math.1.5 Ca.sup.2+.2H.sub.2O. The test was repeated to obtain sufficient doses for pharmacodynamic experiments.

Example 4

[0045] A Pharmacodynamic Study on the Complex in the Canine Chronic Heart Failure Model-Reduced Ejection Fraction

[0046] 4.1 Methods: After the animals arrived at the facility, they were on adaptive feeding, and randomized after echocardiography and ECG, and then the experiment was started. On the day of operation, animals were anesthetized by intramuscular injection of Zoletil (5 mg/kg). The trachea of the anesthetized dogs were connected to the ventilator, and then they were fixed in a supine position, their chest was opened between the third and fourth ribs, the left anterior descending coronary artery was ligated to close the thoracic cavity, and then the skin was sutured. After the animals recovered for 3 days after operation, they were given therapeutic drugs by gavage, once a day for four consecutive weeks. During the experiment, animal's living conditions were observed every day, and their abnormal conditions were recorded. After 42 days of dosing, echocardiography was performed.

[0047] 4.2 Modeling: On the day before operation, the animals were fasted overnight. On the day of operation, the animals were intramuscularly injected with Zoletil (dose: 5 mg/kg) to induce anesthesia, and also were intramuscularly administered with atropine sulfate injection (dose: 0.5 mg/dog). After the animals were anesthetized, their hair on the left chest was shaved clean. Tracheal intubation was quickly performed to connect to the ventilator to provide artificial respiration and provide 1.5% isoflurane gas to maintain the anesthesia state, and also a monitor was used to monitor blood oxygen saturation, heart rate, electrocardiogram, body temperature and respiratory rate, etc. After the skin of the forelimbs was disinfected with 70% alcohol, the cephalic veins were found for intravenous intubation and indwelling intravenously as the dosing access. Iodophor and 70% alcohol were used to sterilize the left chest skin as the aseptic treatment. A sterile surgical drape hole towel was spread. A sterile scalpel was used to cut the skin along the fourth and fifth intercostal space, and after hemostasis, an electric knife was used to cut open the subcutaneous tissues layer by layer and muscular layers, and bleeding was stopped in a timely manner. The pleural membrane was carefully opened to expose the lung tissues, avoiding damage to the lung tissues; the surgical field of view was gradually expanded to 20-25 cm along the lower edge of the fourth rib, and a chest expander was used to expand the surgical window. A sterile gauze soaked in warm normal saline was used to push and protect the lung tissues. A sterile gauze soaked in warm normal saline was used to push the left atrial appendage to expose it between the left ventricle and the left atrium, and a blunt right-angle forceps was used to separate the left anterior descending coronary artery. A 4 #silk thread was used to pass through the artery, avoiding pulling the artery during the separating and threading. A silk thread was used to ligate the left anterior descending coronary artery. During the ligation, the animals were closely observed for blood oxygen saturation, heart rate, electrocardiogram, body temperature and respiratory rate. If an animal had abnormalities such as ventricular fibrillation, the operation would be stopped immediately and lidocaine injection (10 mg/kg) would be quickly administered via cephalic veins for treatment. After it was confirmed to have no bleeding in the thoracic cavity, the protective gauze was removed. A 7 #suture was used to pass through the fourth and fifth ribs to suture the thoracic cavity. The manual compression method was used to recruit lungs. The tissue and skin were sutured layer by layer. After operation, the animals were kept warm and properly replenished with physiological saline, and were closely observed for blood oxygen saturation, heart rate, electrocardiogram, body temperature and respiratory changes; the gas anesthesia machine was turned off, and the tracheal intubation was removed until the animals fully recovered their spontaneous respiration. After operation, a pain-killer (meloxicam, 0.67 mg/kg) was intramuscularly injected for pain relief, and ampicillin sodium 20 mg/kg was intramuscularly injected for anti-infection.

[0048] 4.3 Groups and administration: Before grouping, each dog received echocardiography and ECG monitoring. According to ejection fraction, the dogs were randomized into 5 groups (4-6 animals in each group). Three days after animal modeling, the dogs in each group were given corresponding drugs by gavage once a day for 6 weeks. All operations were performed in 6 batches for the experiment, with 4-5 animals in each batch and 0-1 animal in each group. Information about each group is shown in Table 1:

TABLE-US-00001 TABLE 1 Group Number of Dosing No. Group animals Dose given frequency Duration 1 Sham 4 — p.o qd 3 d after modeling for 6 weeks 2 Model 5 — p.o qd 3 d after modeling for 6 weeks 3 Positive drug 6 100 mg/kg p.o qd 3 d after modeling (LCZ696) for 6 weeks 4 EXP3174 + 6 EXP3174 52 p.o qd 3 d after modeling sacubitril calcium mpk + for 6 weeks salt physical mix Sacubitril 48 mpk 5 Complex of the 6 100 mg/kg p.o qd 3 d after modeling invention for 6 weeks Note: All doses are given based on anhydrous free acid, and the compound obtained in Example 3 is used as the complex of the invention.

[0049] 4.4 Experimental results: An important manifestation of chronic heart failure is reduced left ventricular systolic function, which is the primary endpoint for clinical detection of chronic heart failure. Echocardiography showed that the left ventricular ejection fraction (LVEF) was significantly reduced (<40%) in the dogs of the model group after modeling, with a P value less than 0.001 as compared with the sham group, which could better simulate the chronic heart failure with reduced clinical ejection fraction. Table 2 showed that the endpoint LVEF of dogs was 46.45% in the LCZ696 group, which was significantly higher than that in the model group (P<0.001). LVEF could be increased by both the complex of the invention and the physical mixture, which was statistically significant compared with that in the model group (P<0.001). Also, the 100 mpk (mg/kg) of the complex of the invention and the equimolar dose of LCZ696 had better effects on LVEF. The details are shown in the table below:

TABLE-US-00002 TABLE 2 Effects of compounds on the endpoint-left ventricular ejection fraction in dogs with heart failure (Mean ± SD) Group Number LVEF (%) Sham 4 66.20 ± 2.83 Model 5 35.82 ± 2.02### LCZ696, 100 mpk 6 46.45 ± 3.39*** EXP3174 + sacubitril calcium 6 46.34 ± 2.59*** salt physical mix Complex of the invention, 6 51.87 ± 1.01***.sup.@$ 100 mpk

[0050] Among them, model LVEF is 35.82% (<40%), indicating that the ejection fraction is reduced and the modeling is successful, as shown in FIG. 1.

[0051] ###P<0.001, compared with sham group; *P<0.05, **P<0.01, ***P<0.001, compared with model group; @P<0.05, compared with physical mixture; .sup.$P<0.05, compared with LCZ696 group.

[0052] Note: The compound obtained in Example 3 is used as the complex of the invention. It can be seen from the above results that the supramolecular complexes with dual effects provided by the invention are used as the medicament in heart failure with reduced ejection fraction, which have a significantly better effect at the same dose than 100 mpk of LCZ696.

[0053] The complex of the invention has a better effect than the physical mixture of EXP3174+AHU377, fully demonstrating that use of the complex has significant advantages over the use of a physical combination of drugs.

Example 5

[0054] A Pharmacodynamic Study on the Complex in the Canine Chronic Heart Failure Model-Preserved Ejection Fraction

[0055] 5.1 Methods: After the animals arrived at the facility, they were on adaptive feeding, and randomized after echocardiography and ECG, and then the experiment was started. On the day of operation, animals were anesthetized by intramuscular injection of Zoletil (5 mg/kg). The tracheae of the anesthetized dogs were connected to the ventilator, and then they were fixed in a supine position; their chests were opened between the third and fourth ribs, the left anterior descending coronary artery was ligated to close the thoracic cavity, and then the skin was sutured. After the animals recovered for 3 days after operation, they were given therapeutic drugs by gavage, once a day for two consecutive weeks. During the experiment, the animals' living conditions were observed every day, and their abnormal conditions were recorded. After 14 days of dosing, echocardiography was performed.

[0056] 5.2 Modeling: On the day before operation, the animals were fasted overnight. On the day of operation, the animals were intramuscularly injected with Zoletil (dose: 5 mg/kg) to induce anesthesia, and also were intramuscularly administered with atropine sulfate injection (dose: 0.5 mg/dog). After the animals were anesthetized, their hair on the left chest was shaved clean. Tracheal intubation was quickly performed to connect to the ventilator to provide artificial respiration and provide 1.5% isoflurane gas to maintain the anesthesia state, and also a monitor was used to monitor blood oxygen saturation, heart rate, electrocardiogram, body temperature and respiratory rate, etc. After the skin of the forelimbs was disinfected with 70% alcohol, the cephalic veins were found for intravenous intubation and indwelling intravenously as the dosing access. Iodophor and 70% alcohol were used to sterilize the left chest skin as the aseptic treatment. A sterile surgical drape hole towel was spread. A sterile scalpel was used to cut the skin along the fourth and fifth intercostal space, and after hemostasis, an electric knife was used to cut open the subcutaneous tissues layer by layer and muscular layers, and bleeding was stopped in a timely manner. The pleural membrane was carefully opened to expose the lung tissues, avoiding damage to the lung tissues; the surgical field of view was gradually expanded to 20-25 cm along the lower edge of the fourth rib, and a chest expander was used to expand the surgical window. A sterile gauze soaked in warm normal saline was used to push and protect the lung tissues. A sterile gauze soaked in warm normal saline was used to push the left atrial appendage to expose it between the left ventricle and the left atrium, and a blunt right-angle forceps was used to separate the left anterior descending coronary artery. A 4 #silk thread was used to pass through the artery, avoiding pulling the artery during the separating and threading. A silk thread was used to ligate the left anterior descending coronary artery. During the ligation, the animals were closely observed for blood oxygen saturation, heart rate, electrocardiogram, body temperature and respiratory rate. If an animal had abnormalities such as ventricular fibrillation, the operation would be stopped immediately and lidocaine injection (10 mg/kg) would be quickly administered via cephalic veins for treatment. After it was confirmed to have no bleeding in the thoracic cavity, the protective gauze was removed. A 7 #suture was used to pass through the fourth and fifth ribs to suture the thoracic cavity. The manual compression method was used to recruit lungs. The tissue and skin were sutured layer by layer. After operation, the animals were kept warm and properly replenished with physiological saline, and were closely observed for blood oxygen saturation, heart rate, electrocardiogram, body temperature and respiratory changes; the gas anesthesia machine was turned off, and the tracheal intubation was removed until the animals fully recovered their spontaneous respiration. After operation, a pain-killer (meloxicam, 0.67 mg/kg) was intramuscularly injected for pain relief, and ampicillin sodium 20 mg/kg was intramuscularly injected for anti-infection.

[0057] 5.3 Groups and administration: Before grouping, each dog received echocardiography and ECG monitoring. According to ejection fraction, the dogs were randomized into 5 groups (5-6 animals in each group). Three days after animal modeling, the dogs in each group were given corresponding drugs by gavage once a day for 2 weeks. All operations were performed in 6 batches for the experiment, with 4-5 animals in each batch and 0-1 animal in each group. Information about each group is shown in Table 3:

TABLE-US-00003 TABLE 3 Group Number of Dosing No. Group animals Dose given frequency Duration 1 Sham 5 — p.o qd 3 d after modeling for 2 weeks 2 Model 5 — p.o qd 3 d after modeling for 2 weeks 3 Positive drug 6 100 mpk p.o qd 3 d after modeling (LCZ696) for 2 weeks 4 EXP3174 + 6 EXP3174 p.o qd 3 d after modeling sacubitril 52 mpk + for 2 weeks calcium salt sacubitril physical mix 48 mpk 5 Complex of 6 100 mpk p.o qd 3 d after modeling the invention for 2 weeks Note: All doses are given based on anhydrous free acid, and the compound obtained in Example 3 is used as the complex of the invention.

[0058] 5.4 Experimental results: An important manifestation of chronic heart failure is reduced left ventricular systolic function, which is the primary endpoint for clinical detection of chronic heart failure. Echocardiography showed that the left ventricular ejection fraction (LVEF) was significantly reduced but still higher than 50% in the dogs of the model group after modeling, with a P value less than 0.001 as compared with the sham group, which could better simulate the chronic heart failure with preserved clinical ejection fraction. Table 4 showed that the endpoint LVEF of dogs was 57.98% in the LCZ696 group, which was significantly higher than that in the model group (P<0.001). LVEF could be increased by both the complex of the invention and the physical mixture, which was statistically significant compared with that in the model group (P<0.05). Also, the 100 mg/kg of the complex of the invention and the equimolar dose of LCZ696 had better effects on LVEF and significantly better effects than those in the physical mixture group. The experimental results are shown in Table 4.

TABLE-US-00004 TABLE 4 Effects of compounds on the endpoint-left ventricular ejection fraction in dogs with heart failure (Mean ± SD) Group Number LVEF (%) Sham 4 68.15 ± 1.89 Model 5 51.80 ± 0.80### LCZ696, 100 mpk 6 57.98 ± 2.64** EXP3174 + sacubitril calcium 6 55.18 ± 2.96* salt physical mix Complex of the invention, 6 58.04 ± 1.29***.sup.@ 100 mpk

[0059] Among them, model LVEF is 51.80% (50%), indicating that the ejection fraction is preserved and the modeling is successful, as shown in FIG. 1.

[0060] ###P<0.001, compared with the sham group; *P<0.05, **P<0.01, ***P<0.001, compared with the model group; @P<0.05, compared with the physical mixture Note: The compound obtained in Example 3 is used as the complex of the invention. The above results show that the complex of the invention in a dog model with reduced ejection fraction has a better effect than that in a dog model with preserved ejection fraction. It can be seen that the pharmaceutical composition of the invention has specific selectivity for heart failure with reduced ejection fraction, which is difficult to predict based on the prior art.

[0061] The above-mentioned examples are the preferred embodiments of the invention, but the embodiments of the invention are not limited by the above-mentioned embodiments. Any other changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principle of the invention shall be equivalent replacement methods and be within the scope covered by the invention.