New use of carvedilol or its salts, compositions, and methods of preparation

Abstract

This invention discloses a novel use of carvedilol or its salts and compositions, as well as the composition and its preparation method. The β-blocker or pharmaceutical composition provided by this invention has good efficacy in preventing, treating, and / or alleviating drug-induced liver injury, effectively reducing mortality caused by this disease; simultaneously, it can significantly reduce serum transaminase levels, alleviate symptoms such as liver and spleen edema, and has a good inhibitory effect on pro-inflammatory cytokines. Furthermore, experimental results further indicate that the combination of the β-blocker and pharmaceutically acceptable excipients (steviosides and / or glycyrrhizate) has good safety and is easily dissolved, released, and / or absorbed, improving the bioavailability of carvedilol and exhibiting a synergistic effect, showing promising application prospects.

Description

Technical Field

[0001] This invention belongs to the field of chemical pharmaceuticals, specifically relating to new uses of carvedilol or its salts, compositions thereof, and the composition and its preparation method. Background Technology

[0002] Acetaminophen, also known as paracetamol, is one of the most common antipyretic analgesics. It is mainly used for fever caused by the common cold or influenza, and also for relieving mild to moderate pain. It has a very wide range of applications.

[0003] However, excessive dosage or prolonged use of medication can damage the liver, causing partial hepatocyte necrosis, manifested as abnormal liver function indicators such as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Generally, liver injury caused by drugs and / or their metabolites is termed drug-induced liver injury.

[0004] In recent years, the incidence of drug-induced liver injury has increased significantly in clinical practice, especially in Europe and the United States, where it is showing an increasingly serious trend. Overuse and abuse of acetaminophen is one of the main causes of this type of disease. After onset, serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) rise, leading to hepatocyte necrosis, which in turn can cause cirrhosis and even death.

[0005] Currently, N-acetylcysteine ​​(NAC) is mainly used as a treatment for this type of drug-induced liver injury. Although it is the most effective drug at present, it also has certain limitations and drawbacks (see: CN 112535694 A). There are no other better and more effective treatment options available.

[0006] Carvedilol is a lipophilic aromatic oxidized propanol compound with the structure shown below. This drug is a third-generation non-selective beta-blocker developed by Boehringer Mannheim in Germany. It was first marketed in the United States in 1991 and is mainly used for conditions such as hypertension and congestive heart failure.

[0007]

[0008] Dipotassium glycyrrhizate (DG) is a white or off-white powder with anti-inflammatory, anti-allergic, and moisturizing effects. In the pharmaceutical industry, it is mainly used for cough relief and expectoration, gastric ulcers, acute and chronic gastritis, eczema, pruritus, as well as for the treatment of cancer and the prevention and treatment of AIDS.

[0009] Stevia glycosides are a type of sweetener extracted from stevia leaves. They are highly sweet and low in calories, with a sweetness level far exceeding that of sucrose. They are widely used in the production of food, beverages, and seasonings.

[0010] To date, no literature has been found on the use of carvedilol, or its combination with glycyrrhizic acid (e.g., dipotassium glycyrrhizate) and steviol glycosides, for the prevention, treatment, and / or relief of liver injury, particularly drug-induced liver injury.

[0011] In view of this, the present invention is hereby proposed. Summary of the Invention

[0012] To address the problems and / or shortcomings of existing technologies, one of the main objectives of this invention is to provide a novel use for β-blockers (carvedilol or a pharmaceutically acceptable salt thereof) or pharmaceutical compositions thereof: the prevention, treatment, and / or relief of drug-induced liver injury. The β-blockers (carvedilol or a pharmaceutically acceptable salt thereof) or pharmaceutical compositions thereof provided by this invention can be used to prepare drugs for the prevention, treatment, and / or relief of drug-induced liver injury, or to lower serum transaminase levels, or as inhibitors of pro-inflammatory cytokines, exhibiting good therapeutic effects.

[0013] The technical solution provided by this invention is as follows:

[0014] Use of a β-blocker or a combination thereof in the preparation of a medicament for the prevention, treatment and / or relief of drug-induced liver injury, wherein the β-blocker is carvedilol or a pharmaceutically acceptable salt thereof;

[0015] Alternatively, the use of a β-blocker or a combination thereof in the preparation of a medicament for reducing serum transaminase levels, wherein the β-blocker is carvedilol or a pharmaceutically acceptable salt thereof.

[0016] Alternatively, the use of a β-blocker or a combination thereof in the preparation of a medicament for use as an inhibitor of pro-inflammatory cytokines, wherein the β-blocker is carvedilol or a pharmaceutically acceptable salt thereof.

[0017] In any of the above technical solutions (the described uses),

[0018] The drug-induced liver injury mentioned refers to drug-induced liver injury caused by acetaminophen.

[0019] Alternatively, the transaminase may be alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST);

[0020] Alternatively, the pro-inflammatory cytokine inhibitor may be an HMGB1 inhibitor, an IL-1β inhibitor, an IL-6 inhibitor, an NF-κB inhibitor, or a TNF-α inhibitor.

[0021] In any of the above technical solutions (the described uses), the composition comprises a β-receptor blocker and a pharmaceutically acceptable excipient; wherein the pharmaceutically acceptable excipient comprises steviol glycosides and / or glycyrrhizate;

[0022] Preferably, the pharmaceutically acceptable excipient comprises a combination of steviol glycosides and glycyrrhizic acid salts; wherein the weight ratio of steviol glycosides to glycyrrhizic acid salts is 1:5 to 15 (e.g., 1:5, 1:6, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:12, 1:13, 1:14, 1:15, etc.), for example: the weight ratio of steviol glycosides to glycyrrhizic acid salts is 1:8 to 10 (e.g., 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:9, 1:9.2, 1:9.5, etc.);

[0023] Preferably, the steviol glycoside is riboboroside (e.g., riboboroside A, B, C, D, E, F, etc. disclosed in prior art CN 112512337 A), more preferably riboboroside A; the glycyrrhizate is selected from one or more of sodium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, dipotassium glycyrrhizate, ammonium glycyrrhizate, and diammonium glycyrrhizate, more preferably dipotassium glycyrrhizate or disodium glycyrrhizate;

[0024] Preferably, the weight ratio of the β-receptor blocker to the pharmaceutically acceptable excipient is 1:5 to 50 (e.g., 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.), for example, the weight ratio of the β-receptor blocker to the pharmaceutically acceptable excipient is 1:10 to 25 or 1:15 to 20 (e.g., 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, etc.).

[0025] The present invention also provides a pharmaceutical composition comprising a β-receptor blocker and a pharmaceutically acceptable excipient; wherein the β-receptor blocker is carvedilol or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable excipient comprises steviol glycosides and / or glycyrrhizate;

[0026] Preferably, the steviol glycoside is riboboroside, more preferably riboboroside A; the glycyrrhizate is selected from one or more of sodium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, dipotassium glycyrrhizate, ammonium glycyrrhizate, and diammonium glycyrrhizate, more preferably dipotassium glycyrrhizate or disodium glycyrrhizate.

[0027] Preferably, the pharmaceutically acceptable excipient comprises a combination of steviol glycosides and glycyrrhizic acid salts; wherein the weight ratio of steviol glycosides to glycyrrhizic acid salts is 1:5 to 15 (e.g., 1:5, 1:6, 1:7, 1:7.5, 1:8, 1:8.5, 1:9, 1:9.5, 1:10, 1:10.5, 1:11, 1:12, 1:13, 1:14, 1:15, etc.), for example: the weight ratio of steviol glycosides to glycyrrhizic acid salts is 1:8 to 10 (e.g., 1:8.1, 1:8.2, 1:8.3, 1:8.4, 1:8.5, 1:8.6, 1:8.7, 1:8.8, 1:9, 1:9.2, 1:9.5, etc.).

[0028] In any of the above-described technical solutions (the pharmaceutical composition), the weight ratio of the β-receptor blocker to the pharmaceutically acceptable excipient is 1:5 to 50 (e.g., 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc.); preferably, the weight ratio of the β-receptor blocker to the pharmaceutically acceptable excipient is 1:10 to 25 or 1:15 to 20 (e.g., 1:15, 1:15.5, 1:16, 1:16.5, 1:17, 1:17.5, 1:18, 1:18.5, 1:19, etc.).

[0029] In any of the above-described technical solutions (the pharmaceutical composition), the β-receptor blocker exists in the pharmaceutical composition in an amorphous form.

[0030] In any of the above-described technical solutions (the pharmaceutical composition), the encapsulation efficiency of the β-blocker is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.); preferably, the encapsulation efficiency of the β-blocker is ≥90% or ≥95%.

[0031] In any of the above technical solutions (the pharmaceutical composition), the pharmaceutical composition is a solid dosage form or a liquid dosage form; and / or, the β-receptor blocker in the pharmaceutical composition is a therapeutically effective amount;

[0032] Preferably, the pharmaceutical composition is a liquid formulation, and the solvent of the liquid formulation is selected from pharmaceutically acceptable water, PBS buffer, or an aqueous solution of sodium carboxymethyl cellulose.

[0033] Preferably, when the concentration of carvedilol in the liquid formulation is 1 mg / mL, the liquid formulation satisfies one or more of the following conditions ① to ③:

[0034] ① The average micelle diameter of the liquid formulation is 1-80 nm (e.g., 1 nm, 5 nm, 10 nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, etc.); preferably 10-20 nm (e.g., 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, etc.).

[0035] ② The polydispersity index of the liquid formulation is ≤0.5; preferably ≤0.4;

[0036] ③ The zeta potential of the liquid formulation is -20 to 0 mV (e.g., -20 mV, -19 mV, -18 mV, -17 mV, -16 mV, -15 mV, -14 mV, -13 mV, -12 mV, -11 mV, -10 mV, -9 mV, -8 mV, -7 mV, -6 mV, -5 mV, etc.); preferably -15 to -5 mV;

[0037] More preferably, when the concentration of carvedilol in the liquid formulation is 1 mg / mL, the liquid formulation simultaneously satisfies conditions ① to ③.

[0038] In any of the above technical solutions (the pharmaceutical composition), the pharmaceutical composition is prepared by a method comprising the following steps: dispersing or dissolving a β-receptor blocker and a pharmaceutically acceptable excipient together in an organic solvent, mixing them, and then removing the organic solvent by rotary evaporation at 35-45°C, thereby obtaining the pharmaceutical composition.

[0039] Preferably, the organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the β-blocker is 0.5 to 20 mL (e.g., 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, etc.);

[0040] More preferably, the alcohol solvent is methanol or ethanol; and / or, the amount of the organic solvent used per milligram of the β-blocker is 1.2 to 2.5 mL (e.g., 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 2.2 mL, etc.).

[0041] In any of the above technical solutions (the pharmaceutical composition), the pharmaceutical composition is a pharmaceutical composition for preventing, treating and / or alleviating drug-induced liver injury; preferably, the drug-induced liver injury is drug-induced liver injury caused by acetaminophen.

[0042] Alternatively, the pharmaceutical composition is a pharmaceutical composition for lowering serum transaminase levels; for example, the transaminase is alanine aminotransferase (ALT) and / or aspartate aminotransferase (AST).

[0043] Alternatively, the pharmaceutical composition may be an inhibitor of pro-inflammatory cytokines, such as HMGB1 inhibitors, IL-1β inhibitors, IL-6 inhibitors, NF-κB inhibitors, or TNF-α inhibitors.

[0044] Furthermore,

[0045] The present invention also provides a method for preparing the pharmaceutical composition described in any of the preceding claims, comprising the following steps: dispersing or dissolving a β-receptor blocker and a pharmaceutically acceptable excipient together in an organic solvent, mixing them thoroughly, and then removing the organic solvent by rotary evaporation at 35–45°C to obtain the pharmaceutical composition;

[0046] Preferably, the organic solvent is an alcohol solvent; and / or, the amount of the organic solvent used per milligram of the β-blocker is 0.5 to 20 mL (e.g., 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 11 ml, 12 ml, 13 ml, 14 ml, 15 ml, 16 ml, 17 ml, 18 ml, etc.);

[0047] More preferably, the alcohol solvent is methanol or ethanol; and / or, the amount of the organic solvent used per milligram of the β-blocker is 1.2 to 2.5 mL (e.g., 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2 mL, 2.2 mL, etc.).

[0048] Regarding the definitions of terms used in this invention, unless otherwise stated, the initial definitions provided herein apply to the term throughout the text; for terms not specifically defined herein, the meanings that a person skilled in the art should give them should be based on the disclosure and / or context and common knowledge in the field.

[0049] The term "stevioside" refers to glycosides of steviol; including but not limited to: naturally occurring steviol glycosides, such as: steviol monosaccharide, steviol monosaccharide A, steviol disaccharide, steviol disaccharide D, stevioside, steviol disaccharide A, steviol disaccharide B, ribobadiol B, steviol glycoside, ribobadiol G, steviol glycoside A, steviol glycoside B, steviol glycoside C, ribobadiol glycoside A, ribobadiol glycoside E, ribobadiol glycoside E2, ribobadiol glycoside E4, ribobadiol glycoside E6, ribobadiol glycoside E3, ribobadiol glycoside D, ribobadiol glycoside I, ribobadiol glycoside AM, ribobadiol glycoside D7, ribobadiol glycoside M, ribobadiol glycoside M4, ribobadiol glycoside 1a, ribobadiol glycoside 1b, ribobadiol glycoside 1c, ribobadiol glycoside 1d, ribobadiol glycoside 1e, ribobadiol glycoside 1f, ribobadiol glycoside 1g, ribobadiol glycoside 1h, ribobadiol glycoside 1i, 1j, 1k, 1l, 1m, 1n, 1o, 1p, 1q, 1r, 1s, 1t, 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h, 2i, 2j, 2k, 2l, 2m, 2n, 2o, 2p, 2q, 2r, 2s; and synthetic steviol glycosides, such as enzymatically glycosylated steviol glycosides and combinations thereof (see: Chinese Patent Application CN). 112512337 A, the applicant is Spectrum Inc. (USA).

[0050] The term "pharmaceutical acceptable" generally refers to a drug that is chemically or physically compatible with other components that make up a drug dosage form and physiologically compatible with receptors.

[0051] The terms "salt" and "pharmaceutically acceptable salt" refer to the acidic and / or basic salts formed by the above-mentioned compound (carvedilol: containing a secondary amino group (NH), hydroxyl group (OH), etc., that can form salts) or its stereoisomers with inorganic and / or organic acids and bases, including zwitterionic salts (internal salts) and quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly during the final separation and purification of the compound. Alternatively, they can be obtained by mixing the above-mentioned compound, or its stereoisomers, with an appropriate amount (e.g., equimolar amounts) of an acid or base. These salts may be collected by filtration after precipitating in solution, or recovered after solvent evaporation, or prepared by freeze-drying after reaction in an aqueous medium. The salts described in this invention can be carvedilol hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoric acid, phosphate, acetate, propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate, or trifluoroacetate, etc.

[0052] The term "excipients" refers to substances other than the active ingredient that are included in a dosage form.

[0053] The term "therapeutic effective dose" refers to the amount of a drug compound administered to a patient that is sufficient to effectively treat the disease. Therapeutic effective doses vary depending on the drug compound, the type of disease, the severity of the disease, the patient's age, and other factors, and may be routinely adjusted as appropriate by those skilled in the art.

[0054] There are no particular restrictions on the administration method of the drug of the present invention. Representative administration methods include, but are not limited to, oral, parenteral (intravenous, intramuscular or subcutaneous), and local administration.

[0055] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), or with the following components: (a) filler or compatibilizer; (b) thickener, such as carboxymethyl cellulose and its salts; (c) humectant; (d) disintegrant; (e) slowing agent; (f) absorption accelerator; (g) wetting agent; (h) adsorbent; and (i) lubricant. In capsules, tablets, and pills, the dosage form may also contain a buffer.

[0056] Solid dosage forms such as tablets, sugar pills, capsules, pellets, and granules can be prepared using coatings and shell materials, such as casings and other materials known in the art. They may contain opaque agents, and the release of the active compound in such dosage forms can be delayed in a portion of the digestive tract. If necessary, the active compound may also be formed into microcapsules with one or more of the excipients described above.

[0057] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally used in the art, such as water or other solvents, as well as solubilizers and emulsifiers. Besides these inert diluents, the drug may also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and fragrances. Suspensions may contain suspending agents in addition to the active compound.

[0058] Drugs intended for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients are also included.

[0059] Pharmaceutical dosage forms for topical administration include ointments, powders, patches, sprays, and inhalers. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be necessary.

[0060] Without violating common sense in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0061] The reagents and raw materials used in this invention are all commercially available.

[0062] The positive and progressive effects of this invention are as follows:

[0063] In vitro and / or in vivo test results show that the β-blocker (carvedilol or a pharmaceutically acceptable salt thereof) or its pharmaceutical composition provided by this invention has good efficacy in preventing, treating, and / or alleviating liver injury (especially drug-induced liver injury), effectively reducing mortality and prolonging lifespan caused by this disease; simultaneously, it can significantly reduce serum transaminase levels, alleviate symptoms such as liver and spleen edema, and has a good inhibitory effect on pro-inflammatory cytokines. Furthermore, the test results further indicate that the combination of the β-blocker (carvedilol or a pharmaceutically acceptable salt thereof) and pharmaceutically acceptable excipients (steviosides and / or glycyrrhizate) has good safety and is easily dissolved, released, and / or absorbed, improving the bioavailability of carvedilol (CAR) and exhibiting a synergistic effect, achieving superior efficacy compared to any single component. Attached Figure Description

[0064] Figure 1 This is an appearance diagram of the CAR-DG-RA obtained in Embodiment 1 of the present invention.

[0065] Figure 2 This is an FT-IR comparison image of CAR-DG-RA obtained in Example 1 of the present invention.

[0066] Figure 3 This is a DSC comparison chart of CAR-DG-RA obtained in Example 1 of the present invention.

[0067] Figure 4 This is a comparison XRD pattern of CAR-DG-RA obtained in Example 1 of the present invention.

[0068] Figure 5 This is a comparison chart of the solubility of CAR-DG-RA obtained in Example 1 of the present invention.

[0069] Figure 6 This is a comparison of the in vitro release curves of CAR-DG-RA obtained in Example 1 of the present invention in PBS.

[0070] Figure 7 This is a comparison of the in vitro release curves of CAR-DG-RA obtained in Example 1 of the present invention in SGF / SIF.

[0071] Figure 8This is a comparison chart of the antioxidant activity of CAR-DG-RA obtained in Example 1 of the present invention (ABTS method: different culture times at a fixed concentration).

[0072] Figure 9 This is a comparison chart of the antioxidant activity of CAR-DG-RA obtained in Example 1 of the present invention (ABTS method: different concentrations under fixed culture time).

[0073] Figure 10 This is a comparison chart of the antioxidant activity of CAR-DG-RA obtained in Example 1 of the present invention (FRAP method: different culture times at a fixed concentration).

[0074] Figure 11 This is a comparison chart of the antioxidant activity of CAR-DG-RA obtained in Example 1 of the present invention (FRAP method: different concentrations under fixed culture time).

[0075] Figure 12 This is a comparison image of blood vessels before and after contact between CAR-DG-RA obtained in Example 1 of the present invention and the chicken embryo allantoic membrane (CAM).

[0076] Figure 13 This is a comparison chart of trypan blue absorption of CAR-DG-RA in contact with the chicken embryo allantoic membrane (CAM) obtained in Example 1 of the present invention.

[0077] Figure 14 This is a comparison of the survival rates of mice in different groups after injection of acetaminophen.

[0078] Figure 15 This is a comparison of the ratio of liver tissue to body weight in mice of different groups after injection of acetaminophen.

[0079] Figure 16 This is a comparison of the ratio of spleen tissue to body weight in mice of different groups after injection of acetaminophen.

[0080] Figure 17 This is a comparison of the serum aspartate aminotransferase (AST) levels in mice from different groups after acetaminophen injection.

[0081] Figure 18 This is a comparison of serum alanine aminotransferase (ALT) levels in mice from different groups after acetaminophen injection.

[0082] Figure 19 Images show the appearance of liver tissue in mice after injection of acetaminophen.

[0083] Figure 20 Comparative images of the histopathological observations of the livers of mice in different groups after injection of acetaminophen, stained with hematoxylin and eosin, under a microscope.

[0084] Figure 21This is a comparison chart showing the percentage of hepatocyte necrosis area in each group of mice after injection of acetaminophen.

[0085] Figure 22 This is a comparison of superoxide dismutase (SOD) levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0086] Figure 23 This is a comparison of malondialdehyde (MDA) levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0087] Figure 24 This is a comparison of HMGB1 levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0088] Figure 25 This is a comparison of IL-1β levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0089] Figure 26 This is a comparison of IL-6 levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0090] Figure 27 This is a comparison of NF-κB levels in the liver tissues of mice in different groups after injection of acetaminophen.

[0091] Figure 28 This is a comparison of TNF-α levels in the liver tissues of mice in different groups after injection of acetaminophen. Detailed Implementation

[0092] The present invention will now be clearly and completely described in conjunction with specific embodiments. Those skilled in the art will understand that the embodiments described below are some, but not all, embodiments of the present invention, and are only used to illustrate the present invention, and should not be regarded as a limitation on the scope of protection of the present invention.

[0093] In this invention, unless otherwise specified, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0094] For example:

[0095] Carvedilol (CAR): Purchased from Aladdin Ltd. (Shanghai, China).

[0096] Dipotassium glycyrrhizinate (DG): purity ≥98%, purchased from Shaanxi Fujie Pharmaceutical Co., Ltd.

[0097] Rebaudioside A (abbreviated as RA, CAS: 58543-16-1), with a purity of ≥98%, was purchased from Jining Aoxing Stevia Products Co., Ltd.

[0098] Trypan Blue (TB) and Acetaminophen (APAP): Purchased from Beijing Solarbio Technology Co., Ltd.

[0099] Male Kunming mice (8 weeks old): purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd.; all animals were healthy and had no clinically observable physical abnormalities.

[0100] In this invention, SPSS 11.5 software (SPSS Inc., Chicago) was used for data analysis, and P < 0.05 indicates significance.

[0101] Example 1

[0102] Ethanol evaporation method: 25 mg carvedilol and 400 mg excipients (containing 43.5 mg riboside A and 356.5 mg dipotassium glycyrrhizate) were completely dissolved in ethanol (the volume can be selected from 30 ml to 50 ml; 50 ml was used in this example). Then, the ethanol was completely removed by vacuum evaporation at 40 °C using a rotary evaporator to obtain CAR-DG-RA, the appearance of which is shown in the figure. Figure 1 .

[0103] The obtained FT-IR, DSC, and XRD spectra of CAR-DG-RA are shown in the figure below. Figures 2-4 CAR, DG & RA physical mixture (ratio: 43.5 mg riboflavin A and 356.5 mg dipotassium glycyrrhizate) and CAR & DG & RA physical mixture (ratio: 25 mg carvedilol, 43.5 mg riboflavin A and 356.5 mg dipotassium glycyrrhizate) were used as controls.

[0104] Example 2

[0105] 1. Solubility

[0106] Excess CAR and CAR-DG-RA (obtained in Example 1) were added to 1 ml of test solution: water, phosphate-buffered saline (PBS) (pH = 7.4), simulated intestinal fluid (SIF) (pH = 6.8), and simulated gastric fluid (SGF) (pH = 1.2). The solution was shaken for 24 h at 37 °C and 100 rpm. The sample was then centrifuged for 10 min at 10,000 rpm, filtered through a 0.22 μm filter, diluted with methanol, and the CAR concentration was determined by high performance liquid chromatography (HPLC). The solubility was then calculated.

[0107] The concentration of CAR in the sample was determined by HPLC; chromatographic column: Agilent ZORBAX SB-C18 (250mm×4.60mm, 5μm), column temperature 25℃; mobile phase: methanol-water = 60:40 (volume ratio; pH adjusted to 3.5 with phosphoric acid), flow rate 1.0ml / min; detection wavelength 241nm; injection volume 20μl.

[0108] The results are shown in Table 1 below. Figure 5 As shown.

[0109] Table 1. Solubility data of CAR in CAR-DG-RA

[0110]

[0111]

[0112] 2. Critical micelle concentration (CMC)

[0113] Using 1,6-diphenyl-1,3,5-hextriene (DPH) as a probe, the critical micelle concentration (CMC) of CAR-DG-RA (obtained in Example 1) in water and PBS (pH=7.4) was determined.

[0114] The results showed that the CMC values ​​of CAR-DG-RA in water and PBS (pH=7.4) were 4.638±0.017 mg / ml and 4.244±0.412 mg / ml, respectively.

[0115] 3. Micelle size, polydispersity index, and zeta potential

[0116] The CAR-DG-RA obtained in Example 1 was prepared into an aqueous solution (CAR concentration 1.0 mg / mL), which was a light yellow clear solution. The zeta potential, micelle size and polydispersity index (PDI) were measured at 25 °C using a Zetasizer Nano ZS90 (dynamic light scattering, DLS).

[0117] The results showed that the zeta potential of the CAR-DG-RA aqueous solution was -11.77±0.93mV, the average micelle size was 15.62±0.27nm, and the particle size distribution was narrow (PDI=0.126±0.01).

[0118] 4. Encapsulation rate

[0119] The CAR-DG-RA obtained in Example 1 was prepared into an aqueous solution (CAR concentration 1.0 mg / mL), filtered through a 0.22 μm filter membrane, and the unencapsulated CAR was separated by filtration. The CAR-DG-RA solutions before and after filtration were diluted with methanol to disrupt the micelles. The CAR concentration in the solution was determined by the aforementioned HPLC method; the encapsulation efficiency was the ratio (%) of the CAR concentration detected after filtration to the CAR concentration detected before filtration.

[0120] The results showed that the initial (on the day of preparation) encapsulation efficiency was 99.67 ± 0.02%.

[0121] 5. Storage stability evaluation

[0122] The CAR-DG-RA obtained in Example 1 was sealed in a glass bottle and stored at 25°C in the dark for 6 months. The encapsulation efficiency, average micelle size and PDI were tested according to the aforementioned method.

[0123] The results showed that after 6 months of storage at 25℃, the encapsulation efficiency was >95%, the average micelle size was 17.7±0.1nm, and the PDI was <0.20, demonstrating good stability.

[0124] Example 3

[0125] In vitro release test

[0126] (1) Place 1 ml of CAR-DG-RA (obtained in Example 1) aqueous solution (CAR concentration 1.0 mg / mL) and 1 ml of CAR suspension (CAR concentration 1.0 mg / mL) into dialysis bags (molecular weight cutoff = 12000 Da), immerse in 100 ml of phosphate-buffered saline (PBS powder commercially available from Wuhan Saiwei Biotechnology Co., Ltd., dissolved in distilled water, concentration 0.01 M, pH 7.2-7.4 at 25℃) or PBS (pH = 7.4), and incubate at 37℃ with a shaker at 100 rpm. At different time points, take 1 ml of culture medium (with 1 ml of phosphate-buffered saline added simultaneously), and detect the CAR concentration in the 1 ml of culture medium taken by HPLC as described above. The in vitro release curve in PBS is shown in [Figure number missing]. Figure 6 .

[0127] (2) 1 ml of CAR-DG-RA (obtained in Example 1) aqueous solution (CAR concentration 1.0 mg / mL) and 1 ml of CAR suspension (CAR concentration 1.0 mg / mL) were placed in dialysis bags (molecular weight cutoff = 12000 Da). For the first 2 hours, they were incubated in 100 ml of simulated gastric fluid SGF (pH = 1.2) at 37°C with a shaker at 100 rpm. Then, they were transferred to 100 ml of simulated intestinal fluid SIF (pH = 6.8) and incubated again at 37°C with a shaker at 100 rpm. At different time points, 1 ml of culture medium (SGF or SIF) samples were collected (with an equal volume of SGF or SIF added simultaneously). The CAR concentration in each 1 ml of culture medium was determined by the aforementioned HPLC. The in vitro release curve in SGF / SIF is shown in [Figure number missing]. Figure 7 .

[0128] The results showed that

[0129] (1) The release rate of CAR is greatly affected by pH: the cumulative release of CAR in PBS (pH=7.4) is less than 10% in 24 hours; it is released quickly in acidic SGF (pH=1.2), with a cumulative release of 50.91±3.87% in the first 2 hours, and very slowly in SIF (pH=6.8) in the next 22 hours; this is also an important factor leading to the low bioavailability of CAR (see: Markovic, M., et al., Segmental-Dependent Solubility and Permeability as Key Factors Guiding Controlled Release Drug Product Development. Pharmaceutics, 2020.12(3).).

[0130] (2) The release rate of CAR in CAR-DG-RA is less affected by pH: CAR in CAR-DG-RA shows a continuous and constant release curve (the slope is basically stable) in PBS (pH=7.4) and SGF (pH=1.2) / SIF (pH=6.8), which can expand the time window for CAR absorption and utilization (it can be absorbed through the stomach and intestines, etc.), and help improve the bioavailability of CAR.

[0131] Example 4

[0132] In vitro antioxidant activity

[0133] The antioxidant activities of CAR, DG&RA physical mixture (43.5 mg riboside A and 356.5 mg dipotassium glycyrrhizate) and CAR-DG-RA (obtained in Example 1) were detected by 2,2'-hydrazine-bis(3-ethylbenzothiazoline-6-sulfonic acid) diamine salt (ABTS method) and ferric reduction / antioxidant capacity method (FRAP).

[0134] The results of the ABTS method can be found in... Figure 8 (different culture times at a fixed concentration) and Figure 9 (Different concentrations at a fixed culture time). FRAP detection results are shown below. Figure 10 (different culture times at a fixed concentration) and Figure 11 (Different concentrations under a fixed culture time).

[0135] The results of ABTS and FRAP assays showed that CAR-DG-RA exhibited stronger antioxidant activity than CAR.

[0136] Example 5

[0137] 1. Hemolysis test

[0138] Physiological saline was used as a negative control (0% hemolysis), and 0.1% Triton X-100 was used as a positive control (100% hemolysis) for the hemolysis test.

[0139] The results showed that the hemolysis rate was less than 0.1% when the concentration of CAR in the aqueous solution of CAR-DG-RA (obtained in Example 1) was 0.1-0.2 mg / ml, and less than 1% when the concentration of CAR in the aqueous solution was 0.5-2 mg / ml, both of which were below the safety value of 5%, indicating that CAR-DG-RA has good blood biocompatibility.

[0140] 2. Chicken embryo chorioallantoic membrane (HET-CAM) - trypan blue staining test

[0141] The experimental chicken embryos were randomly divided into the following 6 groups, with 3 embryos in each group:

[0142] ① Negative control: 0.9% NaCl solution (physiological saline);

[0143] ② Positive control: 0.1M NaOH solution;

[0144] ③ DG&RA physical mixing;

[0145] ④, CAR;

[0146] ⑤ Physical mixing of CAR, DG, and RA;

[0147] ⑥ CAR-DG-RA (obtained in Example 1): aqueous solution, CAR concentration 5 mg / ml;

[0148] After the aforementioned sample solution was in contact with the chicken embryo allantoic membrane (CAM) for 5 minutes, the vascular condition of the CAM was as follows: Figure 12 As shown in the figure; then, trypan blue staining was performed and the trypan blue absorption was measured, and the results are as follows. Figure 13 As shown.

[0149] The results showed that the positive control (0.1M NaOH) group exhibited symptoms such as congestion and thrombosis, as well as a high amount of trypan blue absorption, indicating that it was severely irritating; while the CAR-DG-RA group was basically similar to the saline (normal) group and did not show obvious irritation, which, on the other hand, demonstrates the safety of CAR-DG-RA.

[0150] Example 6

[0151] Healthy male Kunming mice were randomly divided into the following 9 groups, with 16 mice in each group:

[0152] ① Healthy control group;

[0153] ② PBS treatment group;

[0154] ③ Positive (NAC: N-acetylcysteine) treatment group (200 mg / kg);

[0155] ④ DG & RA treatment group (52.2 mg / kg RA and 427.8 mg / kg DG);

[0156] ⑤ CAR suspension treatment group (30mg / kg);

[0157] ⑥ CAR&DG&RA physical therapy combination group (52.2mg / kg RA, 427.8mg / kg DG and 30mg / kg CAR);

[0158] ⑦ Low-dose treatment group of CAR-DG-RA (obtained in Example 1) (7.5 mg / kg CAR);

[0159] ⑧. CAR-DG-RA (obtained in Example 1) medium-dose treatment group (15 mg / kg CAR);

[0160] ⑨ High-dose treatment group of CAR-DG-RA (obtained in Example 1) (30mg / kg CAR).

[0161] The mice were administered the drug for 7 consecutive days (healthy control mice were fed an equal volume of physiological saline). The mice were fasted for 12 hours before administration on day 7, and injected with acetaminophen (400 mg / kg, 40 mg / ml, using physiological saline as a solvent) 1 hour after administration on day 7. Healthy control mice were injected with an equal volume of physiological saline.

[0162] 1. Survival rate and the ratio of liver and spleen tissue to body weight.

[0163] After injection of APAP (acetaminophen) or saline for 6 hours (during which time the mice were fasted but allowed to drink water), the survival rate of the mice was recorded (see: Figure 14 Mice in each group were sacrificed, and serum was separated from the blood samples (for subsequent testing). Liver and spleen tissues were separated, rinsed with physiological saline, blotted dry with filter paper, and weighed. The ratios of liver and spleen tissues to body weight were calculated to evaluate the degree of liver and spleen edema (see: Figure 15 and Figure 16 Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&RA group; @ indicates P < 0.05 compared to the CAR group; % indicates P < 0.05 compared to the CAR&DG&RA group; ^ indicates P < 0.05 compared to the low-dose CAR-DG-RA group; + indicates P < 0.05 compared to the medium-dose CAR-DG-RA group. Each liver tissue sample was divided into two parts after photography; one part was stored at -80°C, and the other part was fixed with formalin.

[0164] The results showed that CAR-DG-RA could reduce mortality caused by excessive APAP (acetaminophen), significantly improve the survival rate of mice (the survival rate of mice in the PBS group was only 37.5% VS the survival rate of mice in the medium and high dose CAR-DG-RA treatment groups increased to 87.5%), and could effectively alleviate and / or prevent symptoms such as hepatosplenic edema caused by excessive APAP (acetaminophen).

[0165] 2. Serum Aspartate Aminotransferase (AST) and Alanine Aminotransferase (ALT) Tests

[0166] The levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in mouse serum were measured using a commercially available kit (Nanjing Jiancheng Bioengineering Institute). (AST and ALT are two important indicators reflecting the severity of liver damage.) The results are shown in the table below. Figure 17 and Figure 18(Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&RA group; @ indicates P < 0.05 compared to the CAR group; % indicates P < 0.05 compared to the CAR&DG&RA group; ^ indicates P < 0.05 compared to the low-dose CAR-DG-RA group).

[0167] The results showed that CAR-DG-RA could effectively inhibit or prevent the sudden increase in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) caused by excessive APAP (acetaminophen), and had a good liver-protective effect.

[0168] 3. Histological examination

[0169] Formalin-fixed liver (see) Figure 19 The tissue was embedded in paraffin, sectioned, stained with hematoxylin and eosin, and then evaluated histopathologically under a microscope. (See attached image.) Figure 20 The histopathological images of the liver were obtained using known methods (References: 1. Oke, GO, et al., Zingiber officinale (Roscoe) mitigates CCl4-induced liverhistopathology and biochemical derangements through antioxidant, membrane-stabilizing and tissue-regenerating potentials. Toxicol Rep, 2019.6: P416-425. 2. Peter, AI, et al., Investigating Organ Toxicity Profile of Tenofovir and Tenofovir Nanoparticle on the Liver and Kidney: Experimental Animal Study. Toxicol Res, 2018.34(3): P221-229.). Semi-quantitative analysis was performed using the ratio of hepatocyte necrosis area to total area. The results are shown in […]. Figure 21(Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&RA group; @ indicates P < 0.05 compared to the CAR group; % indicates P < 0.05 compared to the CAR&DG&RA group; ^ indicates P < 0.05 compared to the low-dose CAR-DG-RA group).

[0170] The results showed that CAR-DG-RA can effectively inhibit or prevent symptoms such as hepatocellular necrosis, liver edema, and hemorrhage caused by excessive APAP (acetaminophen), and has a good liver-protecting effect.

[0171] 4. Detection of superoxide dismutase (SOD) and malondialdehyde (MDA)

[0172] Liver tissue was homogenized with physiological saline. The supernatant after centrifugation was used to detect SOD and MDA levels using a commercially available kit (Beyotime Biotechnology, Shanghai, China). Results are expressed as U / mg protein. (See attached table.) Figure 22 and Figure 23 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&RA group; @ indicates P < 0.05 compared to the CAR group; % indicates P < 0.05 compared to the CAR&DG&RA group; ^ indicates P < 0.05 compared to the low-dose CAR-DG-RA group).

[0173] The results showed that CAR-DG-RA could effectively inhibit or prevent the significant decrease in superoxide dismutase (SOD) levels in liver tissue caused by excessive APAP (acetaminophen), and could also effectively inhibit or prevent the significant increase in malondialdehyde (MDA) levels in liver tissue caused by excessive APAP (acetaminophen), thus exhibiting good liver-protective effects.

[0174] 5. Enzyme-linked immunosorbent assay (ELISA)

[0175] The levels of different cytokines (e.g., pro-inflammatory cytokines) in mouse liver were detected using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Shanghai Enzyme-Link Biotechnology Co., Ltd.).

[0176] HMGB1 (referring to high-mobility group box 1, an important late-stage pro-inflammatory factor; studies have shown that HMGB1 is an important late-stage inflammatory mediator of endotoxin lethality and plays an important role in the pathogenesis of various diseases such as sepsis, tumors, and arthritis).

[0177] IL-1β (an important pro-inflammatory cytokine with strong pro-inflammatory activity, capable of inducing various pro-inflammatory mediators);

[0178] IL-6 (an important inflammatory cytokine, mainly expressed in inflammatory responses, etc.);

[0179] NF-κB (an important transcription activator involved in various pathological processes such as inflammation and acute response, cell proliferation, apoptosis, and viral infection);

[0180] TNF-α (a pro-inflammatory cytokine whose dysfunction is believed to be associated with many diseases; TNF-α inhibitors have been reported to be the first-line treatment for autoimmune diseases such as rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis);

[0181] See results Figures 24-28 (Where, * indicates P < 0.05 compared to the healthy control group; # indicates P < 0.05 compared to the PBS group; & indicates P < 0.05 compared to the NAC group; $ indicates P < 0.05 compared to the DG&RA group; @ indicates P < 0.05 compared to the CAR group; % indicates P < 0.05 compared to the CAR&DG&RA group; ^ indicates P < 0.05 compared to the low-dose CAR-DG-RA group; + indicates P < 0.05 compared to the medium-dose CAR-DG-RA group).

[0182] The results showed that CAR-DG-RA can effectively inhibit the expression of cytokines such as HMGB1, IL-1β, IL-6, NF-κB, and TNF-α, and has good preventive and / or therapeutic effects on various inflammations caused by it. It can be used as a potential inhibitor of HMGB1, IL-1β, IL-6, NF-κB or TNF-α, providing more options for clinical drug use.

[0183] Of course, the present invention can also have other various embodiments. Without departing from the spirit and essence of the present invention, those skilled in the art can make various corresponding changes and / or modifications according to the present invention, and these corresponding changes and / or modifications should all fall within the protection scope of the appended claims.

Claims

1. A pharmaceutical composition, characterized by: It comprises a β-receptor blocker and pharmaceutically acceptable excipients; wherein the β-receptor blocker is carvedilol or a pharmaceutically acceptable salt thereof, and the pharmaceutically acceptable excipients comprise steviol glycosides and glycyrrhizate; wherein the weight ratio of steviol glycosides to glycyrrhizate is 1:5 to 15; The weight ratio of the β-receptor blocker to the pharmaceutically acceptable excipient is 1:5 to 50. The encapsulation efficiency of the β-receptor blocker is ≥95%; The stevioside is riboside A; The pharmaceutical composition is prepared by a method comprising the following steps: dispersing or dissolving a β-receptor blocker and a pharmaceutically acceptable excipient together in an organic solvent, mixing them thoroughly, and then removing the organic solvent by rotary evaporation at 35–45°C; wherein the organic solvent is an alcohol solvent.

2. The pharmaceutical composition of claim 1, wherein: The glycyrrhizic acid salt is selected from one or more of sodium glycyrrhizate, disodium glycyrrhizate, potassium glycyrrhizate, dipotassium glycyrrhizate, ammonium glycyrrhizate, and diammonium glycyrrhizate.

3. The pharmaceutical composition of claim 2, wherein: The glycyrrhizic acid salt is dipotassium glycyrrhizate or disodium glycyrrhizate.

4. The pharmaceutical composition of claim 1, wherein: The weight ratio of steviol glycosides to glycyrrhizate is 1:8 to 10.

5. The pharmaceutical composition of claim 1, wherein: The weight ratio of the β-blocker to the pharmaceutically acceptable excipient is 1:10 to 25.

6. The pharmaceutical composition of claim 5, wherein: The weight ratio of the β-blocker to the pharmaceutically acceptable excipient is 1:15 to 20.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein: The pharmaceutical composition is a solid dosage form or a liquid dosage form.

8. The pharmaceutical composition according to any one of claims 1 to 6, wherein: The β-receptor blocker in the pharmaceutical composition is a therapeutically effective amount.

9. The pharmaceutical composition of claim 7, wherein: The pharmaceutical composition is a liquid formulation, and the solvent for the liquid formulation is selected from pharmaceutically acceptable water, PBS buffer, or an aqueous solution of sodium carboxymethyl cellulose.

10. The pharmaceutical composition according to claim 9, characterized in that: When the concentration of carvedilol in the liquid formulation is 1 mg / mL, the liquid formulation satisfies one or more of the following conditions ① to ③: ① The average diameter of the micelles in the liquid formulation is 1–80 nm; ② The polydispersity index of the liquid formulation is ≤0.5; ③ The zeta potential of the liquid formulation is -20 to 0 mV.

11. The pharmaceutical composition of claim 10, wherein: When the concentration of carvedilol in the liquid formulation is 1 mg / mL, the liquid formulation satisfies one or more of the following conditions ① to ③: ① The average diameter of the micelles in the liquid formulation is 10-20 nm; ② The polydispersity index of the liquid formulation is ≤0.4; ③ The zeta potential of the liquid formulation is -15 to -5 mV.

12. The pharmaceutical composition of claim 10, wherein: When the concentration of carvedilol in the liquid formulation is 1 mg / mL, the liquid formulation simultaneously satisfies conditions ① to ③.

13. The pharmaceutical composition according to any one of claims 1 to 6, wherein: The amount of the organic solvent used per milligram of the β-receptor blocker is 0.5 to 20 mL.

14. The pharmaceutical composition of claim 13, wherein: The alcohol solvent is methanol or ethanol.

15. The pharmaceutical composition of claim 13, wherein: The amount of the organic solvent used per milligram of the β-receptor blocker is 1.2 to 2.5 mL.

16. The pharmaceutical composition according to any one of claims 1 to 6, wherein: The pharmaceutical composition described herein is a pharmaceutical composition for the prevention, treatment and / or relief of drug-induced liver injury.

17. The pharmaceutical composition of claim 16, wherein, The drug-induced liver injury mentioned refers to drug-induced liver injury caused by acetaminophen.

18. A process for the preparation of a pharmaceutical composition according to any one of claims 1 to 12, characterized in that, The process includes the following steps: dispersing or dissolving a β-receptor blocker and pharmaceutically acceptable excipients together in an organic solvent, mixing them thoroughly, and then removing the organic solvent by rotary evaporation at 35–45°C; the organic solvent is an alcohol solvent.

19. The method of claim 18, wherein the pharmaceutical composition is prepared by, The amount of the organic solvent corresponding to each milligram of the beta receptor blocker is 0.5-20 mL.

20. The method of claim 19, wherein the pharmaceutical composition is prepared by, The alcohol solvent is methanol or ethanol.

21. The method of claim 19, wherein the pharmaceutical composition is prepared by, The amount of the organic solvent corresponding to each milligram of the beta receptor blocker is 1.2-2.5 mL.

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