CN115068476A - Combination medicine for treating heart failure and application thereof - Google Patents

Abstract

The invention belongs to the biomedical technology, and particularly relates to a pharmaceutical composition capable of effectively resisting heart failure, which comprises active ingredients selected from the following groups: menthol-allethrin combination, nifedipine-verapamil combination, miconazole-allethrin combination, miconazole-butamben combination, nifedipine-allethrin combination, or single drug thereof, or combination thereof.

Description

Combination medicine for treating heart failure and application thereof

Technical Field

The invention belongs to the biomedical technology, and particularly relates to a plurality of combined medicines for treating heart failure and application thereof.

Background

Heart failure (heart failure for short) is a complex clinical syndrome caused by structural and functional abnormalities of the heart induced by various factors, which damages the contraction or relaxation function of the ventricles, and is also the late stage of various cardiovascular diseases (diabetes, hyperglycemia, hyperlipidemia). Although some medicines (beta-receptor blockers, angiotensin converting enzyme inhibitors, aldosterone receptor antagonists, vasodilators, inotropic drugs, diuretics) are currently used for treating heart failure, conventional drug therapy still has the problems of high hospitalization rate and high mortality rate of heart failure patients within five years.

The combined medicine can simultaneously act on a plurality of signal paths of diseases by combining medicine combination, thereby achieving the effective method of increasing the curative effect of the medicine and reducing the side effect of the medicine, and is widely applied to clinical treatment. The existing combination drugs for treating heart failure on the market are only 4, and have obvious difference compared with 51 single drugs on the market. Therefore, the development of novel anti-heart failure combination drugs is urgent in the field.

Disclosure of Invention

The invention mainly aims to provide a novel heart failure resisting combined medicament. Specifically, the invention provides a pharmaceutical composition of menthol-allethrin, nifedipine-verapamil, miconazole-allethrin, miconazole-butamben and nifedipine-allethrin, which is used for preparing a heart failure resistant medicament.

In a first aspect of the present invention, there is provided a pharmaceutical composition comprising:

A) a first active ingredient selected from the group consisting of:

A1) a menthol-allethrin combination;

A2) nifedipine-verapamil combination;

A3) a miconazole-allethrin combination;

A4) a miconazole-butamben-ester combination;

A5) nifedipine-allethrin combinations;

A6) any one of the active ingredients selected from the group consisting of: menthol, allethrin, miconazole, and butamben;

A7) one or more of a1) to a 6); and/or

B) Other pharmaceutically acceptable carriers, excipients or adjuvants.

In another preferred embodiment, the adjuvant is selected from: diluents, binders, disintegrants, lubricants, antioxidants, emulsifiers, colorants, solubilizers, or combinations thereof.

In another preferred embodiment, when the first active ingredient is a menthol-allethrin combination, the molar ratio of the menthol to the allethrin is 0.5-1000: 0.5-100; preferably 0.5-20: 0.5-20;

when the first active ingredient is a nifedipine-verapamil combination, the dosage molar ratio of nifedipine to verapamil is 0.5-500: 0.5-500; preferably 0.5-20: 0.5-20; more preferably 10-15: 10-15;

when the first active ingredient is a miconazole-allethrin combination, the using amount molar ratio of the miconazole to the allethrin is 0.5-200: 10-500; preferably 0.5-10: 0.5-20;

when the first active ingredient is a combination of miconazole and butyl aminobenzoate, the molar ratio of the miconazole to the butyl aminobenzoate is 0.5-200: 0.5-1000; preferably 0.5-10: 0.5-20; more preferably 0.5-10: 10-20;

when the first active component is a nifedipine-allethrin combination, the molar ratio of the nifedipine to the allethrin is 0.5-500: 0.5-500; preferably 0.5-20: 0.5-20; more preferably 10-20: 10-20.

In a second aspect of the invention, there is provided a use of a pharmaceutical composition according to the first aspect for the preparation of: a) a calcium channel blocker; and/or b) an anti-heart failure agent.

In another preferred embodiment, the calcium ion is an L-type calcium ion.

In another preferred embodiment, the pharmaceutical composition is used for preparing a calcium ion blocker or a medicament having one or more of the following effects:

(1) blocking Ca ²⁺ Internal flow;

(2) reduction of cytoplasmic Ca ²⁺ Concentration;

(3) improving diastolic function;

(4) protecting cardiac muscle cells.

In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from: oral liquid, tablet, capsule, injection or granule.

In a third aspect of the invention, there is provided an in vitro non-diagnostic, non-therapeutic block of Ca ²⁺ Influx and/or decrease cytoplasmic Ca ²⁺ A method of concentration comprising the steps of:

s1) administering a medically effective amount of the pharmaceutical composition according to the first aspect to the cardiomyocytes;

s2) the pharmaceutical composition binds to the allosteric site and the central cavity of the calcium channel.

In another preferred embodiment, the calcium ion channel is located on a cardiomyocyte.

In another preferred embodiment, the calcium channel is an L-type calcium channel.

In another preferred embodiment, the cardiomyocytes are selected from mammals, preferably humans, rats or mice.

In another preferred embodiment, the cardiomyocytes are H9C2 cells.

In a fourth aspect of the invention, there is provided a method of treating heart failure, comprising the steps of: administering to a subject in need thereof a medically effective amount of the pharmaceutical composition of the first aspect.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the protection of cardiomyocytes by menthol and allethrin in example 1

FIG. 2 shows that menthol-allethrin synergistically protects cardiomyocytes in example 2

FIG. 3 shows the effect of menthol-allethrin on LDH release and crystal violet staining in cardiomyocytes in example 3

FIG. 4 shows that in example 4, menthol-allethrin synergistically inhibits Ca ²⁺ Internal flow

FIG. 5 shows that nifedipine-verapamil synergistically protects cardiomyocytes in example 5

FIG. 6 shows that Miconazole-allethrin synergistically protects cardiomyocytes in example 6

FIG. 7 shows that in example 7, miconazole-butamben-amide synergistically protects cardiomyocytes

FIG. 8 shows that nifedipine-allethrin synergistically protects cardiomyocytes in example 8

FIG. 9 shows that nifedipine-verapamil synergistically inhibit Ca in example 9 ²⁺ Internal flow

Detailed Description

The present inventors have made extensive and intensive studies and, as a result, have unexpectedly found a series of pharmaceutical compositions effective against heart failure, and have completed the present invention.

Specifically, the research of the invention finds that menthol and allethrin both have the effect of resisting heart failure, and accordingly, a novel combined drug menthol-allethrin and other combined drugs for resisting heart failure are developed, wherein the novel combined drug menthol-allethrin comprises nifedipine-verapamil, miconazole-allethrin, miconazole-butamben and nifedipine-allethrin.

Term(s) for

As used herein, "pharmaceutical composition of the present invention", "a series of pharmaceutical compositions effective against heart failure" are used interchangeably and refer to a pharmaceutical composition comprising:

A) a first active ingredient selected from the group consisting of:

A1) a menthol-allethrin combination;

A2) nifedipine-verapamil combination;

A3) a miconazole-allethrin combination;

A4) a miconazole-butamben-ester combination;

A5) nifedipine-allethrin combinations;

A6) any one of the active ingredients selected from the group consisting of: menthol, allethrin, miconazole, and butamben;

A7) one or more of a1) to a 6); and/or

B) Other pharmaceutically acceptable carriers, excipients or adjuvants.

In another preferred embodiment, the adjuvant is selected from: diluents, binders, disintegrants, lubricants, antioxidants, emulsifiers, colorants, solubilizers, or combinations thereof.

Pharmaceutical compositions of the invention

The pharmaceutical composition comprises the components as described above, and the compatibility is as follows:

when the first active component is a menthol-allethrin combination, the molar ratio of the menthol to the allethrin is 0.5-1000: 0.5-100; preferably 0.5-20: 0.5-20;

when the first active ingredient is a nifedipine-verapamil combination, the dosage molar ratio of nifedipine to verapamil is 0.5-500: 0.5-500; preferably 0.5-20: 0.5-20; more preferably 10-15: 10-15;

when the first active ingredient is a miconazole-allethrin combination, the using amount molar ratio of the miconazole to the allethrin is 0.5-200: 10-500; preferably 0.5-10: 0.5-20;

when the first active ingredient is a combination of miconazole and butyl aminobenzoate, the molar ratio of the miconazole to the butyl aminobenzoate is 0.5-200: 0.5-1000; preferably 0.5-10: 0.5-20; more preferably 0.5-10: 10-20;

when the first active component is a nifedipine-allethrin combination, the molar ratio of the nifedipine to the allethrin is 0.5-500: 0.5-500; preferably 0.5-20: 0.5-20; more preferably 10-20: 10-20.

Application method

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity (i.e., anti-aging function) in a human and/or animal and is acceptable to the human and/or animal.

As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The pharmaceutical composition of the invention contains a safe and effective amount of the first active ingredient of the invention, and pharmaceutically acceptable carriers and excipients. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Such excipients include, but are not limited to, diluents, binders, disintegrants, lubricants, antioxidants, emulsifiers, colorants, solubilizers, or combinations thereof.

The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the pharmaceutical composition of the present invention is administered at a dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight) per day. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

Typically, when the pharmaceutical composition of the present invention is orally administered, the daily average dose of a subject (human) of 60kg body weight is usually 10 to 500mg, preferably 20 to 300mg, more preferably 50 to 250 mg. The daily dose may be administered in one, two or more divided doses.

The pharmaceutically acceptable carrier of the present invention includes (but is not limited to): water, saline, liposomes, lipids, peptidic substances, cellulose, nanogels, or combinations thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

Heart failure & L-type calcium ion channel

L-type calcium channel-associated proteins are associated with the adrenergic and cardiac muscle contraction signaling pathways in cardiomyocytes. The calcium channel blocker can reduce the concentration of calcium ions in cells, cause the relaxation of cardiac muscle, directly improve the relaxation function of cardiac muscle and further relieve heart failure.

The main advantages of the present invention include:

(1) the invention discovers a series of pharmaceutical compositions capable of effectively resisting heart failure for the first time.

(2) The pharmaceutical composition of the invention can play a role in resisting heart failure synergistically.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not indicated in the following examples, are generally carried out according to conventional conditions, for example as described in Sambrook et al, molecular cloning, A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the conditions as recommended by the manufacturer. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Example 1 protection of cardiomyocytes by menthol and allethrin based on heart failure disease model

(1) Rat cardiomyocytes (H9C2) were inoculated uniformly into 96-well plates at a cell concentration of 10000-.

(2) H9C2 cells were cultured in a cell incubator for 36H, the medium was aspirated, 100. mu.L PBS was added to each well for rinsing, and PBS was aspirated.

(3) The experiment was divided into a Normal Group (NG), a model Group (GD), an empagliflozin group (EMPA), and a dosing group. The normal group is added with a high-sugar DMEM medium, and the model group is added with a serum-free and sugar-free DMEM medium. The engagliflozin group and the administration group were added with serum-free and sugar-free DMEM medium in which the drugs were dissolved. After 36h of drug treatment, cell viability was measured using the CCK8 kit.

The test results show that 100 mu M-400 mu M menthol has protective effect on myocardial cells to different degrees. The 10 mu M-25 mu M allethrin myocardial cells have protective effects in different degrees.

Example 2 synergistic protective Effect of menthol and allethrin on myocardial cells

The inventors tested the effect of 0-20 μ M menthol, 0-20 μ M allethrin, and their combination on the cell viability of H9C2, while the cell viability of the single and combination drugs was entered into CompuSyn (http:// www.combosyn.com) to calculate the synergy coefficient.

The results show that combinations of 0-20 μ M menthol and 0-20 μ M allethrin exhibit varying degrees of cytoprotective activity. 10 mu M menthol and 10 mu M allethrin show excellent cell protection activity (72.1 percent), and the synergistic coefficient is 0.01, thereby showing obvious synergistic effect.

Example 3 menthol and allethrin LDH and Crystal Violet staining experiments

Lactate Dehydrogenase (LDH) is released from dead cells and is therefore used as a measure of cell damage. 36h H9C2 cells GD, 50 uL of supernatant and 50 uL of reaction solution (b)

reagent) were added to 96-well plates. Incubate at room temperature in the dark, and add 50. mu.L of stop buffer after 30 minutes. The UV absorbance of 490nM was collected and the LDH release calculated.

The inventors found that 10 μ M menthol +10 μ M allethrin can significantly reduce LDH release compared to the model group, which indicates that 10 μ M menthol +10 μ M allethrin can protect cardiomyocytes.

Crystal violet staining can visually reflect the viability of cells. H9C2 cells were seeded into 12-well plates at 100,000 cells per well. After 36h drug treatment and injury, cells were placed on ice and rinsed twice with pre-cooled PBS. Then, a pre-cooled methanol solution was added and placed in a-20 ℃ freezer for 10 min. The methanol was aspirated and incubated for 10min with 0.5% crystal violet solution. After dyeing, with ddH ₂ Cells were washed and photographed.

The inventors found that the menthol + allethrin group stained more area than the GD group, indicating that it had a significant cytoprotective effect.

Example 4 menthol and allethrin affect Ca ²⁺ Intracellular experiments

The calcium channel blocker reduces the concentration of cytoplasmic calcium, causes the relaxation of cardiac muscle, and directly improves the relaxation function, thereby achieving the effect of resisting heart failure. Menthol and allethrin are calcium channel blockers, and the inventor guesses that the synergistic anti-heart-failure effect of menthol and allethrin is that the menthol and allethrin simultaneously act on different sites of calcium channels, so that the synergistic protection effect is achieved.

Ca for the inventors ²⁺ Measurement of intracellular Ca by fluorescent probe Fluo-4/AM ²⁺ And (4) concentration. H9C2 cells were seeded into 96-well plates and after 12H overnight incubation, washed three times with HBSS buffer. mu.M Fluo-4/AM solvent in HBSS buffer and incubation at 37 ℃ for 10 min. Cells were then rinsed three times with HBSS buffer. Test compounds were dissolved in HBSS and added to cells for incubation for 15min, followed by Ca priming with 50mM KCl ²⁺ And (4) internal flow. The fluorescence signal was measured using a microplate reader. Fluo-4/AM excitation wavelength is 488nM, emission wavelength is 520 nM.

The inventors found that 10. mu.M allethrin, Ca, compared to the control group ²⁺ The fluorescence signal decreased significantly, whereas 10. mu.M peppermintThe alcohol did not change significantly. Whereas 10 μ M allethrin +10 μ M menthol compares with Ca, a single drug ²⁺ The fluorescence signal decreased significantly, indicating that allethrin and menthol can synergistically block Ca ²⁺ The internal flow, thereby achieving the effect of resisting heart failure.

Example 5 synergistic protective Effect of nifedipine and verapamil on myocardial cells based on Heart failure disease models

The inventors tested the effect of 0-20. mu.M nifedipine, 0-20. mu.M allethrin, and their combinations on the cell viability of H9C2, while the cell viability of the single and combination drugs was input to CompuSyn (http:// www.combosyn.com) to calculate the synergy coefficient.

The test result shows that nifedipine and verapamil can synergistically protect H9C2 cells. Wherein 20 mu M nifedipine and 20 mu M verapamil show excellent cell protection activity (66.4%), the synergistic coefficient is 0.02, and obvious synergistic effect is shown.

Example 6 synergistic protective action of miconazole and allethrin on myocardial cells based on heart failure disease model

The inventors tested the effect of miconazole at 0-10. mu.M, allethrin at 0-20. mu.M, and their combination on the cell viability of H9C2, while the cell viability of the single and combination drugs was entered into CompuSyn (http:// www.combosyn.com) to calculate the synergy coefficient.

The test results show that miconazole and allethrin can synergistically protect H9C2 cells. Wherein 10 mu M miconazole and 20 mu M allethrin show excellent cell protection activity (63.8 percent), the synergistic coefficient of the miconazole and the allethrin is 0.01, and obvious synergistic effect is shown.

Example 7 synergistic protective Effect of Miconazole and Aminophenylbutyl on myocardial cells based on Heart failure disease models

The inventors tested the effect of 0-10. mu.M miconazole, 0-20. mu.M butamben-amide, and their combinations on the cell viability of H9C2, while the cell viability of the single and combination drugs was entered into CompuSyn (http:// www.combosyn.com) to calculate the synergy coefficient.

The test results show that miconazole and phenylbutyramine can protect H9C2 cells synergistically. Wherein 10 mu M miconazole and 20 mu M aminobenzene butyl ester show excellent cell protection activity (68.7%), the synergistic coefficient is 0.01, and obvious synergistic effect is shown.

Example 8 synergistic protective action of nifedipine and allethrin on myocardial cells based on heart failure disease model

The inventor tests the influence of 0-20 mu M nifedipine, 0-20 mu M allethrin and the combination on the cell activity of H9C2, and simultaneously inputs the cell activity of the single drug and the combination drug into CompuSyn (http:// www.combosyn.com) to calculate the synergistic coefficient.

The test result shows that nifedipine and allethrin can synergistically protect H9C2 cells. Wherein 20 mu M nifedipine and 20 mu M allethrin show excellent cytoprotective activity (76.6%), the synergy coefficient is 0.009, and obvious synergistic effect is shown.

Example 9 nifedipine and verapamil affect Ca ²⁺ Intracellular experiments

The inventors found that 20. mu.M nifedipine and 20. mu.M verapamil contained Ca compared to the control group ²⁺ The fluorescence signal decreased significantly. While 20 μ M nifedipine +20 μ M verapamil compared to Ca alone ²⁺ The fluorescence signal is obviously reduced, which shows that nifedipine and verapamil can act synergistically to prevent Ca through an L-type calcium ion channel ²⁺ The internal flow, thereby achieving the effect of resisting heart failure.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims (8)

1. A pharmaceutical composition, comprising:

A) a first active ingredient selected from the group consisting of:

A1) a menthol-allethrin combination;

A2) nifedipine-verapamil combination;

A3) a miconazole-allethrin combination;

A4) a miconazole-butamben-ester combination;

A5) nifedipine-allethrin combinations;

A6) any one of the active ingredients selected from the group consisting of: menthol, allethrin, miconazole, and butamben;

A7) one or more of a1) to a 6); and/or

B) Other pharmaceutically acceptable carriers, excipients or adjuvants.

2. The pharmaceutical composition of claim 1, wherein the excipient is selected from the group consisting of: diluents, binders, disintegrants, lubricants, antioxidants, emulsifiers, colorants, solubilizers, or combinations thereof.

3. The pharmaceutical composition of claim 1, wherein when the first active ingredient is a menthol-allethrin combination, the molar ratio of the menthol to the allethrin is 0.5-1000: 0.5-100; preferably 0.5-20: 0.5-20;

when the first active ingredient is a nifedipine-verapamil combination, the dosage molar ratio of nifedipine to verapamil is 0.5-500: 0.5-500; preferably 0.5-20: 0.5-20; more preferably 10-15: 10-15;

when the first active ingredient is a miconazole-allethrin combination, the using amount molar ratio of the miconazole to the allethrin is 0.5-200: 10-500; preferably 0.5-10: 0.5-20;

when the first active ingredient is a combination of miconazole and butyl aminobenzoate, the molar ratio of the miconazole to the butyl aminobenzoate is 0.5-200: 0.5-1000; preferably 0.5-10: 0.5-20; more preferably 0.5-10: 10-20;

when the first active component is a nifedipine-allethrin combination, the molar ratio of the nifedipine to the allethrin is 0.5-500: 0.5-500; preferably 0.5-20: 0.5-20; more preferably 10-20: 10-20.

4. Use of a pharmaceutical composition according to any one of claims 1 to 3 for the preparation of: a) a calcium channel blocker; and/or b) an anti-heart failure agent.

5. The use of claim 4, wherein the pharmaceutical composition is for the preparation of a calcium blocker or medicament having one or more of the following effects:

(1) blocking Ca ²⁺ Internal flow;

(2) reduction of cytoplasmic Ca ²⁺ Concentration;

(3) improving diastolic function;

(4) protecting cardiac muscle cells.

6. The use of claim 4, wherein the pharmaceutical composition is in a dosage form selected from the group consisting of: oral liquid, tablet, capsule, injection or granule.

7. In vitro non-diagnostic non-therapeutic block Ca ²⁺ Influx and/or decrease cytoplasmic Ca ²⁺ A method of concentration, comprising the steps of:

s1) administering a medically effective amount of the pharmaceutical composition of claim 1 to the cardiomyocytes;

s2) the pharmaceutical composition binds to the allosteric site and the central cavity of the calcium channel.

8. A method of treating heart failure, comprising the steps of: administering to a subject in need thereof a medically effective amount of the pharmaceutical composition of claim 1.

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