Clevidipine butyrate micelle injection and preparation method thereof

Abstract

The invention relates to the technical field of medicine, in particular to a clevidipine butyrate micelle injection and a preparation method thereof.The clevidipine butyrate micelle injection is prepared from clevidipine butyrate, phospholipid, cholic acid or salt thereof, a pH regulator and water for injection and further contains a nano-micelle stabilizer and a nano-micelle solubilizing agent, oil for injection is abandoned in a prescription, and the preparation method is simple. Clevidipine butyrate, such as soybean oil, triglyceride and the like, meets the medication requirements of people with lipid metabolism deficiency, not only greatly improves the solubility of clevidipine butyrate, but also can further improve the stability and drug loading capacity of the micelle injection by creatively adding the nano-micelle stabilizer and the nano-micelle solubilizer. The blank micellar method is adopted for preparation, so that the use of organic solvents can be abandoned, organic solvents with large toxic and side effects, such as dichloromethane, chloroform and propylene glycol, are not needed, the toxicity of the medicine is reduced, the stimulation to blood vessels is small, and the adverse reaction of the medicine is reduced; and a production process of a common water injection is selected, so that production amplification is facilitated.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly to a clevidipine butyrate micelle injection and a preparation method thereof. Background Art

[0002] Clevidipine butyrate is a mixture of optical isomers, and its chemical name is methyl (1-butyryloxy) methyl 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate. Its molecular formula: C 21 H 23 Cl 2 NO 6 , with a molecular weight of 456.32, and the structural formula is as follows: .

[0003] Clevidipine butyrate is a dimethyl pyridine dicarboxylate, which is a white to off-white crystalline powder with a melting point of 138 °C. It is soluble in ethanol, chloroform, ethyl acetate and dimethyl sulfoxide, but insoluble in water. Two polymorphic forms have been found, among which, the proportion of type A is >95%.

[0004] As a dihydropyridine derivative, clevidipine butyrate is a very short-acting intravenous calcium channel blocker. As a short-term control drug for hypertension during the perioperative period, it has high selectivity for blood vessels and myocardium, and can be rapidly metabolized into inactive substances in the body. Clevidipine butyrate has a strong activity of reducing the pulse rate and also has a dilating effect on systemic blood vessels and pulmonary vessels. Clevidipine butyrate has a fast onset and a fast elimination of action, and can accurately control blood pressure by increasing the dose. Different from many current intravenous antihypertensive drugs that are metabolized by the kidney and / or liver, it is metabolized in the blood and tissues, and thus does not accumulate in the body. As an ideal therapeutic drug for perioperative hypertension, it should have the characteristics of parenteral administration, fast onset time, and easy rapid adjustment. The advent of clevidipine butyrate injection has filled this gap.

[0005] Clevidipine butyrate is a water-insoluble drug, and its solubility in water is only 0.0001 mg / mL, which is lower than that of existing dihydropyridine drugs. It is very difficult to prepare injection preparations, especially injection preparations for intravenous injection. In order to overcome the solubility problem of clevidipine butyrate, the American pharmaceutical company (Medicine) made an oil-in-water emulsion Cleviprox® containing 0.5 mg / mL with soybean oil for injection.

[0006] Domestic and foreign pharmaceutical companies or research institutions have conducted a large number of studies on clevidipine butyrate injection emulsion.

[0007] Patent document CN103110580A discloses clevidipine butyrate injection, which includes clevidipine butyrate, oil phase, phospholipid and organic solvent. The organic solvents are ethanol, propylene glycol, glycerol, polyethylene glycol, etc. The phospholipid is used as an emulsifier, forming an anhydrous oil phase system. High-concentration organic solvents such as ethanol and propylene glycol may cause pain and irritation at the injection site.

[0008] Patent document CN103237446 (US2011055617) discloses a clevidipine emulsion preparation containing an antimicrobial agent. The emulsion preparation contains clevidipine, antimicrobial agent, lipid, emulsifier, osmotic pressure regulator and water. The osmotic pressure of the emulsion is adjusted with glycerol and also contains an amount of EDTA sufficient to inhibit the growth of microorganisms in the preparation in case of accidental external contamination. The specific gravity of the antimicrobial agent EDTA is 0.001 to 0.01% w / v. The pharmaceutical preparation containing clevidipine and an antimicrobial agent shows a reduced tendency of microbial growth, and for administration to patients, the clevidipine-containing preparation provides increased convenience for healthcare workers. However, in the above technology, lipids such as soybean oil and safflower oil are selected, and emulsifiers such as egg yolk phospholipid are selected, forming an oil-in-water emulsion with poor stability and greater safety risks.

[0009] Patent CN104473870A discloses a clevidipine butyrate lipid microsphere injection and its preparation method, which includes clevidipine butyrate, injection oil, emulsifier, stabilizer, isotonic agent, pH regulator, and injection water. The injection oil is soybean oil for injection, and the isotonic agent is glycerol. The high-pressure microfluidic rapid preparation method is selected for high-pressure homogenization, with high equipment cost and difficulty in realizing large-scale production amplification.

[0010] Patent document CN113197853 discloses a clevidipine butyrate injection emulsion, its preparation method and application. The preparation method includes: shearing and mixing the oil phase and the water phase to obtain a primary emulsion; and after the primary emulsion is subjected to first homogenization and second homogenization, mixing it with a pH value regulator. Among them, the oil phase includes clevidipine butyrate or its pharmaceutically acceptable salt or hydrate, oil phase solvent, emulsifier and co-emulsifier; the water phase includes osmotic pressure regulator, stabilizer and water. Using this preparation method, a homogenizer is used for homogenization, with complex equipment, high cost and difficulty in production amplification.

[0011] At present, the butylchloride clevidipine fat emulsion injection provides a solution to the problem of the hydrolysis instability of butylchloride clevidipine. The technical routes for solving the oxidation of drugs mainly include adding antioxidants, filling inert gases, and changing the packaging, etc. However, in clinical applications, since emulsion injections need to be disposed of, such as diluting the emulsion injection before administration, the above protection measures may face the risk of failure, exacerbating the hydrolysis of butylchloride clevidipine and further oxidation. Therefore, how to adopt more effective methods to improve the stability of butylchloride clevidipine, reduce or inhibit the hydrolysis and oxidative degradation of butylchloride clevidipine, remains a difficult problem that urgently needs to be solved in the preparation.

[0012] As a special preparation, the particle size distribution of the butylchloride clevidipine injection emulsion is directly related to its pharmacokinetics in vivo, and thus affects the drug efficacy. Since the diameter of human microvessels is about 4 - 9 μm, and the diameter of pulmonary microvessels is about 5 μm, if a large number of emulsion particles have a diameter greater than 5 μm, it may lead to pulmonary embolism, alveolar tissue damage, and even death of the patient.

[0013] The disclosed patent literature has paid attention to the problem of large emulsion particles, but based on the way of using multiple, even 8 high-pressure homogenizers in series for homogenization, the cost of one homogenizer is easily over one million, and the overall cost of the equipment is relatively high; secondly, to ensure that the emulsion particles larger than 5 μm are qualified, an excessive amount of co-emulsifier (i.e., oleic acid) and bacteriostatic agent (i.e., disodium edetate) are used in the formulation. However, oleic acid is easily oxidized to form free fatty acids or other oxides, which can cause harm to the body. At the same time, excessive use of disodium edetate will cause a complexation reaction with calcium ions in the blood, resulting in a decrease in blood calcium.

[0014] Since the soybean oil used in the currently marketed butylchloride clevidipine injection is long-chain triglyceride (LCT), long-chain triglyceride is hydrolyzed into long-chain fatty acids in the blood. Long-chain fatty acids are insoluble in water and need to enter cells after binding to proteins. The long-chain fatty acids entering the cells need to be activated in the cytoplasm, that is, activated into the corresponding acyl coenzyme A by fatty acid thiokinase on the outer membrane of mitochondria or endoplasmic reticulum, and then enter the mitochondria for β-oxidation under the action of carnitine acyltransferase on the inner membrane of mitochondria. Therefore, long-chain triglyceride is easily phagocytosed and deposited by reticuloendothelial cells in organs such as the liver, spleen, and lungs, which can harm the immune function. Using medium-chain triglyceride (MCT) instead of long-chain triglyceride (LCT) can reduce the safety hazards brought by long-chain triglyceride. However, using medium-chain triglyceride (MCT) will also cause effects on the central nervous system, such as drowsiness, nausea, and sleepiness.

[0015] Therefore, it is necessary to develop a new type of butylchloride clevidipine preparation to overcome the above various disadvantages and provide a more safe injection with a higher concentration of butylchloride clevidipine, which has become an urgent technical problem to be solved in this field. Summary of the Invention

[0016] To solve the problems in the prior art that in the butylchloride clevidipine preparation, due to the low solubility of butylchloride clevidipine, its concentration is relatively low, and injection oils such as soybean oil need to be used as raw materials, which may cause embolism due to large milk particles, too high oleic acid content, poor solubility and stability, etc., the present invention provides a high-concentration butylchloride clevidipine micelle injection for intravenous drip and its preparation method. The injection contains butylchloride clevidipine, phospholipid, cholalic acid or its salt, pH regulator and injection water, and can also selectively add nano-micelle stabilizer and nano-micelle solubilizer. This injection selects physiologically compatible excipients phospholipid and cholalic acid or its salt, and uses a micelle system with good safety for solubilization. In the prescription, injection oils such as soybean oil and triglyceride are abandoned, meeting the medication needs of patients with lipid metabolism defects, such as patients with pathological hyperlipidemia, lipoid nephrosis, acute pancreatitis accompanied by hyperlipidemia, etc. It not only greatly improves the solubility of butylchloride clevidipine, but also creatively adds nano-micelle stabilizer and nano-micelle solubilizer, which can further improve the stability and drug loading capacity of the micelle injection. The preparation process of the butylchloride clevidipine micelle injection of the present invention selects the blank micelle method to abandon the use of organic solvents, does not need to use organic solvents with large toxic and side effects such as dichloromethane, chloroform, and propylene glycol, reduces the toxicity of the drug, has little irritation to blood vessels, and reduces the adverse reactions of the drug; selects the production process of ordinary aqueous injection, which is conducive to production scale-up.

[0017] The present invention first provides a butylchloride clevidipine micelle injection, the main raw materials of which include butylchloride clevidipine, phospholipid, cholalic acid or its salt, and pH regulator, and the solvent used is injection water.

[0018] In the above injection, the content of butylchloride clevidipine is 1-10 mg / mL, the content of cholalic acid or its salt is 30-100 mg / mL, the mass ratio of phospholipid to cholalic acid or its salt is 1:(0.5-1.5), and the pH regulator adjusts the pH of the injection to 6.0-7.5.

[0019] As a preferred technical solution, the injection also contains nano-micelle stabilizer and nano-micelle solubilizer; wherein the content of nano-micelle stabilizer is 0-20 mg / mL, and the content of nano-micelle solubilizer is 0-20 mg / mL.

[0020] After being made into a finished product, the volume specification of the prepared butylchloride clevidipine micelle injection is 1-10 mg / mL, the pH is 6.0-7.5, the particle size is 2-10 nm, the Zeta potential is -20~-60 mV, and the light transmittance >90%; the volume specification is preferably 5 mL, and it is placed in an ampoule bottle or a vial.

[0021] In some preferred embodiments, the content of phospholipid is 30-100 mg / mL.

[0022] When the injection also contains a nanomicelle stabilizer and a nanomicelle solubilizer, the mass ratio of preferred clevidipine butyrate to the nanomicelle stabilizer is 1:(0.1 - 0.5); the mass ratio of clevidipine butyrate to the nanomicelle solubilizer is 1:(0.1 - 0.5).

[0023] The nanomicelle stabilizer includes one or more of polyethylene glycol 15 - hydroxystearate, sodium oleate, poloxamer, polyoxyethylene castor oil, polysorbate, preferably polyethylene glycol 15 - hydroxystearate; the nanomicelle solubilizer includes one or more of vitamin E, oleic acid, ethyl oleate; the nanomicelle stabilizer can form a hydrophilic layer on the surface of the micelle or increase the potential, thereby improving the stability of the clevidipine butyrate micelle injection, and the nanomicelle solubilizer can further increase the drug loading of the clevidipine butyrate micelle injection; the phospholipid is one or more of soybean phospholipid, egg yolk phospholipid, hydrogenated soybean phospholipid, hydrogenated egg yolk phospholipid, synthetic phospholipid, preferably soybean phospholipid; optionally, the bile acid or its salt is one or two of deoxycholic acid, glycine cholate, sodium glycine cholate, sodium deoxycholate.

[0024] In the present invention, a pH regulator is added to adjust the pH of the injection to 6.0 - 7.5, and the pH regulator includes at least one of an acidic pH regulator and a basic pH regulator; the acidic pH regulator includes one or more of citric acid, acetic acid, hydrochloric acid, phosphoric acid, more preferably citric acid, and the basic pH regulator is sodium hydroxide and / or sodium carbonate.

[0025] The present invention further provides a preparation method of the clevidipine butyrate micelle injection, and the preparation method is the thin - film dispersion method, which specifically includes the following steps: (1) Thin - film preparation: Weigh the prescribed amount of clevidipine butyrate, phospholipid, bile acid or its salt, add an appropriate amount of organic solvent to dissolve, and place it on a rotary evaporator to obtain a loose thin - film through rotary evaporation; the bile acid or its salt is one or two of deoxycholic acid, glycine cholate.

[0026] (2) Medicinal solution preparation: Add a basic pH regulator with a molar ratio of 1:(0.8 - 1.2) to the bile acid or its salt added in step (1), dissolve it completely with injection water filled with a protective gas accounting for 50 - 85% of the total volume of the injection preparation, transfer the obtained solution to the loose thin - film in step (1), after complete hydration, adjust the pH to 6.0 - 7.5 with a pH regulator to obtain a medicinal solution.

[0027] (3) Volume or weight determination: The medicinal solution obtained in step (2) is volume - determined or weight - determined, filtered, and then filled with a protective gas, and the filtration uses a 0.22 μm filter membrane.

[0028] (4) Sealing and sterilization: Sub - pack, seal after filling the headspace with a protective gas, and sterilize by autoclaving at 121°C for 15 min.

[0029] When the formulation contains a nanomicelle stabilizer and / or a nanomicelle solubilizer, weigh and add them in step (1).

[0030] When the cholate or its salt used in step (1) is one or both of sodium deoxycholate and sodium glycicholate, the inventor adjusts step (2) to directly transfer the water for injection filled with protective gas, which accounts for 50 - 85% of the total volume of the injection preparation, to the loose film for complete hydration, and then adjust the pH to 6.0 - 7.5 with a pH regulator to obtain the medicinal solution.

[0031] The rotary evaporation parameters in step (1) are: water bath temperature 40 - 60°C, time 2 - 6 h; the organic solvent in step (1) is one or more of methanol, ethanol, and isopropanol in any proportion combination; the dosage of the organic solvent is 2 - 8% of the total volume of the injection preparation; as a further preference, the organic solvent is a mixed solvent of methanol and ethanol, and the volume ratio of methanol to ethanol is 2:1.

[0032] The protective gas is any one of nitrogen, helium, carbon dioxide, and argon. The time for introducing the protective gas is 0.5 - 2 h, the residual range of dissolved oxygen is 0 - 5 mg / L, and the residual headspace oxygen content is controlled within 0 - 5%.

[0033] The present invention also provides another preparation method of the clevidipine butyrate micelle injection, and the preparation method is the blank micelle method, which specifically includes the following steps: (1) Preparation of cholate solution: When the cholate or its salt is selected as deoxycholic acid and / or glycicholic acid, add a basic pH regulator with a molar ratio of 1:(0.8 - 1.2) to the cholate, dissolve it completely with the water for injection filled with protective gas, which accounts for 50 - 85% of the total volume of the injection preparation, and then adjust the pH of the solution to 6.0 - 7.5 with a pH regulator to obtain the cholate solution.

[0034] (2) Preparation of blank micelle solution: Weigh the phospholipid in the prescription, add it to the cholate solution in step (1), and stir in an oil bath at 90 - 110°C until the solution becomes clear to obtain the blank micelle solution.

[0035] (3) Preparation of medicinal solution: Weigh clevidipine butyrate according to the prescription, slowly add it to the blank micelle solution obtained in step (2), heat and stir in an oil bath at 90 - 110°C until the solution becomes clear and transparent, cool it to room temperature, and then adjust the pH to 6.0 - 7.5 with a pH regulator.

[0036] (4) Volume or weight determination: Volume or weight determine and filter the medicinal solution obtained in step (3), and then fill it with a protective gas. The filtration uses a 0.22 μm filter membrane.

[0037] (5) Sealing and sterilization: After filling and purging the headspace with a protective gas, seal it, and sterilize it by autoclaving at 121 °C for 15 min.

[0038] When the formulation contains a nanomicelle stabilizer and / or a nanomicelle solubilizer, weigh and add them together with the phospholipids in step (2).

[0039] When the cholic acid or its salt used in step (1) is one or both of sodium deoxycholate and sodium glycicholate, the inventor adjusts step (1) to directly dissolve the cholic acid or its salt in injection water filled with a protective gas, accounting for 50 - 85% of the total volume of the injection preparation, and then adjust the pH to 6.0 - 7.5 with a pH regulator to obtain a cholate solution.

[0040] Preferably, among the injection water filled with a protective gas, the liquid medicine filled with a protective gas after volume fixing, and the protective gas filled before sealing, the protective gas is any one of nitrogen, helium, carbon dioxide, and argon. The time for introducing the protective gas is 0.5 - 2 h, the residual range of dissolved oxygen is 0 - 5 mg / L, and the residual oxygen content in the headspace is controlled within 0 - 5%. As a further preference, the protective gas is nitrogen.

[0041] The obtained clevidipine butyrate micelle injection, as a dihydropyridine calcium channel blocker, is applicable to the treatment of hypertension when oral antihypertensive drugs are not applicable or cannot achieve satisfactory efficacy.

[0042] In specific applications, the compatible and dilutable injection solutions that can be used with the clevidipine butyrate micelle injection provided by the present invention include but are not limited to injection water, sodium chloride injection (0.9%), glucose injection (5%), glucose (5%) - sodium chloride (0.9%) injection, 10% amino acid, and lactated Ringer's injection. And as one of the preferred embodiments, glucose injection (5%) or sodium chloride injection (0.9%) is preferred.

[0043] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) Improve the safety of medication: The system provided by the present invention selects a phospholipid and bile salt mixed micelle system with good physiological compatibility and high safety. The micelles are formed by mixing two natural materials. The mixed micelle particles are composed of phospholipids, bile salts, and drug molecules, and the drug is encapsulated in the hydrophobic core. On this basis, a nanomicelle stabilizer and a nanomicelle solubilizer are creatively added. The addition of the nanomicelle stabilizer can form a hydrophilic layer on the micelle surface or increase the Zeta potential, reduce the aggregation between particles or increase the Zeta potential of the micelle particles, so as to further improve the stability of the micelle system. The addition of the solubilizer can further improve the drug - loading capacity of the micelles.

[0044] For fat emulsion injections in other literature patents such as commercially available injections (e.g., Cleviprex®), an oil-phase solvent is selected, and phospholipids, bile acids, etc. are added as emulsifiers or stabilizers, and vitamin E is used as an antioxidant. The functions of such components in the present invention are significantly different (the present invention is a mixed micelle). In the present invention, the use of a large amount of soybean oil as a solvent is avoided, thereby avoiding the increase in blood lipid levels of hyperlipidemic patients caused by the infusion of the injection, and further causing cardiovascular harm to hyperlipidemic patients, meeting the medication needs of patients with lipid metabolism defects, and significantly improving the medication safety of special patients with hyperlipidemia.

[0045] (2) Avoid large milk particles and reduce the risk of medication: The prepared clevidipine butyrate micelle injection of the present invention is a sterilized clear solution, without phenomena such as the rupture of milk particles, the aggregation and merger of milk droplets, the increase in particle size, and local demulsification of the marketed emulsion, avoiding large milk particles and avoiding adverse reactions caused by large milk particles. It is mainly manifested in: a) Avoid that the large particles in the injected fat emulsion enter the human lungs during the injection process, causing the formation of granulomas in the lungs; b) Avoid that the large particles entering the blood vessels can cause vascular granulomas, phlebitis, thrombosis, etc.; c) Avoid that the large particles can enter other organs of the body along with the blood circulation, such as the liver, kidneys, etc., which are damaged; d) The micelle injection provided by the present invention has a clear solution, which is easier to observe visible foreign matters and insoluble particles than the emulsion, can predict medication in advance, and reduce the risk of medication; e) The particle size of the micelles of the prepared clevidipine butyrate micelle injection of the present invention is very small, 2 - 10 nm, which can penetrate biological membranes and remain in tissues, thereby enhancing its aggregation in tissues and improving the drug efficacy.

[0046] (3) Improve compliance: When the prepared clevidipine butyrate micelle injection of the present invention is clinically used, it can be directly diluted with sterilized water for injection, 0.9% sodium chloride injection, 5% glucose injection, etc. to obtain a dosing concentration of 0.01 - 1 mg / mL. By formulating the drug concentration of the prepared injection and controlling the dosing rate, there is no need for a Y-type infusion set and a "special precision-adjustable dosing infusion device", and the dosing dose is controlled within the range of 1 - 32 mg / h. The clinical dosing method is more flexible and convenient, and the dosing dose is more accurately controllable.

[0047] (4) Improve the drug loading capacity: In the system of the present invention, by dissolving clevidipine butyrate in phospholipid-bile salt mixed micelles, and creatively adding a lipid-soluble nano-micelle solubilizer to the formulation, the drug loading capacity of the mixed micelles is further improved; The concentration of the clevidipine butyrate micellar injection prepared by the present invention is as high as 10 mg / mL, which significantly improves the drug solubility compared with the commercially available injection (0.5 mg / mL). During clinical administration, the total infusion volume of this preparation is 1 / 2 - 1 / 20 of Cleviprex®, eliminating or reducing the risk of extravasation of hypertonic drug fat emulsion injection, greatly reducing the occurrence of adverse reactions such as phlebitis, and having higher clinical value.

[0048] (5) Improving stability: Through a large number of experiments, it is proved that adding a nano-micelle stabilizer to the system of the present invention can achieve unexpected effects. The addition of the stabilizer can form a hydrophilic layer on the surface of the micelles, reducing the aggregation between particles, or the addition of sodium oleate, etc. can increase the Zeta potential of the micelle particles, both of which can achieve the purpose of improving the stability of the micellar injection, especially significantly improving the compatibility stability of the injection.

[0049] Intravenous injection emulsions belong to the category of submicron emulsions in pharmaceutics, with an average particle size mostly between 100 - 500 nm, and they are a thermodynamically unstable system. The shelf life of Cleviprex® at room temperature is no more than 2 months. However, the clevidipine butyrate micellar injection prepared by the present invention has passed the freeze-thaw cycle test, low-temperature cycle test and compatibility stability experiment, and after being placed at room temperature for 3 months, the solution is clear and no crystals are precipitated, overcoming the disadvantage that fat emulsions cannot be stored frozen for a long time or temporarily, and improving the convenience of transportation, storage and use. The clevidipine butyrate micellar injection prepared by the present invention creatively adds a nano-micelle stabilizer, greatly improving the stability of the preparation. By terminal sterilization at 121 °C for 15 minutes, a clear solution with good stability is obtained, which can effectively avoid phenomena such as droplet aggregation, increased particle size and local demulsification after sterilization and long-term storage of intravenous injection emulsions.

[0050] (6) The preparation process is simple and easy to scale up production: The existing fat emulsion preparation process requires complex processes such as high-pressure homogenizers to obtain milk particles, and the process-related quality control is difficult and costly; while in the system of the present invention, no special equipment is required, and the process is easy to control. Especially for the blank micelle method, the production route of ordinary aqueous injections can be selected. Therefore, the process provided by the present invention is simple, with low production cost and easy to implement. Moreover, the existing publicly disclosed patent literatures use organic solvents with large toxic and side effects such as dichloromethane, chloroform, and propylene glycol, which are not friendly to the environment and hinder scale-up production. The present invention does not require or uses a small amount of methanol and ethanol, which is conducive to scale-up production. Description of the Drawings

[0051] Figure 1 It is the appearance morphology diagram of the clevidipine butyrate mixed micellar injection prepared by the thin film dispersion method in Example 1; Figure 2Particle size distribution diagram of clevidipine butyrate micelle injection prepared by the thin film dispersion method in Example 1, where Figure 2 a is Figure 2 the average particle size, aggregation index and intercept of the particle size distribution shown in b, Figure 2 b is the particle size distribution diagram; Figure 3 Zeta potential diagram of clevidipine butyrate micelle injection prepared by the blank micelle method in Example 7, where Figure 3 a is Figure 3 the Zeta potential, standard deviation of Zeta potential and conductivity of the Zeta potential distribution shown in b, Figure 3 b is the Zeta potential distribution diagram; Figure 4 High performance liquid chromatogram for the determination of related substances of clevidipine butyrate micelle injection prepared by the blank micelle method in Example 3 of Experimental Example 1; Figure 5 Content change curves (0.25 mg / mL and 0.1 mg / mL) measured during the compatibility stability study (0.9% sodium chloride injection) of clevidipine butyrate micelle injection prepared by the blank micelle method in Example 11 of Experimental Example 2; Figure 6 Content change curves (0.25 mg / mL and 0.1 mg / mL) measured during the compatibility stability study (5% glucose injection) of clevidipine butyrate micelle injection prepared by the blank micelle method in Example 11 of Experimental Example 2; Figure 7 Zeta potential diagram of the freeze-thaw cycle test of clevidipine butyrate micelle injection prepared by the blank micelle method in Example 11 of Experimental Example 5, where Figure 7 a is Figure 7 the Zeta potential, standard deviation of Zeta potential and conductivity of the Zeta potential distribution shown in b, Figure 7 b is the Zeta potential distribution diagram; Figure 8 Particle size distribution diagram of clevidipine butyrate micelle injection prepared by the blank micelle method in Example 9 of Experimental Example 6 for accelerated / 3 months, where Figure 8 a is Figure 8 the average particle size, aggregation index and intercept of the particle size distribution shown in b, Figure 8 b is the particle size distribution diagram. Detailed implementation manners

[0052] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. For those not specified in the examples regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications.

[0053] In the examples, the water for injection and the liquid medicine after volume fixation are filled with protective gas, and the dissolved oxygen is controlled within the range of 0 - 5 mg / L. The residual oxygen content in the headspace before melting and sealing after sub-packaging is controlled within the range of 0 - 5%.

[0054] Example 1 A clevidipine butyrate micelle injection, and its raw material prescription is as follows: Clevidipine butyrate 500 mg, soybean phospholipid 5.33 g, glycocholic acid 6.67 g, sodium hydroxide 0.57 g, add water for injection to 100 mL.

[0055] The thin film dispersion method is adopted, and the preparation method includes the following steps: (1) Weigh the prescribed amounts of clevidipine butyrate, soybean phospholipid, and glycocholic acid, add 5 mL of methanol and 2 mL of absolute ethanol to dissolve completely, and rotary evaporate at 40 °C in a water bath for 4 h to obtain a loose thin film.

[0056] (2) Take the prescribed amount of sodium hydroxide, add 80 mL of nitrogen-filled water for injection, stir to dissolve, transfer it to the loose thin film obtained in step (1), stir at a speed of 300 rpm in a 40 °C water bath for 30 min to make it completely hydrated, and adjust the pH value to 6.5 with 1 mol / L sodium hydroxide solution and 0.5 mol / L citric acid solution.

[0057] (3) Fix the volume of the liquid medicine obtained in (2) to 100 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0058] (4) Fill the ampoules with nitrogen (20 s / ampoule), sub-package (5 mL / ampoule), melt and seal, and sterilize by autoclaving at 121 °C for 15 min.

[0059] As Figure 1 shown, the clevidipine butyrate micelle injection obtained in Example 1 is clear and transparent, light yellowish green in color, and has a pH of 6.6.

[0060] As Figure 2 shown, the Z-Average of the mixed micelle solution is 2.655 nm, the PDI is 0.296; the Zeta potential is -36.8 mV.

[0061] Example 2 A clevidipine butyrate micelle injection, and its raw material prescription is as follows: Clevidipine butyrate 2 g, soybean phospholipid 25.66 g, deoxycholic acid 25.66 g, sodium hydroxide 2.32 g, add water for injection to 400 mL.

[0062] The thin film dispersion method is adopted, and the preparation method includes the following steps: (1) Weigh the prescribed amounts of clevidipine butyrate, soybean phospholipid, and deoxycholic acid, add 20 mL of methanol and 10 mL of absolute ethanol, dissolve completely, place on a rotary evaporator, and evaporate under a water bath at 40 °C for 4 h to obtain a loose film.

[0063] (2) Weigh the prescribed amount of sodium hydroxide, add 350 mL of nitrogen-purged and deoxygenated injection water, stir until completely dissolved, transfer to the loose film obtained in (1), stir at 300 rpm under a water bath at 35 °C for 30 min to completely hydrate it, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0064] (3) Make up the volume of the medicinal liquid obtained in (2) to 400 mL, filter through a 0.22 μm pore size filter membrane, and purge with nitrogen.

[0065] Step (4) is the same as in Example 1.

[0066] This injection is clear, transparent, and light yellowish-green. The Z-Average of the mixed micelle solution is 2.560 nm, the PDI is 0.264, the Zeta potential is -36.5 mV, and the pH is 6.7.

[0067] Example 3 A clevidipine butyrate micelle injection, the raw material prescription is as follows: Clevidipine butyrate 1.5 g, soybean phospholipid 23.98 g, sodium glycocholate 17.38 g, add injection water to 300 mL.

[0068] Using the blank micelle method, the preparation method includes the following steps: (1) Weigh the prescribed amount of sodium glycocholate, add 160 mL of nitrogen-purged and deoxygenated injection water, stir for 15 min to dissolve, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0069] (2) Weigh the soybean phospholipid in the prescription, add it to the solution in step (1), heat in an oil bath at 100 °C and stir at 400 rpm for 1 h until the solution is clear to obtain a blank micelle solution.

[0070] (3) Weigh the prescribed amount of clevidipine butyrate, add it to the blank micelle solution obtained in step (2), heat in an oil bath at 100 °C, heat and stir until the API is completely dissolved and the solution is clear, cool to room temperature, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0071] (4) Make up the volume of the medicinal liquid in step (3) to 300 mL, filter through a 0.22 μm filter membrane, and purge with nitrogen.

[0072] (5) Sub-packaging (5 mL / vial), filling nitrogen into the headspace of ampoules (10 s / vial), melting and sealing, heat-press sterilization at 121 °C for 15 min.

[0073] This injection solution is clear, transparent, light yellowish-green. The Z-Average of the mixed micelle solution is 2.763 nm, the PDI is 0.261, the Zeta potential is -39.0 mV, and the pH is 7.0.

[0074] Example 4 A clevidipine butyrate micelle injection solution, with the following raw material prescription: Clevidipine butyrate 1.25 g, soybean phospholipid 17.96 g, glycocholic acid 22.45 g, sodium hydroxide 2.02 g, add injection water to make up 250 mL.

[0075] Using the blank micelle method, the preparation method includes the following steps: (1) Weigh the prescribed amounts of glycocholic acid and sodium hydroxide, add 160 mL of nitrogen-filled and deoxygenated injection water, stir for 15 min to dissolve, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0076] Steps (2) and (3) are carried out in the same manner as described in Example 3.

[0077] (4) Adjust the liquid medicine in step (3) to 250 mL, filter through a 0.22 μm filter membrane, and fill with nitrogen.

[0078] (5) Sub-packaging (5 mL / vial), filling nitrogen into the headspace of ampoules (10 s / vial), melting and sealing, heat-press sterilization at 121 °C for 15 min.

[0079] This injection solution is clear, transparent, light yellowish-green. The Z-Average of the mixed micelle solution is 2.725 nm, the PDI is 0.293, the Zeta potential is -38.3 mV, and the pH is 7.0.

[0080] Example 5 A clevidipine butyrate micelle injection solution, with the following raw material prescription: Clevidipine butyrate 500 mg, soybean phospholipid 6.34 g, glycocholic acid 5.07 g, sodium hydroxide 0.46 g, add injection water to make up 100 mL.

[0081] The thin film dispersion method is selected for the preparation method, and the specific implementation steps are the same as those in Example 1.

[0082] This injection solution is clear, transparent, light yellowish-green. The Z-Average of the mixed micelle solution is 2.065 nm, the PDI is 0.203, the Zeta potential is -39.6 mV, and the pH is 6.5.

[0083] Example 6. A cinepazide maleate micellar injection, with the following raw material prescription: Cinepazide maleate 500 mg, soybean phospholipid 5.71 g, glycocholic acid 5.71 g, sodium hydroxide 0.52 g, add water for injection to make 100 mL.

[0084] Using the blank micelle method, the preparation method includes the following steps: (1) Weigh the prescribed amount of glycocholic acid and sodium hydroxide, add 80 mL of nitrogen-filled and deoxygenated water for injection, stir to dissolve, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0085] (2) Weigh the soybean phospholipid in the prescription, add it to the solution in step (1), set the oil bath temperature at 100 °C, heat and stir until the solution is clear to obtain a blank micelle solution.

[0086] (3) Weigh the prescribed amount of cinepazide maleate, add it to the blank micelle solution in step (2), set the oil bath temperature at 90 °C, heat and stir until the API is completely dissolved and the solution is clear, cool to room temperature, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0087] (4) Make the solution in step (3) up to 100 mL, filter through a 0.22 μm filter membrane, and fill with nitrogen.

[0088] (5) Subpackage (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal by melting, and sterilize by autoclaving at 121 °C for 15 min.

[0089] This injection is clear, transparent, and light yellowish green. The Z-Average of the mixed micelle solution is 1.985 nm, the PDI is 0.209, the Zeta potential is -40.8 mV, and the pH is 6.5.

[0090] Example 7. A cinepazide maleate micellar injection, with the following raw material prescription: Cinepazide maleate 1 g, soybean phospholipid 11.79 g, sodium glycocholate 9.43 g, vitamin E 200 mg, add water for injection to make 200 mL.

[0091] Using the blank micelle method, the preparation method includes the following steps: (1) Weigh the prescribed amount of sodium glycocholate, add 160 mL of nitrogen-filled and deoxygenated water for injection, stir for 20 min to dissolve, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0092] (2) Weigh the soybean phospholipid and vitamin E in the prescription, add them to the solution prepared in step (1), heat in an oil bath at 100 °C, stir at 300 rpm until the solution is clear, and obtain a blank micelle solution.

[0093] (3) Weigh the prescribed amount of clevidipine butyrate, add it to the blank micelle solution prepared in step (2), heat in an oil bath at 100 °C, heat and stir until the solution is clear and transparent, cool to room temperature, and then adjust the pH value to 6.5.

[0094] (4) Fix the liquid medicine in step (3) to 200 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0095] (5) Subpackage (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it by melting, and sterilize it by hot pressing at 121 °C for 15 min.

[0096] The injection is clear, transparent, light yellowish green. The Z-Average of the mixed micelle solution is 2.766 nm, the PDI is 0.239, and the pH is 6.5; as Figure 3 shown, the Zeta potential is -48.6 mV.

[0097] Example 8 A clevidipine butyrate micelle injection, the raw material prescription is as follows: Clevidipine butyrate 1 g, soybean phospholipid 9.5 g, sodium glycocholate 9.5 g, oleic acid 200 mg, add injection water to 200 mL.

[0098] Using the thin film dispersion method, the preparation method includes the following steps: (1) Weigh the prescribed amounts of clevidipine butyrate, soybean phospholipid, sodium glycocholate, and oleic acid, dissolve them completely in 5 mL of methanol and 2 mL of absolute ethanol, place them in a rotary evaporator, and obtain a loose film by rotary evaporation in a water bath at 50 °C for 3 h.

[0099] (2) Add 160 mL of nitrogen-filled injection water to the obtained loose film, stir to make it completely hydrated, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0100] (3) After adjusting the pH, fix the volume of the liquid medicine to 200 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0101] (4) Subpackage (10 mL / vial), fill the headspace of the vial with nitrogen and then press the stopper and crimp the cap, and sterilize it by hot pressing at 121 °C for 15 min.

[0102] The injection is clear, transparent, light yellowish green. The Z-Average of the mixed micelle solution is 2.986 nm, the PDI is 0.293, the Zeta potential is -45.6 mV, and the pH is 6.4.

[0103] Example 9 A clevidipine butyrate micellar injection, and its raw material prescription is as follows: Clevidipine butyrate 1 g, egg yolk phospholipid 10.31 g, glycocholic acid 10.31 g, sodium hydroxide 0.93 g, vitamin E 200 mg, polyethylene glycol 15-hydroxystearate 400 mg, add injection water to make 200 mL.

[0104] Using the blank micelle method, the preparation method includes the following steps: (1) Weigh the prescribed amount of glycocholic acid and sodium hydroxide, add 160 mL of nitrogen-filled and deoxygenated injection water, stir for 20 min to dissolve, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0105] (2) Weigh the egg yolk phospholipid, vitamin E, and polyethylene glycol 15-hydroxystearate in the prescription, add them to the solution prepared in step (1), heat in an oil bath at 100 °C and stir at 300 rpm until the solution is clear to obtain a blank micelle solution.

[0106] The remaining steps are the same as those in Example 7.

[0107] The obtained injection is clear, transparent, and light yellowish green. The Z-Average of the mixed micelle solution is 2.726 nm, the PDI is 0.287, the Zeta potential is -37.6 mV, and the pH is 6.5.

[0108] Example 10 A clevidipine butyrate micellar injection, and its raw material prescription is as follows: Clevidipine butyrate 1 g, soybean phospholipid 9.5 g, glycocholic acid 9.5 g, sodium hydroxide 0.87 g, vitamin E 200 mg, sodium oleate 200 mg, add injection water to make 200 mL.

[0109] Using the thin film dispersion method, the preparation method includes the following steps: (1) Weigh the clevidipine butyrate, soybean phospholipid, glycocholic acid, vitamin E, and sodium oleate in the prescription according to the quantity, add 5 mL of methanol and 2 mL of absolute ethanol to dissolve completely, place it on a rotary evaporator, and rotate and evaporate in a water bath at 50 °C for 4 h to obtain a loose film.

[0110] (2) Take the prescribed amount of sodium hydroxide, add 160 mL of nitrogen-filled injection water, stir to dissolve, transfer it to the loose film obtained in step (1), stir at 30 °C for 1 h to make it completely hydrated, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0111] The remaining steps are the same as those in Example 8.

[0112] The injection solution is clear, transparent, light yellowish-green. The Z-Average of the mixed micelle solution is 2.853 nm, the PDI is 0.275, the Zeta potential is -36.7 mV, and the pH is 6.5.

[0113] Example 11 A micellar injection of clevidipine butyrate, the raw material prescription thereof is as follows: Clevidipine butyrate 5 g, soybean phospholipid 51.53 g, glycocholic acid 41.22 g, sodium hydroxide 3.72 g, vitamin E 1.0 g, polyethylene glycol 15-hydroxystearate 1.25 g, add injection water to 1000 mL.

[0114] The blank micelle method is adopted, and the preparation method includes the following steps: (1) Weigh glycocholic acid and sodium hydroxide in the prescription, add 800 mL of nitrogen-filled and deoxygenated injection water, stir for 20 min to dissolve it, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0115] (2) Weigh soybean phospholipid, polyethylene glycol 15-hydroxystearate, and vitamin E in the prescription, add them to the solution prepared in step (1), heat in an oil bath at 100 °C and stir at 400 rpm until the solution is clear to obtain a blank micelle solution.

[0116] (3) Weigh the prescribed amount of clevidipine butyrate, add it to the blank micelle solution prepared in step (2), heat in an oil bath at 100 °C, heat and stir until the solution is clear and transparent, cool to room temperature, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0117] (4) Fix the medicinal liquid in step (3) to 1000 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0118] (5) Subpackage (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it by melting, and sterilize it by autoclaving at 121 °C for 15 min.

[0119] The injection solution is clear, transparent, light yellowish-green. The Z-Average of the mixed micelle solution is 2.906 nm, the PDI is 0.330, the Zeta potential is -38.6 mV, and the pH is 6.6.

[0120] Example 12 A micellar injection of clevidipine butyrate, the raw material prescription thereof is as follows: Clevidipine butyrate 1 g, soybean phospholipid 11.79 g, sodium deoxycholate 9.43 g, poloxamer 300 mg, vitamin E 200 mg, add injection water to 200 mL.

[0121] The thin film dispersion method is adopted, and the preparation method includes the following steps: (1) Weigh the prescribed amounts of clevidipine butyrate, soybean phospholipid, sodium deoxycholate, poloxamer, and vitamin E, dissolve them completely in 5 mL of methanol and 2 mL of absolute ethanol, place them on a rotary evaporator for rotary evaporation, and perform rotary evaporation in a water bath at 50 °C for 5 h to obtain a loose film.

[0122] (2) Add 160 mL of nitrogen-filled injection water to the obtained loose film, stir at 25 °C for 1 h to completely hydrate it, and adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0123] (3) After adjusting the pH, make the volume of the medicinal liquid up to 200 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0124] (4) Subpackage (5 mL per vial), after filling the headspace of the ampoule with nitrogen, seal it by melting, and sterilize it by autoclaving at 121 °C for 15 min.

[0125] This injection is clear, transparent, and light yellowish green. The Z-Average of the mixed micelle solution is 3.065 nm, the PDI is 0.196, the Zeta potential is -48.5 mV, and the pH is 6.5.

[0126] Example 13 A clevidipine butyrate micelle injection, the raw material prescription is as follows: Clevidipine butyrate 1 g, soybean phospholipid 9.5 g, deoxycholic acid 9.5 g, sodium hydroxide 0.87 g, polyoxyl castor oil 500 mg, ethyl oleate 200 mg, add injection water to make 200 mL.

[0127] The blank micelle method is adopted, and the preparation method includes the following steps: (1) Weigh the deoxycholic acid and sodium hydroxide in the prescription, add 160 mL of nitrogen-filled and deoxygenated injection water, stir for 20 min to dissolve, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0128] (2) Weigh the soybean phospholipid, polyoxyl castor oil, and ethyl oleate in the prescription, add them to the solution prepared in step (1), heat in an oil bath at 100 °C and stir at 300 rpm until the solution is clear to obtain a blank micelle solution.

[0129] (3) Weigh the prescribed amount of clevidipine butyrate, add it to the blank micelle solution prepared in step (2), heat in an oil bath at 100 °C, heat and stir until the solution is clear and transparent, cool it to room temperature, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0130] (4) Make the liquid medicine in step (3) reach 200 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0131] (5) Subpackage (5 mL per vial), fill the headspace of the ampoule with nitrogen, seal it by melting, and sterilize it by autoclaving at 121 °C for 15 min.

[0132] This injection solution is clear, transparent, light yellowish green. The Z-Average of the mixed micelle solution is 3.012 nm, the PDI is 0.129, the Zeta potential is -45.2 mV, and the pH is 7.0.

[0133] Example 14 A clevidipine butyrate micelle injection solution, the raw material prescription thereof is as follows: Clevidipine butyrate 2.5 g, hydrogenated soy phosphatide 27.95 g, sodium glycocholate 18.63 g, vitamin E 0.300 g, polysorbate 80 0.625 g, add injection water to make up to 333 mL.

[0134] Using the blank micelle method, the preparation method comprises the following steps: (1) Weigh sodium glycocholate in the prescription, add 280 mL of nitrogen-filled and deoxygenated injection water, stir to completely dissolve it, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0135] (2) Weigh hydrogenated soy phosphatide, polysorbate 80, and vitamin E in the prescription, add them to the solution prepared in step (1), heat in an oil bath at 100 °C, stir at 300 rpm until the solution is clear, and obtain a blank micelle solution.

[0136] (3) Weigh the prescribed amount of clevidipine butyrate, add it to the blank micelle solution prepared in step (2), heat in an oil bath at 100 °C, heat and stir until the solution is clear and transparent, cool it to room temperature, and adjust the pH value to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0137] (4) Make the liquid medicine in step (3) reach 333 mL, filter it through a 0.22 μm filter membrane, and fill it with nitrogen.

[0138] (5) Subpackage (5 mL per vial), fill the headspace of the ampoule with nitrogen, seal it by melting, and sterilize it by autoclaving at 121 °C for 15 min.

[0139] This injection solution is clear, transparent, light yellowish green. The Z-Average of the mixed micelle solution is 2.506 nm, the PDI is 0.189, the Zeta potential is -40.6 mV, and the pH is 7.0.

[0140] Experimental Example 1 Determination Methods for Drug Loading, Content, Related Substances, and Residual Solvents: For the determination of drug loading, content, related substances, and residual solvents, the following methods were referred to.

[0141] 1. Drug Loading: Clevidipine butyrate / (sum of the masses of clevidipine butyrate and added excipients) × 100%.

[0142] 2. Content Determination Method (the following ratios in the mobile phase, reference solution, and test solution are all volume ratios): Chromatographic conditions: Octadecylsilane chemically bonded silica gel was used as the filler (Sunniest C18 specification: 250×4.6 mm, 5 μm); methanol - acetonitrile - 10 mM NaH 2 PO 4 (Weigh about 1.2 g of anhydrous sodium dihydrogen phosphate, dissolve it in water and dilute to 1000 mL, adjust the pH value to 3.0 with phosphoric acid) (25:25:50) was used as mobile phase A, and acetonitrile - water (90:10) was used as mobile phase B (mobile phase A - mobile phase B, 20:80); the detection wavelength was 239 nm; the injection volume was 10 μL.

[0143] Reference solution: Take about 25 mg of clevidipine butyrate reference substance, place it in a 50 mL volumetric flask, add methanol and shake to dissolve, then dilute to the scale, and shake well; accurately measure 2 mL and place it in a 20 mL volumetric flask, dilute to the scale with the solvent (methanol - acetonitrile - water, 40:40:20), and shake well.

[0144] Test solution: Accurately measure 2 mL of the test solution under the item of related substances, place it in a 20 mL volumetric flask, dilute to the scale with the solvent (methanol - acetonitrile - water, 40:40:20), and shake well.

[0145] 3. Related Substances Test Method: Chromatographic conditions: Octadecylsilane chemically bonded silica gel was used as the filler (Sunniest C18 specification: 250×4.6 mm, 5 μm); methanol - acetonitrile - 10 mM NaH 2 PO 4 (Weigh about 1.2 g of anhydrous sodium dihydrogen phosphate, dissolve it in water and dilute to 1000 mL, adjust the pH value to 3.0 with phosphoric acid) (25:25:50) was used as mobile phase A, and acetonitrile - water (90:10) was used as mobile phase B; the detection wavelength was 239 nm; the injection volume was 20 μL, and gradient elution was carried out according to the method shown in Table 1.

[0146] Table 1 Gradient Elution Table for the Related Substances Test Method of Clevidipine Butyrate Micellar Injection Test solution: Accurately measure 1 mL of this product, place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well.

[0147] 4. Determination method for residual solvents: (1) Chromatographic conditions: A capillary column with 6% cyanopropylphenyl - 94% dimethylpolysiloxane (or similar polarity) as the stationary phase (Agilent DB - 624, 0.53 mm × 30 m, 3 μm or a chromatographic column with equivalent efficiency is recommended); the initial temperature is 50°C, hold for 10 minutes, then increase the temperature to 150°C at a rate of 50°C per minute, and hold for 5 min; the injection port temperature is 200°C; the detector is a flame ionization detector, and the detector temperature is 300°C; the column flow rate is 2 mL per minute; the split ratio is 20:1; the headspace equilibrium temperature is 80°C; the quantitative loop temperature is 90°C; the equilibrium time is 30 minutes.

[0148] System suitability requirements: In the chromatogram of the reference solution, methanol and ethanol elute in sequence, and the resolution between each chromatographic peak should meet the requirements.

[0149] (2) Preparation of solutions Test solution: Accurately measure 2 mL of this product, place it in a 20 mL headspace vial, stopper and seal.

[0150] Reference solution: Weigh appropriate amounts of methanol and ethanol respectively, accurately, quantitatively dilute with ultrapure water to prepare a mixed solution containing approximately 3 mg of methanol and 5 mg of ethanol per 1 mL. Accurately measure 2 mL of this solution, place it in a 20 mL headspace vial, stopper and seal.

[0151] Test Examples 1 - 14 were detected according to the above methods for drug loading, content, related substances, and residual solvents. The results are shown in Table 2; the chromatogram of related substances of the micellar injection prepared in Test Example 3 after sterilization is as Figure 4 shown. It can be seen from the results in Table 2 that the drug loadings of Test Examples 7 - 14 are all higher than those of Test Examples 1 - 6. It can be seen that adding a nano - micelle solubilizer to the micellar injection formulation can further increase the drug loading; the content of the micellar injection prepared in the above test examples has no obvious change before and after autoclaving.

[0152] Table 2 Detection results of drug loading, content, related substances, etc. of Test Examples 1 - 14 .

[0153] Experimental Example 2 Compatibility stability Take the micelle injection solutions prepared in Example 1 and Examples 4 - 11, and dilute them with 0.9% sodium chloride injection solution and 5% glucose injection solution to different concentrations (such as 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL), and place them at room temperature (25 °C) for 24 h to investigate the stability under clinical use conditions. Combining with the content and related substance analysis and detection methods in Experimental Example 1, the dilution stability was investigated. The results are shown in Table 3 - 7, and the content change curve is shown in Figure 5 and Figure 6 .

[0154] Table 3 Physical Stability Test Results of Sample Compatibility Solutions Note: ① "-" indicates "no crystal precipitation", "+" indicates "crystal precipitation", and "++" indicates "severe crystal precipitation"; ② The compatibility reagent is 5% glucose injection solution, and the concentration of the compatibility solution is 0.5 mg / mL.

[0155] Table 4 Detection Results of Particle Size (Z - Average) of Compatibility Solution of Example 11 .

[0156] Table 5 Detection Results of Particle Size (Z - Average) of Compatibility Solution of Example 7 .

[0157] Table 6 Content Determination Results of Compatibility Solution of Example 11 .

[0158] Table 7 Related Substance Determination Results of Compatibility Solution of Example 11 As can be seen from the results in Table 3, the micelle injection prepared in the examples shown in the table was formulated with 5% glucose injection and placed at room temperature (25°C) for 24 hours. For Examples 1 and 4, they were stable after being placed for 12 hours, which could meet the clinical needs. Therefore, without adding nano-micelle solubilizers and stabilizers, when the drug loading was up to 4.0%, there was no precipitation during the 12-hour compatibility stability test, but crystals precipitated after 20 hours. For Examples 5 and 6, precipitation occurred after being placed for 8 hours. It can be seen that without adding nano-micelle stabilizers and nano-micelle solubilizers, when the drug loading was increased to 4.2%, the compatibility stability of the injection decreased significantly. For Examples 7 - 8, crystals precipitated after being placed for 20 hours, while for Examples 9 - 11, no crystals precipitated after being placed for 24 hours. It can be seen that adding nano-micelle solubilizers (vitamin E, oleic acid were added in Examples 7 - 8 respectively, and the drug loadings were 4.5% and 5%) in the formulation not only increased the drug loading but also improved the stability to a certain extent. However, when adding nano-micelle stabilizers (sodium oleate, polyethylene glycol 15-hydroxystearate were added in Examples 9 - 11 respectively, and the drug loadings were 4.5%, 4.9%, and 5.0%), the stability could be further improved, and there was no precipitation after being placed at 25°C for 24 hours.

[0159] As can be seen from the results in Table 4 to Table 7, the compatibility solutions of Examples 7 and 11 with 5% glucose injection were placed at room temperature (25°C) for 24 hours. Compared with 0 hour, there were no significant changes in particle size, content, and related substances in Example 11, while the particle size in Example 7 increased significantly. It can be seen that adding nano-micelle stabilizer in Example 11 could further improve the compatibility stability of the injection.

[0160] Conclusion: Compared with the samples without adding nano-micelle stabilizer in the formulation, the compatibility solutions of the samples prepared by adding nano-micelle stabilizer in the formulation had no crystal precipitation after being placed at room temperature for 24 hours, and the content had no significant change. It could further improve the stability of the micelle injection while increasing the drug-loading capacity.

[0161] Experimental Example 3 Investigation of the Influence of Adding Nano-Micelle Stabilizer For the clevidipine butyrate micelle injection prepared by the present invention, adding nano-micelle stabilizers such as polyethylene glycol 15-hydroxystearate in the formulation can, on the one hand, shorten the dissolution time of clevidipine butyrate in the blank micelle preparation process and improve the physical and chemical stability of the mixed micelles.

[0162] By comparing Example 4 (not added), Example 5 (not added), Example 7 (added nano micelle solubilizer), and Example 11 (added nano micelle stabilizer on the basis of Example 7), the filtered liquid medicine was placed at room temperature. The physical stability was evaluated by observing whether crystals precipitated, and the dissolution time of clevidipine butyrate was compared. The results are shown in Table 8 and Table 9 below. The liquid medicine in Example 4 precipitated after being placed at room temperature for 8 h, and the liquid medicine in Example 5 precipitated after being placed for 4 h; while in Example 7, a small amount precipitated after being placed for 14 h, and in Example 11, no precipitation occurred after being placed for 24 h. Adding nano micelle solubilizer to the prescription can improve the drug loading capacity and stability at the same time, but adding nano micelle stabilizer on this basis can further improve the physical stability of the micelle injection. Compared with Example 4, the dissolution time of clevidipine butyrate in Example 7 and Example 11 was short, which can reduce the time-consuming of the preparation process by the blank micelle method.

[0163] Table 8 Results of Physical Stability Investigation Note: "-" indicates "no crystal precipitation", "+" indicates "crystal precipitation", and "N / A" indicates "not applicable".

[0164] Table 9 Results of Dissolution Time Investigation of Clevidipine Butyrate 。

[0165] Experimental Example 4 Low Temperature Cycling Test Take the clevidipine butyrate micelle injection prepared in Example 11 for the low temperature cycling test. The test should include 3 cycles. Each cycle is placed at 2 - 8 °C for 2 days, and then placed at 25 °C for 2 days. Samples are taken for testing after each round. The results are shown in Table 10.

[0166] It can be seen from the results in Table 10 that after 3 rounds, there was no precipitation phenomenon; compared with day 0, there were no significant changes in pH value, clarity and color, particle size, potential, content, related substances, etc., indicating that the prepared clevidipine butyrate micelle injection can withstand the low temperature cycling test and has good low temperature tolerance.

[0167] Note: Information of the color comparison solution used under the item of color: Name: Pharmacopoeia Standard Color Comparison Solution 2020 Edition, Number: TM - 2020 - 0901, Batch Number: C2011013, Manufacturer: Tanmo Quality Inspection Technology Co., Ltd.; The turbidity standard solution used for clarity was prepared with reference to the Chinese Pharmacopoeia 2020 Edition.

[0168] Table 10 Results of Low Temperature Cycling Test of Clevidipine Butyrate Micelle Injection in Example 11 。

[0169] Experimental Example 5 Freeze - Thaw Test The butyryl clonidine hydrochloride micellar injection prepared in Example 1, Example 6 (without adding nano micelle stabilizer and solubilizer), and Examples 7 - 11 was subjected to freeze - thaw test. The test should include 3 cycles. Each cycle was placed at - 10 to - 20 °C for 2 days, and then placed at 25 °C for 2 days. Samples were taken for detection after each round. The results are shown in Tables 11 and 12.

[0170] After 3 rounds of cycles, as can be seen from the results in Table 11, in Example 1, without adding nano micelle stabilizer and nano micelle solubilizer, crystals had precipitated in the 3rd round of freeze - thaw cycle; in Example 6, based on Example 1, the drug loading was increased to 4.2%, and crystals had precipitated in the 2nd round of freeze - thaw cycle; in Examples 7 - 8, only nano micelle solubilizer was added without adding nano micelle stabilizer, and crystals had precipitated in the 3rd round of freeze - thaw cycle, which could significantly increase the drug loading but had limited improvement in stability; when a certain amount of nano micelle stabilizer was added on the basis of containing nano micelle solubilizer in the prescription composition (Examples 9 - 11), no crystals had precipitated in 3 rounds. It can be seen that nano micelle stabilizer can further improve the freeze - thaw stability of the injection.

[0171] As can be seen from the results in Table 12, for the sample prepared in Example 11, no crystals had precipitated; compared with day 0, there were no significant changes in pH value, clarity and color, particle size, potential, content, related substances, etc. The Zeta potential diagrams of 3 rounds of Example 11 are as Figure 7 shown. The above results indicate that the butyryl clonidine hydrochloride micellar injection of the present invention can withstand the freeze - thaw test.

[0172] Table 11 Results of freeze - thaw test Note: "-" indicates "no crystal precipitation", and "+" indicates "crystal precipitation".

[0173] Table 12 Results of freeze - thaw test of Example 11 .

[0174] Experimental Example 6 Accelerated test and long - term test The stability of the butyryl clonidine hydrochloride mixed micellar injection in Examples 9 - 14 was studied under the following conditions. The appearance, properties, pH and Zeta potential changes were recorded, and the content and related substances of butyryl clonidine hydrochloride were determined according to Experimental Example 1 above. The results are shown in Table 13.

[0175] Long - term test: 5 °C ± 3 °C; Accelerated test: 25 °C ± 2 °C / 60% RH ± 5% RH.

[0176] Table 13 Results of stability investigation of accelerated test and long - term test Figure 8 Shown is the particle size distribution diagram of clevidipine butyrate micelle injection prepared in Example 9 under the accelerated condition for 3 months, indicating that after the prepared micelle injection was placed for 3 months under the accelerated condition, there was no significant change in the particle size compared with that at day 0.

[0177] Conclusion: As can be seen from the above table, after the clevidipine butyrate micelle injections of Examples 9 - 14 were placed for 6 months under the conditions of long-term stability and accelerated stability, there were no significant changes in indicators such as appearance, pH, potential, content, and related substances. The above results indicate that the clevidipine butyrate micelle injections of Examples 9 - 14 have good stability. The designed storage condition for the clevidipine butyrate micelle injection is to be stored at 2 - 8°C. According to the results of the accelerated stability experiment, the tentative expiration date of the clevidipine butyrate micelle injection is 2 years.

[0178] For those of ordinary skill in the art, the specific embodiments are only exemplary descriptions of the present invention. Obviously, the specific implementation of the present invention is not limited by the above methods. As long as various non-substantial improvements are made by adopting the method concept and technical solution of the present invention, or the concept and technical solution of the present invention are directly applied to other occasions without improvement, they are all within the protection scope of the present invention.

Claims

1. A clevidipine butyrate micellar injection, characterized in that: The invention comprises clevidipine butyrate, phospholipid, bile acid or its salt, and a pH adjuster.

2. The clevidipine butyrate micellar injection according to claim 1, characterized in that: The content of clevidipine butyrate in the injection is 1-10 mg / mL, bile acid or its salt is 30-100 mg / mL, the mass ratio of phospholipid to bile acid or its salt is 1:0.5-1.5, the pH regulator adjusts the pH of the injection to 6.0-7.5, and the solvent is water for injection.

3. The clevidipine butyrate micellar injection according to claim 1, characterized in that: The injection solution also contains a nanomicelle stabilizer and a nanomicelle solubilizer; the nanomicelle stabilizer is 0-20 mg / mL, and the nanomicelle solubilizer is 0-20 mg / mL.

4. The clevidipine butyrate micellar injection according to claim 3, characterized in that: The mass ratio of clevidipine butyrate to the nanomicelle stabilizer is 1:0.1-0.5; the mass ratio of clevidipine butyrate to the nanomicelle solubilizer is 1:0.1-0.

5.

5. The clevidipine butyrate micellar injection according to any one of claims 1 to 4, characterized in that: The specifications of clevidipine butyrate micellar injection are 1-10 mg / mL, pH is 6.0-7.5, particle size is 2-10 nm, Zeta potential is -20~-60mV, and transmittance is >90%.

6. The clevidipine butyrate micellar injection according to any one of claims 1 to 4, characterized in that: The phospholipid is one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, and synthetic lecithin; the bile acid or its salt is one or two of deoxycholic acid, glycocholic acid, sodium glycocholate, and sodium deoxycholate; the pH regulator is selected from at least one of an acidic pH regulator and an alkaline pH regulator; the acidic pH regulator includes one or more of citric acid, acetic acid, hydrochloric acid, and phosphoric acid, and the alkaline pH regulator is sodium hydroxide and / or sodium carbonate; the nanomicelle stabilizer is selected from one or more of 15-hydroxystearate polyethylene glycol ester, sodium oleate, poloxamer, polyoxyethylene castor oil, and polysorbate; the nanomicelle solubilizer is selected from one or more of vitamin E, oleic acid, and ethyl oleate.

7. The method for preparing the clevidipine butyrate micellar injection according to claim 1, characterized in that: The preparation method is a thin film dispersion method, which specifically comprises the following steps: (1) Film preparation: weigh the prescribed amount of clevidipine butyrate, phospholipids, bile acid or its salt, add an appropriate amount of organic solvent to dissolve, place on a rotary evaporator and perform rotary evaporation to obtain a loose film; the bile acid or its salt is one or both of deoxycholic acid and glycocholic acid; (2) Preparation of drug solution: adding an alkaline pH regulator in a molar ratio of 1:0.8-1.2 to the bile acid or its salt added in step (1), dissolving it completely with water for injection filled with protective gas accounting for 50-85% of the total volume of the injection solution, transferring the resulting solution to the loose film in step (1), and after complete hydration, adjusting the pH to 6.0-7.5 with a pH regulator to obtain a drug solution; (3) Volume or weight determination: The drug solution obtained in step (2) is volume or weight determined, filtered, and then filled with protective gas. The filtration is performed using a 0.22 μm filter membrane; (4) Sealing and sterilization: Packaging, filling the headspace with protective gas and then sealing, and autoclaving at 121°C for 15 min; When the formulation contains a nanomicelle stabilizer and / or a nanomicelle solubilizer, they are weighed and added in step (1).

8. The method for preparing the clevidipine butyrate micellar injection according to claim 7, characterized in that: When the bile acid or salt thereof used in step (1) is sodium deoxycholate or sodium glycocholate, or both, in step (2), the water for injection filled with protective gas accounting for 50-85% of the total volume of the injection solution is directly transferred to the loose film for complete hydration, and then the pH is adjusted to 6.0-7.5 with a pH regulator to obtain a drug solution; The rotary evaporation parameters in step (1) are: water bath temperature 40-60°C, time 2-6h; the organic solvent in step (1) is one or more of methanol, ethanol, and isopropanol in any proportion; the amount of the organic solvent is 2-8% of the total volume of the injection solution; the protective gas is any one of nitrogen, helium, carbon dioxide and argon, the time for passing the protective gas is 0.5-2h, the dissolved oxygen residual range is 0-5 mg / L, and the headspace residual oxygen content is controlled at 0-5%.

9. The method for preparing the clevidipine butyrate micellar injection according to claim 1, characterized in that: The preparation method is a blank micelle method, which specifically comprises the following steps: (1) Preparation of bile salt solution: When the bile acid or its salt is deoxycholic acid and / or glycocholic acid, add an alkaline pH regulator at a molar ratio of 1:0.8-1.2 to the bile acid, and completely dissolve it in water for injection filled with protective gas accounting for 50-85% of the total volume of the injection solution, and then adjust the pH of the solution to 6.0-7.5 with the pH regulator to obtain a bile salt solution; (2) Preparation of blank micelle solution: weigh the phospholipid in the prescription, add the bile salt solution in step (1), and stir in an oil bath at 90-110°C until the solution becomes clear, thereby obtaining a blank micelle solution; (3) Preparation of drug solution: Weigh clevidipine butyrate according to the prescription, slowly add it to the blank micelle solution obtained in step (2), heat and stir in an oil bath at 90-110°C until the solution is clear and transparent, cool to room temperature, and adjust the pH to 6.0-7.5 with a pH adjuster; (4) Fixing the volume or weight: Fixing the volume or weight of the drug solution obtained in step (3), filtering it, and then filling it with protective gas. The filtration is performed using a 0.22 μm filter membrane; (5) Sealing and sterilization: Packaging, sealing after filling the headspace with protective gas, and autoclave sterilization at 121°C for 15 min; When the formulation contains a nanomicelle stabilizer and / or a nanomicelle solubilizer, they are weighed and added together with the phospholipids in step (2).

10. The method for preparing the clevidipine butyrate micellar injection according to claim 9, characterized in that: When the bile acid or salt thereof used in step (1) is one or both of sodium deoxycholate and sodium glycocholate, the bile acid or salt thereof is directly dissolved in water for injection filled with protective gas accounting for 50-85% of the total volume of the injection solution in step (1), and then the pH is adjusted to 6.0-7.5 with a pH regulator to obtain a bile salt solution; The protective gas is any one of nitrogen, helium, carbon dioxide and argon. The time of introducing the protective gas is 0.5-2 h, the residual range of dissolved oxygen is 0-5 mg / L, and the residual oxygen content in the headspace is controlled at 0-5%.

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