Levosimendan injection and preparation method thereof

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, and in particular to a levosimendan injection and a preparation method thereof. The levosimendan injection of the present invention is composed of levosimendan, carboxymethyl chitosan, seleno-L-cysteine, a buffer solution, an antibacterial agent, an isotonic regulator, and water for injection. The defect of levosimendan being unstable when exposed to light, heat, and humidity is overcome, and excellent stability is shown in experiments of high temperature and high humidity under light, thereby improving the safety of medication and extending the shelf life. In addition, the levosimendan injection of the present invention has a significant effect in treating heart failure, significantly improving the rat cardiac index and left ventricular index, and reducing the rat myocardial cell apoptosis rate.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical preparations, and particularly relates to a levosimendan injection and a preparation method thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Heart failure is a condition in which the heart is unable to pump enough blood to the body to meet its needs. This may be due to weakened heart muscle contraction, impaired cardiac relaxation, or obstructed cardiac output. Heart failure often results in limited physical activity, dyspnea, fatigue, edema, and other uncomfortable symptoms.

[0004] Levosimendan is a positive inotropic drug mainly used to treat acute decompensated heart failure. It improves cardiac function by enhancing myocardial contractility and vasodilation. The mechanisms of levosimendan include: (1) Calcium sensitization: Levosimendan increases the muscle's sensitivity to calcium ions by interacting with calcium ion binding sites in the myocardium, thereby enhancing myocardial contractility. This mechanism does not increase intracellular calcium ion concentration, thereby reducing the risk of causing calcium overload and related adverse cardiac events (such as arrhythmias). (2) Phosphodiesterase III (PDE III) inhibition: Levosimendan can inhibit PDEIII, increase cAMP levels, lead to increased calcium ion concentration in myocardial cells, and further enhance myocardial contraction. (3) Opening ATP-sensitive potassium channels: Levosimendan can also open ATP-sensitive potassium channels on the vascular smooth muscle cell membrane, leading to vasodilation, reducing systemic vascular resistance and cardiac preload and afterload.

[0005] The main clinical applications of levosimendan include: (1) Acute decompensated heart failure: Levosimendan is mainly used to treat acute decompensated heart failure caused by various reasons, especially when other treatments (such as diuretics, vasodilators and positive inotropic drugs) are ineffective or ineffective. (2) Perioperative heart failure: It is sometimes also used to manage heart failure during or after surgery, especially in high-risk cardiac surgery patients.

[0006] Generally, levosimendan is usually administered by intravenous injection. Currently, the levosimendan injection available on the market needs to be stored in a sealed, light-proof, low-temperature (2-8°C) environment, and the storage conditions are harsh.

[0007] Chinese patent publication number CN116473930A discloses a levosimendan for injection and a preparation method thereof. The levosimendan for injection comprises the active ingredient levosimendan, povidone, anhydrous sodium carbonate or choline hydroxide or sodium hydroxide, mannitol or sucrose and a citric acid aqueous solution. The composition does not contain ethanol to avoid vascular irritation by ethanol, and a citric acid aqueous solution is used to improve stability, thereby improving the safety of medication.

[0008] The Chinese patent with publication number CN105106113A discloses a levosimendan injection and a preparation method thereof. The formula is: levosimendan active ingredient, solubilizer, pH buffer pair and non-aqueous solvent, wherein the pH buffer pair adopts citric acid-sodium citrate buffer pair or acetic acid-acetate buffer pair to maintain the pH stability of the injection and reduce the risk of physical precipitation impurities and chemical degradation impurities generated by pH fluctuations during storage and use of the levosimendan injection. Summary of the invention

[0009] The invention overcomes the shortcomings of the prior art and provides a levosimendan injection with high stability and good effect in treating heart failure. The levosimendan injection is composed of the following ingredients: 3 to 8 parts by weight of levosimendan, 0.2 to 0.8 parts by weight of carboxymethyl chitosan (CAS: 83512-85-0), 0.1 to 0.5 parts by weight of seleno-L-cysteine ​​(CAS: 10236-58-5), a buffer solution, 0.02 to 0.06 parts by weight of an antibacterial agent, an isotonic regulator, and water for injection.

[0010] Furthermore, the buffer is selected from citrate buffer solution, phosphate buffer solution, and acetate buffer solution.

[0011] Furthermore, the antibacterial agent is selected from chlorobutanol, chlorocresol, and methylparaben.

[0012] Furthermore, the isotonicity adjusting agent is selected from sodium chloride, glucose and glycerol.

[0013] The present invention also provides a method for preparing the above-mentioned levosimendan injection, comprising the following steps:

[0014] (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 3° C. to 10° C. for 1 to 3 hours, and bubbling with nitrogen for 15 to 30 minutes to obtain a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use;

[0015] (2) Levosimendan is dissolved in a buffer solution, uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is conditioned by an isotonicity regulator, water for injection is added to the full amount, filtered, and filled.

[0016] Furthermore, the pH of the buffer solution is 5.0-6.5.

[0017] The use of the levosimendan injection of the present invention in preparing a medicine for treating heart failure is specifically embodied in a rat experiment.

[0018] Compared with the prior art, the technical effects of the present invention are:

[0019] The levosimendan injection of the present invention overcomes the defect that levosimendan is unstable when exposed to light, heat and humidity. In experiments under light, high temperature and high humidity, the levosimendan injection of the present invention exhibits excellent stability, the content of its effective ingredient remains substantially unchanged, and the content of related substances is low, thereby greatly improving the safety of medication and extending the shelf life.

[0020] The levosimendan injection of the present invention has a significant effect in treating heart failure, is slightly better than the commercially available levosimendan injection, can significantly improve the cardiac index and left ventricular index of rats, and reduce the apoptosis rate of myocardial cells in rats. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Changes in the content of active ingredients in levosimendan injections of Examples 1 to 3 and Comparative Examples 1 to 7 in the strong light accelerated test.

[0022] Figure 2 : Strong light accelerated test on the changes of related substance contents in levosimendan injections of Examples 1 to 3 and Comparative Examples 1 to 7. DETAILED DESCRIPTION

[0023] In order to make the purpose and technical solution of the present invention clearer, the present invention is further described below in conjunction with embodiments, but the protection scope of the present invention is not limited to these embodiments, and the embodiments are only used to explain the present invention. It should be understood by those skilled in the art that any changes or equivalent substitutions that do not deviate from the concept of the present invention are included in the protection scope of the present invention.

[0024] Example 1 Levosimendan injection

[0025] formula:

[0026]

[0027] Preparation method:

[0028] (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 5° C. for 2 h, and bubbling with nitrogen for 20 min to obtain a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use;

[0029] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0030] Example 2 Levosimendan injection

[0031] formula:

[0032]

[0033] Preparation method:

[0034] (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 3° C. for 1 hour, and bubbling with nitrogen for 15 minutes to obtain a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use;

[0035] (2) Levosimendan is dissolved in a citrate buffer solution with a pH of 5.0, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0036] Example 3 Levosimendan injection

[0037] formula:

[0038]

[0039] Preparation method:

[0040] (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 10° C. for 3 h, and bubbling with nitrogen for 30 min to obtain a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use;

[0041] (2) Levosimendan is dissolved in an acetate buffer solution at pH 6.5, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is conditioned by an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0042] Comparative Example 1 Levosimendan Injection

[0043] formula:

[0044]

[0045] Preparation method:

[0046] (1) dissolving seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 5° C. for 2 h, and bubbling with nitrogen for 20 min to obtain a seleno-L-cysteine ​​treatment solution for later use;

[0047] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0048] Comparative Example 2 Levosimendan Injection

[0049] formula:

[0050]

[0051] Preparation method:

[0052] (1) dissolving carboxymethyl chitosan in an appropriate amount of water for injection, refrigerating at 5° C. for 2 h, and bubbling with nitrogen for 20 min to obtain a carboxymethyl chitosan treatment solution for later use;

[0053] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0054] Comparative Example 3 Levosimendan Injection

[0055] formula:

[0056]

[0057] Preparation method:

[0058] (1) Dissolve chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerate at 5° C. for 2 h, and bubble with nitrogen for 20 min to obtain a mixed solution of chitosan and seleno-L-cysteine ​​for later use;

[0059] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0060] Comparative Example 4 Levosimendan Injection

[0061] formula:

[0062]

[0063] Preparation method:

[0064] (1) dissolving carboxymethyl chitosan and cysteine ​​in an appropriate amount of water for injection, refrigerating at 5° C. for 2 h, and bubbling with nitrogen for 20 min to obtain a mixed solution of carboxymethyl chitosan and cysteine ​​for later use;

[0065] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0066] Comparative Example 5 Levosimendan Injection

[0067] formula:

[0068]

[0069] Preparation method:

[0070] (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, bubbling with nitrogen for 20 min, and obtaining a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use;

[0071] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0072] Comparative Example 6 Levosimendan Injection

[0073] formula:

[0074]

[0075] Preparation method:

[0076] (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 5° C. for 2 h to obtain a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use;

[0077] (2) Levosimendan is dissolved in a phosphate buffer solution with a pH of 5.8, and uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is adjusted with an isotonicity regulator, water for injection is added to the full amount, filtered through a 0.2 μm filter membrane, and filled.

[0078] Comparative Example 7 National Medicine Standard H20100043

[0079] Strong light accelerated test to verify the stability of levosimendan injection

[0080] The levosimendan injections of Examples 1 to 3 and Comparative Examples 1 to 7 were subjected to accelerated tests at a temperature of 40°C ± 2°C, a relative humidity of 75% ± 5%, and an illumination of 4500 lx ± 500 lx. Samples were taken on the first day, at the end of the second month, at the end of the third month, and at the end of the sixth month, respectively, to determine the levosimendan content and the content of levosimendan-related substances.

[0081] Determination of levosimendan content

[0082] Determine according to the high performance liquid chromatography method in accordance with the general rules of Part IV of the Chinese Pharmacopoeia 2020. Solvent: water-acetonitrile (70:30). Test solution: Take Examples 1 to 3 and Comparative Examples 1 to 7 as samples, add 5 ml of water to dissolve, and quantitatively dilute with solvent to make a solution containing about 0.25 mg per 1 ml. Reference solution: Take an appropriate amount of levosimendan reference substance, accurately weigh it, dissolve it with solvent, and quantitatively dilute it to make a solution containing about 0.25 mg per 1 ml. Chromatographic conditions: Use octylsilane bonded silica gel as filler (recommend using Kromasil C8, 4.6mm×250mm, 3.5μm or chromatographic column with equivalent performance); use phosphate buffer (take 1.8g of sodium dihydrogen phosphate, add 1000ml of water to dissolve, and adjust the pH value to 2.1 with phosphoric acid) as mobile phase A, acetonitrile as mobile phase B, and perform linear gradient elution according to Table 1; flow rate is 0.8ml per minute; detection wavelength is 380nm; injection volume is 5μl. Determination method: Accurately measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record the chromatogram. Calculate by peak area according to the external standard method.

[0083] Table 1 Linear gradient elution program

[0084]

[0085] Figure 1 The changes in the content of the active ingredient in the levosimendan injections of Examples 1 to 3 and Comparative Examples 1 to 7 under the strong light accelerated test show that the content of the levosimendan injections of Examples 1 to 3 of the present invention is stable, and the active ingredient can be kept basically unchanged under the experimental conditions of strong light, high temperature and high humidity, thereby extending the shelf life.

[0086] Determination of related substances in levosimendan

[0087] Determine according to the high performance liquid chromatography method in accordance with the general rules of the fourth part of the Chinese Pharmacopoeia 2020. Solvent: water-acetonitrile (70:30); Test solution: Take Examples 1 to 3 and Comparative Examples 1 to 7 as samples, add 5 ml of water to dissolve, and quantitatively dilute with solvent to make a solution containing about 0.25 mg per 1 ml; Chromatographic conditions: Same as under the content determination method; Determination method: Accurately measure the test solution, inject it into the liquid chromatograph, and record the chromatogram; Limit: If there is an impurity peak in the chromatogram of the test solution, the total amount of impurities shall not exceed 2.5%.

[0088] Figure 2 The changes in the contents of related substances in the levosimendan injections of Examples 1 to 3 and Comparative Examples 1 to 7 of the strong light accelerated test are shown. Compared with Comparative Examples 1 to 7, the levosimendan injections of Examples 1 to 3 of the present invention have lower contents of related substances and higher safety.

[0089] Quality Evaluation

[0090] The quality of the levosimendan injections of Examples 1 to 3 and Comparative Examples 1 to 7 was evaluated, including visible foreign matter inspection, sterility inspection, and pH, and the standards were in accordance with the provisions of the 2020 edition of the Chinese Pharmacopoeia.

[0091] Table 2 Quality evaluation of levosimendan injection of Examples 1 to 3 and Comparative Examples 1 to 7

[0092]

[0093]

[0094] As shown in Table 2, the various indicators of the levosimendan injection of the present invention meet the requirements of the Chinese Pharmacopoeia, and the pH is more stable.

[0095] Pharmacological experiments

[0096] The following experimental studies are all conducted on the basis of the safety of the drugs proved by acute toxicity tests and long-term toxicity tests. The dosages used in the experimental studies are all within the safe dosage range.

[0097] Experimental animals

[0098] SD rats, SPF grade, 180-220 g, experimental animal license number: SYXK(Lu)20180008, were provided by Lunan Pharmaceutical Group Co., Ltd. and were adaptively fed under standard conditions for 1 week before the experiment.

[0099] Modeling method and grouping

[0100] Several rats were taken, fasted but not watered for 12h, anesthetized, the rats were fixed with limbs, intubated and connected to a ventilator, the intercostal space was longitudinally opened to expose the heart, the left anterior descending coronary artery was ligated with 6-0 polypropylene sutures, and the incision was layered and sutured, and a chest tube was placed to evacuate the pneumothorax until the rat could walk and then remove the chest tube. During the operation, the electrocardiogram and cardiac color Doppler ultrasound monitored that the ST segment of the rat was elevated or depressed and the left ventricular ejection fraction (LVEF) was ≤50%, indicating that the rat modeling was successful. The blank group of rats was only treated with open chest treatment without any injury treatment, 10. All rats were treated with penicillin after surgery to prevent infection. The rats with successful modeling were divided into a model group, an embodiment 1 group, an embodiment 2 group, an embodiment 3 group, and a comparative example 7 group, 10 in each group.

[0101] Drugs and Dosages

[0102] Example 1 Injection: initial loading dose is 6 μg / kg, 10 min, continuous infusion is 0.1 μg / kg / min, 1 h.

[0103] Example 2 injection: initial loading dose of 6 μg / kg, 10 min, continuous infusion of 0.1 μg / kg / min, 1 h. Example 3 injection: initial loading dose of 6 μg / kg, 10 min, continuous infusion of 0.1 μg / kg / min, 1 h. Comparative Example 7 injection: initial loading dose of 6 μg / kg, 10 min, continuous infusion of 0.1 μg / kg / min, 1 h.

[0104] The rats in each treatment group were intravenously injected with the corresponding drugs, and the rats in the blank group and model group were intravenously injected with an equal amount of normal saline for 7 consecutive days.

[0105] Statistical processing

[0106] SPSS 22.0 software was used to analyze the data. The comparison among multiple groups was performed by one-way analysis of variance, and the comparison between two groups was performed by independent sample T test. P < 0.05 was considered statistically significant.

[0107] Observation indicators and experimental results

[0108] Cardiac index and left ventricular index in rats

[0109] Rats were anesthetized and killed, and the intact heart was removed by thoracotomy. The heart was rinsed to remove excess surrounding tissue, and the water on the surface of the heart and in the ventricle was absorbed with filter paper. The weight of the intact heart was weighed, and the cardiac index (cardiac index = heart mass / body mass) and the left ventricular index (left ventricular index = left ventricular mass / body mass) were calculated.

[0110] Table 3 Cardiac index and left ventricular index of rats in each group ( n=10)

[0111]

[0112] Compared with the model group, *P<0.01.

[0113] TUNEL staining to detect apoptosis rate of rat cardiomyocytes

[0114] According to the TUNEL staining kit operating procedures, the sections were placed in the TUNEL solution prepared in advance and incubated in the dark at 37°C for 1 hour, washed with distilled water and then counterstained with DAPI for 10 minutes. Finally, a fluorescence microscope was used to observe and calculate the cell apoptosis rate.

[0115] Table 4 Myocardial cell apoptosis rate of rats in each group ( n=10)

[0116]

[0117]

[0118] Compared with the model group, *P<0.01.

[0119] The levosimendan of the present invention has a significant therapeutic effect on rat heart failure, and the data show that it is slightly better than the commercially available preparation. Compared with the model group, the apoptosis rate of myocardial cells in rats in Example 1 to 3 groups was significantly reduced (P < 0.01); the cardiac index and left ventricular index of rats in Example 1 to 3 groups were significantly increased (P < 0.01).

Claims

1. A levosimendan injection, characterized in that: The levosimendan injection is composed of the following ingredients: 3-8 parts by weight of levosimendan, 0.2-0.8 parts by weight of carboxymethyl chitosan, 0.1-0.5 parts by weight of seleno-L-cysteine, a buffer solution, 0.02-0.06 parts by weight of an antibacterial agent, an isotonicity regulator, and water for injection; the method for preparing the levosimendan injection comprises the following steps: (1) dissolving carboxymethyl chitosan and seleno-L-cysteine ​​in an appropriate amount of water for injection, refrigerating at 3° C. to 10° C. for 1 to 3 hours, and bubbling with nitrogen for 15 to 30 minutes to obtain a mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​for later use; (2) Levosimendan is dissolved in a buffer solution, uniformly mixed with the mixed solution of carboxymethyl chitosan and seleno-L-cysteine ​​in step (1), an antibacterial agent is added and stirred evenly, the osmotic pressure is conditioned by an isotonicity regulator, water for injection is added to the full amount, filtered, and filled.

2. The levosimendan injection according to claim 1, characterized in that The buffer solution is selected from citrate buffer solution, phosphate buffer solution and acetate buffer solution.

3. The levosimendan injection according to claim 1, characterized in that: The antibacterial agent is selected from chlorobutanol, chlorocresol and methylparaben.

4. The levosimendan injection according to claim 1, characterized in that: The isotonicity adjusting agent is selected from sodium chloride, glucose and glycerol.

5. The levosimendan injection according to claim 1, characterized in that: The pH of the buffer solution is 5.0-6.

5.

6. The levosimendan injection according to claim 1, characterized in that: The refrigeration temperature is 5°C.

7. The levosimendan injection according to claim 1, characterized in that: The filtration is carried out by 0.2 μm filter membrane filtration.

8. The levosimendan injection according to claim 1, characterized in that: The pH of the buffer solution is 5.

8.

9. Use of the levosimendan injection according to claim 1 in the preparation of a medicament for treating heart failure.

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