TWI905319B - Use of complexes of ARB metabolites and NEP inhibitors in the preparation of drugs for the prevention and/or treatment of kidney disease. - Google Patents

Abstract

This invention belongs to the field of pharmaceutical application technology and relates to the use of a complex of ARB metabolites and NEP inhibitors in the preparation of drugs for the prevention and/or treatment of kidney diseases.

Description

Use of complexes of ARB metabolites and NEP inhibitors in the preparation of medicines for the prevention and/or treatment of kidney disease.

This invention belongs to the field of pharmaceutical application technology and relates to the use of a complex of ARB metabolites and NEP inhibitors in the preparation of drugs for the prevention and/or treatment of kidney diseases.

WO2007056546A1 discloses a sodium salt complex of Valsartan and Sacubitril (LCZ696) and its preparation method, which was approved for marketing in China in 2017 under the brand name ^Noxinto® , for the treatment of heart failure. Its molecular structural unit is as follows:

In addition, WO2017125031A1 discloses a series of complexes consisting of angiotensin receptor antagonist metabolites (EXP3174) and NEP inhibitors (Sacubitril), which show certain effects on heart failure with preserved ejection fraction (HFpEF). The molecular structural units are as follows:

Chronic kidney disease (CKD) is characterized by high prevalence, low awareness, poor prognosis, and high medical costs. Following cardiovascular and cerebrovascular diseases, diabetes, and malignant tumors, it is another serious threat to human health. In recent years, the prevalence of CKD has been rising annually, reaching as high as 14.3% in the global general population. Cross-sectional epidemiological studies in my country show that the prevalence of CKD in people aged 18 and above is 10.8%. With the aging of my country's population and the increasing incidence of diseases such as diabetes and hypertension, the incidence of CKD is also showing a continuous upward trend.

Therefore, it is crucial to find a targeted drug that has a good therapeutic effect on kidney disease.

In view of the technical problems existing in the prior art, the present invention provides the use of a complex of ARB metabolites and NEP inhibitors (or "supramolecular complex") in the preparation of drugs for the prevention and/or treatment of kidney diseases, the structural unit of said complex being as follows: (aEXP3174·bAHU377)·xCa·nA. Wherein, the structural formulas of EXP3174 and AHU377 are as follows, EXP3174 AHU377

As a preferred technical solution of the present invention, the kidney disease includes chronic kidney disease, and further includes chronic kidney disease complicated with hypertension, chronic kidney disease complicated with heart failure, etc.

As a preferred embodiment of the present invention, the kidney disease includes CKD patients classified as A1, A2, and A3.

As a preferred technical solution of the present invention, the composite of the present invention can effectively inhibit the increase of urinary albumin, and/or urinary albumin/urinary creatinine ratio, and/or urinary creatinine and serum creatinine in CKD patients.

Specifically, the chronic kidney disease mentioned is referred to in the "Guidelines for Screening, Diagnosis and Prevention of Chronic Kidney Disease", Vol. 37, No. 1, January 2017, Chinese Journal of Practical Internal Medicine, Shanghai Expert Group for Early Detection and Standardized Diagnosis and Treatment of Chronic Kidney Disease.

As a preferred embodiment of the present invention, the drug refers to a drug applied to patients suffering from the aforementioned kidney disease; based on the experimental results of the present invention and the dosage of the prodrug, the single-dose form of the drug refers to the complex containing approximately 60 mg to 1000 mg of the total mass of (aEXP3174·bAHU377), including but not limited to 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 1... 50mg, 160mg, 170mg, 180mg, 190mg, 200mg, 210mg, 220mg, 230mg, 240mg, 250mg, 260mg, 270mg, 280mg, 290 mg, 300mg, 310mg, 320mg, 330mg, 340mg, 350mg, 360mg, 370mg, 380mg, 390mg, 400mg, 410mg, 420mg, 430mg , 440mg, 450mg, 460mg, 470mg, 480mg, 490mg, 500mg, 510mg, 520mg, 530mg, 540mg, 550mg, 560mg, 570mg, 5 80mg, 590mg, 600mg, 610mg, 620mg, 630mg, 640mg, 650mg, 660mg, 670mg, 680mg, 690mg, 700mg, 710mg, 720 mg, 730mg, 740mg, 750mg, 760mg, 770mg, 780mg, 790mg, 800mg, 810mg, 820mg, 830mg, 840mg, 850mg, 860mg , 870mg, 880mg, 890mg, 900mg, 910mg, 920mg, 930mg, 940mg, 950mg, 960mg, 970mg, 980mg, 990mg, 1000mg.

As a preferred embodiment of the present invention, the single-dose form of the drug contains 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, and 960 mg of the complex.

In one implementation, a single-dose form refers to a daily-dose form, in which the patient is given the complex containing 60 mg/day to 1000 mg/day, and the frequency of administration includes, but is not limited to, once a day, twice a day, three times a day, four times a day, etc.

The drug is a solid preparation suitable for oral administration, preferably an oral tablet or capsule, and may be a total amount of 60 mg to 1000 mg in multiple tablets and multiple capsules.

The complex of the drug can be obtained by methods known in the prior art, wherein the complex and its preparation method disclosed in WO2017125031A1 are incorporated herein by reference.

As a better technical solution of the present invention, 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, 1:4.

As a preferred embodiment of the present invention, the structural unit of the composite is as follows: (EXP3174·AHU377)·xCa· _nH2O or Where x is a value between 0.5 and 2; and n is a value between 0 and 3.

As a better technical solution of the present invention, x is selected from 0.5, 1, 1.5, 2.

As a preferred technical solution of the present invention, the structural unit of the composite is as follows: (EXP3174·AHU377)·1.5Ca· _nH2O or (EXP3174·AHU377)·2Ca· _nH2O , where n is any value between 1 and 3.

As a better technical solution of the present invention, n is selected from 0.5, 1, 1.5, 2, 2.5, 3.

As a preferred embodiment of the present invention, the composite is selected from: (EXP3174·AHU377)·1.5Ca· _1H2O ; (EXP3174·AHU377)·1.5Ca· _1.5H2O ; (EXP3174·AHU377)·1.5Ca· _2H2O ; (EXP3174·AHU377)·1.5Ca· _2.5H2O ; (EXP3174·AHU377)·1.5Ca· _3H2O ; (EXP3174·AHU377)·2Ca· _1H2O ; (EXP3174·AHU377)·2Ca· _1.5H2O ; (EXP3174·AHU377)·2Ca· _2H2O ; (EXP3174·AHU377)·2Ca·2.5H ₂ O; (EXP3174·AHU377)·2Ca·3H ₂ O.

Those skilled in the art will understand that in the unit cell of the supramolecular complex, the alisartan ester metabolite (EXP3174), AHU377, calcium ions (Ca2 ⁺ ) and solvent molecules are filled in as several structural units.

The supramolecular complex (complex) described in this invention differs from a mixture obtained by simply physically mixing two active ingredients. The XRD pattern of the resulting supramolecular complex (complex) is significantly different from that of the EXP3174 and AHU377 calcium salts, and its solubility in various solvents (such as water, ethanol, ethanol-water, etc.) also shows significant differences. Other physicochemical properties, such as hygroscopicity, melting point, and infrared spectrum, also show significant differences.

The present invention offers the following advantages and beneficial effects compared to prior art: The present invention provides a series of supramolecular complexes (complexes) with dual action of alisartan medoxomil metabolites (EXP3174) and enkephalinase inhibitors (AHU377) for pharmaceutical use in the prevention and/or treatment of kidney diseases, showing significantly better efficacy at the same dosage compared to the use of LCZ696; The superior efficacy of the present invention's complexes compared to the physical mixture of EXP3174 and AHU377 fully demonstrates the significant advantages of using complexes over physical drug combinations.

The invention will now be described in further detail with reference to embodiments, but the embodiments of the invention are not limited thereto.

In the following embodiment: X-ray powder diffraction was performed using an Empyrean X-ray diffractometer. The detection conditions were: Cu target Kα radiation, voltage 40 kV, current 40 mA, emission slit 1/32°, antiscattering slit 1/16°, antiscattering slit 7.5 mm, 2θ range: 3°-60°, step size 0.02°, and dwell time per step 40 s.

Differential scanning calorimetry (DSC) spectra were obtained using a NETZSCH DSC204F1 differential scanning calorimeter from Germany. The test conditions were: atmosphere: _N2 , 20 mL/min; scan program: temperature increased from room temperature to 250℃ at a rate of 10℃/min, and the temperature curve was recorded.

Moisture content was measured using a NETZSCH TG209 thermogravimetric analyzer from Germany. The test conditions were: atmosphere: _N2 , 20 mL/min; scan program: room temperature - 700℃, heating rate: 10℃/min.

The EXP3174 used in the implementation example is manufactured by the company and has a purity of 98.3%.

The AHU377 calcium salt used in the implementation example is manufactured in-house and has a purity of 99.4%.

Implementation Example 1

Preparation of AHU377 free acid:

Add 2.1 g of AHU377 calcium salt and 40 mL of isopropyl acetate to a 250 mL single-necked flask. At room temperature, add 4.5 mL of 2 mol/L hydrochloric acid and stir until dissolved. Separate the solutions, collect the organic layer, and wash the organic layer twice with 20 mL of water. Desolvate under reduced pressure at 35 °C to obtain free AHU377 acid.

Implementation Example 2

Preparation of the composite: (Prepared according to Embodiment 2 of Patent WO2017125031A1)

At room temperature, 2.36 g of AHU377 free acid, 2 g of EXP3174, and 40 mL of acetone obtained according to Example 1 were added to a 250 mL three-necked flask and dissolved. At room temperature, 1.3 equivalents of calcium hydroxide solid and 1 mL of water were added, and the mixture was stirred at room temperature for 10 h. Another 40 mL of acetone was added, and the reaction was continued for 8 h. The mixture was filtered through a Buchner funnel under nitrogen protection, and the solid was washed with acetone to obtain a white solid. This solid was then vacuum-dried at 35 °C for 8 h to obtain 3.5 g of solid (EXP3174·AHU377) ^³⁻ · ^1.5Ca²⁺ · _2.5H₂O . HPLC analysis showed a purity of 99%. The experiment was repeated to obtain a sufficient amount for the efficacy test.

Implementation Example 3

Preparation of the composite: (Prepared according to Example 3 of Patent WO2017125031A1)

At room temperature, 2.36 g of AHU377 free acid obtained according to the method of Example 1, 2 g of EXP3174, and 40 mL of acetone were added to a 250 mL three-necked flask and dissolved. At room temperature, 1.6 equivalents of calcium hydroxide solid and 0.6 mL of water were added, and the mixture was stirred at 35°C for 6 h. 40 mL of acetone was added, and the reaction was continued for another 8 h. The mixture was filtered through a Buchner funnel under nitrogen protection, and the solid was washed with acetone to obtain a white solid. This solid was then dried under vacuum at 50°C for 8 h to obtain 3.1 g of solid (EXP3174•AHU377) ^³⁻ • ^1.5Ca²⁺ • _2H₂O (named compound A). The experiment was repeated to obtain a sufficient amount for the pharmacodynamic experiment.

Implementation Example 4

4.1 Information on Reagents, Test Samples, and Instruments Sodium Carboxymethyl Cellulose: Batch No.: B1707016, Shanghai Aladdin Biochemical Technology Co., Ltd.; EXP3174: Batch No.: 20190501Z, Huizhou Sinopharm Pharmaceutical Co., Ltd.; Sacubitril Calcium Salt: Batch No.: DYF20003, Huizhou Sinopharm Pharmaceutical Co., Ltd.; LCZ696: Batch No.: DYF19005, Huizhou Sinopharm Pharmaceutical Co., Ltd.; S086: Batch No.: SWV20001, Huizhou Sinopharm Pharmaceutical Co., Ltd.; Cretinol Assay Kit (Croscine Oxidase Method): Batch No.: 141120017, Shenzhen Mairui Biomedical Electronics Co., Ltd.; Albumin Assay Kit (Bromocresol Chloride Method): Batch No.: 148320004, Shenzhen Mairui Biomedical Electronics Co., Ltd. Fully Automated Biochemical Analyzer: Model BS-240VET, Shenzhen Mairui Biomedical Electronics Co., Ltd.

4.2 Experimental Animals

Eighty healthy SPF-grade male SD rats, weighing 180–220g, with laboratory animal quality certificate number No. 110011201106160342, were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., with laboratory animal production license number SCXK(京)2016-0006. They were housed in the SPF-grade laboratory animal center of Shenzhen Sinopharm Pharmaceutical Co., Ltd., with laboratory animal use license number SYXK(笵)2019-0142.

4.3 Preparation and grouping of rat 5/6 nephrectomy CKD model

After one week of acclimatization, the rats were randomly divided into two groups: one group (6 rats) was the sham surgery group, and the other group (72 rats) was the surgery group, which underwent 5/6 nephrectomy. Rats in the surgical group were anesthetized by intraperitoneal injection of 1.5% sodium pentobarbital (30 mg/kg). The rats were fixed in a prone position on a board. After skin preparation, routine disinfection, and the wearing of sterile gloves, sterile gauze was laid. A longitudinal incision of approximately 1 cm was made on the rat's back, approximately 0.5 cm below the left costovertebral junction and approximately 1 cm to the left of the spine. The incision was made layer by layer to access the abdomen, exposing the kidneys located beside the spine. The renal capsule was dissected, and one-third of each end of the kidney was removed. Gelatin sponge was applied to the surgical site to stop bleeding. The incision was sutured layer by layer. Intramuscular injection of sodium penicillin was administered for infection control. Rats were observed daily and received routine nursing care. One week later, the right kidney was removed using the same surgical method. A total of 5/6 of the kidney was removed in the two surgeries. The sham surgery group only separated the fat capsules and did not remove the kidney tissue.

After the second surgery, surviving animals were rehabilitated and reared for 3 weeks before being divided into groups. Each rat was numbered, weighed, and had its blood collected with heparin anticoagulation. Plasma creatinine levels were measured. Using SPSS software, rats were randomly assigned to blocks based on their plasma creatinine levels. The groups were: model group, EXP3174 group (35 mg/kg), sacubitril group (33 mg/kg), LCZ696 low-dose group (34 mg/kg), LCZ696 high-dose group (68 mg/kg), S086 low-dose group (34 mg/kg), and S086 high-dose group (68 mg/kg), with 5-6 rats in each group. Gavage administration of the drugs began the day after group assignment, at a volume of 10 mL/kg, once daily for 4 weeks. Both the sham surgery group and the model group were given 0.5% CMC-Na solvent daily at a volume of 10 mL/kg. Blood was collected from the tail vein and 24-hour urine was collected from the metabolic cage at week 4 of the experiment.

4.4 Detection Indicators and Methods

At week 4 after medication, plasma creatinine, urinary albumin and urinary creatinine concentrations were measured, and 24-hour urinary albumin levels and the urinary albumin/urinary creatinine ratio were calculated.

4.5 Statistical Methods

All experimental data are expressed as mean ± standard deviation. Statistical analysis was performed using SPSS 24.0 software, following these methods: First, normality (Shapiro-Wilk test) and homogeneity of variance (Lvene's test) analyses were conducted. If the data conformed to normality (P > 0.05) and homogeneity of variance (P > 0.05), the SNK test in one-way ANOVA was performed. If the result P < 0.05, the data was considered statistically significant. If the data did not conform to normality (P < 0.05) or heterogeneity of variance (P < 0.05), the two independent samples test in nonparametric tests was used. If the result P < 0.05, the data was considered statistically significant.

4.6 Experimental Results Urinary albumin: ALB ( mg ) /24h Serial Number fake surgery Model EXP3174 (35mg/kg) n=6 AHU377 (33mg/kg) n=6 LCZ696 (34mg/kg) n=5 LCZ696 (68mg/kg) n=6 Compound A (34 mg/kg) n=6 Compound A (68 mg/kg) n=6 average value 38.65±12.94 94.95±63.95 ^# 60.23±13.06 80.03±35.10 55.55±9.12 47.31±15.02 37.38±6.05 ^*$$ 32.19±8.52 ^{*$&&^^} Inhibition rate / / 36.57% 15.72% 41.50% 50.18% 60.63% 66.10% Notes: Compared with the sham surgery group, ^# P <0.05; compared with the model group, ^* P <0.05; compared with the same mole LCZ696, ^$ P < 0.05, ^$$ P <0.01; compared with the same mole EXP3174, ^&& P <0.01; compared with the same mole AHU377, ^{^^} P < 0.01. Urinary albumin / creatinine ratio: UACR ( mg/mmol ) Serial Number fake surgery Model EXP3174 (35mg/kg) n=6 AHU377 (33mg/kg) n=6 LCZ696 (34mg/kg) n=5 LCZ696 (68mg/kg) n=6 Compound A (34 mg/kg) n=6 Compound A (68 mg/kg) n=6 average value 3.02±0.80 7.69±4.51 ^# 5.19±0.95 6.41±2.65 4.65±0.61 4.23±1.11 3.07±0.49 ^{* $$} 2.95±0.70 ^{* $ &&^^} Inhibition rate / / 32.56% 16.74% 39.55% 44.98% 60.14% 61.65% Notes: Compared with the sham surgery group, ^# P <0.05; compared with the model group, ^* P <0.05; compared with the same mole LCZ696, ^$ P < 0.05, ^$$ P <0.01; compared with the same mole EXP3174, ^&& P <0.01; compared with the same mole AHU377, ^{^^} P < 0.01. Serum creatinine: CREA ( µM ) Serial Number fake surgery Model EXP3174 (35mg/kg) n=6 AHU377 (33mg/kg) n=6 LCZ696 (34mg/kg) n=5 LCZ696 (68mg/kg) n=6 Compound A (34 mg/kg) n=6 Compound A (68 mg/kg) n=6 average value 25.28±7.58 81.38±6.92 ^## 63.45±8.27 ^** 76.40±12.53 71.06±13.21 60.50±8.62 ^** 61.43±8.06 ^** 51.07±12.70 ^{** ^} Inhibition rate / / 22.04% 6.12% 12.68% 25.66% 24.51% 37.25% Note: Compared with the sham surgery group, ^## P <0.01; compared with the model group, ^** P <0.01; compared with the same molar AHU377, ^{^} P < 0.05.

The results above show that the compound of the present invention has significantly better effects on the indicators of 24-hour urinary albumin and urinary albumin/creatinine ratio in the CKD model than the same molar dose of LCZ696. At the same time, the compound of the present invention also has better effects than the same molar doses of EXP3174 and AHU377 administered alone, which fully demonstrates that the compound has obvious advantages.

Furthermore, the compound of this invention showed significantly better efficacy in serum creatinine levels in the CKD model compared to LCZ696 at an equal molar dose; at the same time, the compound of this invention also showed better efficacy than EXP3174 and AHU377 administered alone at equal molar doses, fully demonstrating the significant advantages of the compound.

The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit, essence, and principles of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

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Claims (5)

The use of a complex of an ARB metabolite and a NEP inhibitor in the preparation of a medicament for the prevention and/or treatment of chronic kidney disease by reducing urinary albumin levels, the urinary albumin/creatinine ratio, and plasma creatinine concentration, wherein the structural unit of the complex is as follows: (aEXP3174．bAHU377)．xCa．nA where the structural formulas of EXP3174 and AHU377 are as follows, respectively. Where a:b = 1:0.25~4; x is a value between 0.5 and 3; A represents water, methanol, ethanol, 2-propanol, acetone, ethyl acetate, methyl tert-butyl ether, acetonitrile, toluene, and dichloromethane; n is a value between 0 and 3, and the structure of the complex is as follows: As described in claim 1, wherein the drug, in a single-dose form, contains between 60 mg and 1000 mg of the complex, based on the total mass of EXP3174 and AHU377. As described in claim 1, wherein, based on the total mass of EXP3174 and AHU377, the drug is provided in single-dose form in quantities of 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, and 960 mg of the complex. As described in claim 1, wherein the drug is a solid preparation suitable for oral administration. As described in claim 4, the oral solid preparation is an oral tablet or capsule.