Clevidipine butyrate micellar injection and preparation method thereof

Abstract

The present invention relates to the field of medical technology, and in particular to a clevidipine butyrate micelle injection and a preparation method thereof. The clevidipine butyrate micelle injection comprises clevidipine butyrate, phospholipids, bile acid or a salt thereof, a pH regulator and water for injection, and further contains a nano-micelle stabilizer and a nano-micelle solubilizer. Injection oils, such as soybean oil and triglycerides, are omitted from the prescription, thereby meeting the medication needs of patients with lipid metabolism defects. The solubility of clevidipine butyrate is greatly improved, and the nano-micelle stabilizer and the nano-micelle solubilizer are creatively added to further improve the stability and drug loading of the micelle injection. The blank micelle method is adopted to prepare the clevidipine butyrate micelle injection, thereby eliminating the use of organic solvents, and eliminating the need to use organic solvents with large toxic and side effects, such as dichloromethane, chloroform, and propylene glycol, thereby reducing the toxicity of the drug, minimizing vascular stimulation, and reducing adverse drug reactions. The production process of ordinary water injections is selected to facilitate production scale-up.

Description

Technical Field

[0001] The present invention relates to the technical field of medicine, and in particular to a clevidipine butyrate micellar injection and a preparation method thereof. Background Art

[0002] Clevidipine butyrate is a mixture of optical isomers. Its chemical name is 4-(2,3-dichlorophenyl)-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid methyl (1-butyryloxy) methyl ester. Its molecular formula is: C 21 H 23 Cl2NO6, molecular weight 456.32, structural formula is as follows:

[0003] .

[0004] Clevidipine butyrate is a dimethyl pyridinedicarboxylate, 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 identified, of which Form A accounts for >95%.

[0005] Clevidipine butyrate, a dihydropyridine derivative, is an ultra-short-acting intravenous calcium channel blocker. It is used as a short-term treatment for perioperative hypertension. It has high vascular and myocardial selectivity and is rapidly metabolized into inactive substances. Clevidipine butyrate has a strong pulse rate-lowering effect and also has a dilating effect on systemic and pulmonary vasculature. Clevidipine butyrate has a rapid onset of action and rapid elimination of its effects, allowing for precise blood pressure control with escalating doses. Unlike many current intravenous antihypertensive drugs that are metabolized by the kidneys and / or liver, it is metabolized in the blood and tissues and therefore does not accumulate in the body. An ideal treatment for perioperative hypertension requires parenteral administration, a rapid onset of action, and easy, rapid titration. The introduction of clevidipine butyrate injection fills this gap.

[0006] Clevidipine butyrate is a water-insoluble drug with a solubility of only 0.0001 mg / mL in water, which is even lower than the solubility of existing dihydropyridines. This makes the preparation of injectable formulations, especially those for intravenous administration, extremely difficult. To overcome the solubility issue of levidipine butyrate, a US pharmaceutical company, Medicine, developed Cleviprox®, a water-in-oil emulsion containing 0.5 mg / mL, using injectable soybean oil and other ingredients.

[0007] Domestic and foreign pharmaceutical companies or research institutions have conducted extensive research on clevidipine butyrate injection emulsion.

[0008] Patent document CN103110580A discloses a clevidipine butyrate injection, which includes clevidipine butyrate, an oil phase, a phospholipid, and an organic solvent. The organic solvent is ethanol, propylene glycol, glycerol, polyethylene glycol, etc. The phospholipid acts as an emulsifier, creating an anhydrous oil phase system. High concentrations of ethanol and propylene glycol organic solvents may cause pain and irritation at the injection site.

[0009] Patent document CN103237446 (US2011055617) discloses a clevidipine emulsion formulation containing an antimicrobial agent. The emulsion comprises clevidipine, an antimicrobial agent, a lipid, an emulsifier, an osmotic pressure regulator, and water. The osmotic pressure of the emulsion is adjusted with glycerol and the emulsion further comprises EDTA in an amount sufficient to inhibit microbial growth in the formulation in the event of accidental external contamination, with the specific gravity of the antimicrobial EDTA being 0.001 to 0.01% w / v. Pharmaceutical formulations containing clevidipine and an antimicrobial agent exhibit reduced propensity for microbial growth and provide increased convenience for healthcare workers when administering to patients. However, the lipids used in the above-mentioned technology are soybean oil, safflower seed oil, etc., and the emulsifiers are egg yolk phospholipids, etc., resulting in an oil-in-water emulsion with poor stability and significant safety risks.

[0010] Patent CN104473870A discloses a clevidipine butyrate lipid microsphere injection and its preparation method. The solution comprises clevidipine butyrate, an injection oil, an emulsifier, a stabilizer, an isotonic agent, a pH adjuster, and water for injection. The injection oil is soybean oil, and the isotonic agent is glycerol. The high-pressure microfluidization method is used for rapid preparation, involving high-pressure homogenization. This method results in high equipment costs and is difficult to scale up for large-scale production.

[0011] Patent document CN113197853 discloses a clevidipine butyrate injectable emulsion, its preparation method, and use. The preparation method comprises: shear mixing an oil phase and an aqueous phase to produce colostrum; and, after homogenizing the colostrum for a first time and a second time, mixing it with a pH adjuster. The oil phase comprises clevidipine butyrate or a pharmaceutically acceptable salt or hydrate thereof, an oil phase solvent, an emulsifier, and an emulsifier co-agent; and the aqueous phase comprises an osmotic pressure regulator, a stabilizer, and water. This preparation method utilizes a homogenizer for homogenization, which results in complex equipment, high costs, and difficulty in scale-up.

[0012] Currently, clevidipine butyrate fat emulsion injection offers a solution to the hydrolytic instability of clevidipine butyrate. Technical approaches to addressing drug oxidation primarily involve adding antioxidants, filling with inert gas, and changing packaging. However, in clinical applications, these protective measures may become ineffective due to the need to handle the emulsion injection, such as diluting the emulsion injection before administration, exacerbating the hydrolysis and further oxidation of clevidipine butyrate. Therefore, finding more effective methods to improve the stability of clevidipine butyrate and reduce or inhibit its hydrolytic and oxidative degradation remains a pressing challenge for formulations.

[0013] As a special preparation, the distribution of the emulsion size of clevidipine butyrate injection emulsion is directly related to its pharmacokinetics in the body, which in turn affects the efficacy. Since the diameter of human microvessels is about 4 to 9 μm and the diameter of pulmonary microvessels is about 5 μm, if a large number of emulsions are larger than 5 μm in diameter, it may cause pulmonary embolism, alveolar tissue damage, and even worse, the patient's death.

[0014] Publicly available patent documents address the issue of large milk particles, but the homogenization is performed using multiple or even eight high-pressure homogenizers connected in series, with the cost of one homogenizer often exceeding one million yuan, making the overall equipment cost relatively high. Secondly, ensuring that milk particles larger than 5 μm meet the standards is achieved by using excessive amounts of an emulsifier (i.e., oleic acid) and an antibacterial agent (i.e., disodium edetate) in the prescription. Oleic acid is easily oxidized to produce free fatty acids or other oxides, which are harmful to the body. Excessive use of disodium edetate can react with calcium ions in the blood, leading to a decrease in blood calcium.

[0015] The soybean oil used in the currently marketed clevidipine butyrate injection is long-chain triglycerides (LCTs). These long-chain triglycerides are hydrolyzed in the blood into long-chain fatty acids. These fatty acids are insoluble in water and require protein binding for cell entry. Once inside cells, these fatty acids must be activated in the cytoplasm. This is achieved by fatty acid sulfhydrylases on the outer membrane of the mitochondria or endoplasmic reticulum to convert them into the corresponding acyl-CoA. These fatty acids then undergo β-oxidation by carnitine acyltransferase on the inner mitochondrial membrane before entering the mitochondria. Therefore, long-chain triglycerides are easily phagocytosed and deposited in reticuloendothelial cells of organs such as the liver, spleen, and lungs, potentially compromising immune function. Medium-chain triglycerides (MCTs) are being used instead of long-chain triglycerides (LCTs) to mitigate the safety risks associated with long-chain triglycerides. However, the use of MCTs can also cause central nervous system effects, such as drowsiness, nausea, and sleepiness.

[0016] Therefore, it is necessary to develop a new type of clevidipine butyrate preparation to overcome the above-mentioned shortcomings and provide a safer injection with a higher concentration of clevidipine butyrate, which has become a technical problem to be solved urgently in this field. Summary of the Invention

[0017] In order to solve the problems in the prior art of clevidipine butyrate preparations, such as low solubility leading to low concentration of clevidipine butyrate, the need to use injection oils such as soybean oil as a raw material, the presence of large emulsions that easily lead to embolism, excessive oleic acid content, poor solubility and stability, etc., the present invention provides a high-concentration clevidipine butyrate micelle injection for intravenous drip and a preparation method thereof. The injection contains clevidipine butyrate, phospholipids, bile acid or a salt thereof, a pH regulator and water for injection, and can optionally contain a nanomicelle stabilizer and a nanomicelle solubilizer. The injection uses physiologically compatible excipients phospholipids and bile acid or its salts, adopts a safe micellar system for solubilization, and abandons injection oils such as soybean oil and triglycerides in the prescription, meeting the medication needs of patients with lipid metabolism defects, such as pathological hyperlipidemia, lipoid nephropathy, and acute pancreatitis accompanied by hyperlipidemia. It not only greatly improves the solubility of clevidipine butyrate, but also creatively adds nano-micelle stabilizers and nano-micelle solubilizers to further improve the stability and drug loading of the micelle injection. The preparation process of the clevidipine butyrate micelle injection of the present invention, the selected blank micelle method abandons the use of organic solvents, does not require the use of 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; the production process of ordinary water injection is used to facilitate production and scale-up.

[0018] The present invention first provides a clevidipine butyrate micellar injection, the main raw materials of which include clevidipine butyrate, phospholipid, bile acid or its salt, and a pH regulator, and the solvent used is water for injection.

[0019] In the above injection, the concentration of clevidipine butyrate is 1-10 mg / mL, the concentration of 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), and the pH regulator is used to adjust the pH of the injection to 6.0-7.5.

[0020] As a preferred technical solution, the injection solution also contains a nanomicelle stabilizer and a nanomicelle solubilizer; wherein the nanomicelle stabilizer is 0-20 mg / mL, and the nanomicelle solubilizer is 0-20 mg / mL.

[0021] After being made into the finished product, the volume specification of the prepared clevidipine butyrate micellar injection is 1-10 mg / mL, pH is 6.0-7.5, particle size is 2-10 nm, Zeta potential is -20~-60 mV, and transmittance is greater than 90%; the volume specification is preferably 5 mL, and it is placed in an ampoule or a vial.

[0022] In certain preferred embodiments, the phospholipid is 30-100 mg / mL.

[0023] When the injection further contains a nanomicelle stabilizer and a nanomicelle solubilizer, the preferred mass ratio of clevidipine butyrate to the nanomicelle stabilizer is 1:(0.1-0.5); the preferred mass ratio of clevidipine butyrate to the nanomicelle solubilizer is 1:(0.1-0.5).

[0024] The nanomicelle stabilizer includes one or more of 15-hydroxystearate polyethylene glycol ester, sodium oleate, poloxamer, polyoxyethylene castor oil, and polysorbate, preferably 15-hydroxystearate polyethylene glycol ester; the nanomicelle solubilizer includes one or more of vitamin E, oleic acid, and ethyl oleate; the nanomicelle stabilizer can form a hydrophilic layer on the micelle surface or increase the potential, thereby improving the stability of the clevidipine butyrate micelle injection, and the nanomicelle solubilizer can further increase the drug loading capacity of the clevidipine butyrate micelle injection; the phospholipid is one or more of soybean lecithin, egg yolk lecithin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin, and synthetic phospholipid, preferably soybean lecithin; optionally, the bile acid or its salt is one or two of deoxycholic acid, glycocholic acid, sodium glycocholate, and sodium deoxycholate.

[0025] In the present invention, a pH adjuster is added to adjust the pH of the injection to 6.0-7.5. The pH adjuster includes at least one of an acidic pH adjuster and an alkaline pH adjuster. The acidic pH adjuster includes one or more of citric acid, acetic acid, hydrochloric acid, and phosphoric acid, more preferably citric acid. The alkaline pH adjuster is sodium hydroxide and / or sodium carbonate.

[0026] The present invention further provides a method for preparing the clevidipine butyrate micellar injection, which is a thin film dispersion method and specifically comprises the following steps:

[0027] (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.

[0028] (2) Preparation of drug solution: add 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), and completely dissolve it with water for injection filled with protective gas accounting for 50-85% of the total volume of the injection solution. Transfer the resulting solution to the loose film in step (1). After complete hydration, adjust the pH to 6.0-7.5 with a pH regulator to obtain a drug solution.

[0029] (3) Volume or weight determination: The liquid 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.

[0030] (4) Sealing and sterilization: Pack the contents separately, fill the headspace with protective gas and then seal, then sterilize by autoclave at 121°C for 15 min.

[0031] When the formulation contains a nanomicelle stabilizer and / or a nanomicelle solubilizer, they are weighed and added in step (1).

[0032] When the bile acid or its salt used in step (1) is one or both of sodium deoxycholate and sodium glycocholate, the inventors adjust step (2) by directly transferring the protective gas-filled water for injection, which accounts for 50-85% of the total volume of the injection solution, to the loose film for complete hydration, and then adjusting the pH to 6.0-7.5 with a pH regulator to obtain the drug solution.

[0033] 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; the amount of the organic solvent is 2-8% of the total volume of the injection preparation; as a further preferred embodiment, the organic solvent is a mixed solvent of methanol and ethanol, and the volume ratio of methanol to ethanol is 2:1.

[0034] 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 dissolved oxygen range is 0-5 mg / L, and the residual oxygen content in the headspace is controlled at 0-5%.

[0035] The present invention also provides another method for preparing the clevidipine butyrate micellar injection, which is a blank micelle method and specifically comprises the following steps:

[0036] (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 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. Then, adjust the pH of the solution to 6.0-7.5 with the pH regulator to obtain a bile salt solution.

[0037] (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 to obtain a blank micelle solution.

[0038] (3) Preparation of drug solution: Weigh clevidipine butyrate according to the prescription and 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. After cooling to room temperature, adjust the pH to 6.0-7.5 with a pH adjuster.

[0039] (4) Volume or weight determination: The liquid obtained in step (3) is volume or weight determined, filtered, and then filled with protective gas. The filtration is performed using a 0.22 μm filter membrane.

[0040] (5) Sealing and sterilization: Divide the contents into smaller pieces, fill the headspace with protective gas, and then seal the contents. Sterilize by autoclave at 121°C for 15 min.

[0041] When the formulation contains a nanomicelle stabilizer and / or a nanomicelle solubilizer, they are weighed and added together with the phospholipids in step (2).

[0042] When the bile acid or its salt used in step (1) is one or both of sodium deoxycholate and sodium glycocholate, the inventors adjusted the step (1) by directly dissolving the bile acid or its salt in water for injection filled with protective gas accounting for 50-85% of the total volume of the injection preparation, and then adjusting the pH to 6.0-7.5 with a pH regulator to obtain a bile salt solution.

[0043] Preferably, the protective gas used for injection, the protective gas used after the liquid is fixed to volume, and the protective gas used before sealing is any one of nitrogen, helium, carbon dioxide and argon. The time for passing the protective gas is 0.5-2 h, the residual dissolved oxygen range is 0-5 mg / L, and the residual oxygen content in the headspace is controlled at 0-5%. As a further preference, the protective gas is nitrogen.

[0044] The obtained clevidipine butyrate micellar injection is a dihydropyridine calcium channel blocker and is suitable for treating hypertension when oral antihypertensive drugs are not applicable or cannot achieve satisfactory therapeutic effects.

[0045] In specific applications, the dilution injections that can be used in combination with the clevidipine butyrate micellar injection provided by the present invention include but are not limited to water for injection, sodium chloride injection (0.9%), glucose injection (5%), glucose (5%) sodium chloride (0.9%) injection, 10% amino acids, and lactated Ringer's injection. As one of the preferred embodiments, glucose injection (5%) or sodium chloride injection (0.9%) is preferred.

[0046] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0047] (1) Improve medication safety:

[0048] 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 drugs are wrapped in a hydrophobic core. On this basis, nano-micelle stabilizers and nano-micelle solubilizers are creatively added. The addition of the nano-micelle stabilizer can form a hydrophilic layer on the micelle surface or increase the Zeta potential, reduce aggregation between particles or increase the Zeta potential of the micelle particles, thereby achieving the purpose of further improving the stability of the micelle system. The addition of the solubilizer can further improve the drug loading capacity of the micelle.

[0049] Fat emulsion injections in commercially available injections (such as Cleviprex®) and other literature patents use oil-phase solvents and add phospholipids, bile acid, etc. as emulsifiers or stabilizers, and vitamin E is used as an antioxidant. The functions of these components in the present invention are significantly different (the present invention is a mixed micelle). The present invention avoids the use of large amounts of soybean oil as a solvent, thereby avoiding the increase in blood lipid levels in hyperlipidemia patients caused by injection infusion, thereby avoiding cardiovascular damage to hyperlipidemia patients, meeting the medication needs of patients with lipid metabolism defects, and significantly improving the medication safety of special patients with hyperlipidemia.

[0050] (2) Avoiding large emulsions and reducing medication risks: The clevidipine butyrate micellar injection prepared by the present invention is a sterilized clear solution. It does not have the phenomena of emulsion rupture, droplet aggregation and merging, particle size increase, and local demulsification that are common in marketed emulsions. It avoids large emulsions and the adverse reactions caused by large emulsions. The main manifestations are:

[0051] a) To prevent large particles in the injected fat emulsion from entering the lungs during the injection process and causing granulomas in the lungs;

[0052] b) Avoid large particles entering blood vessels that may cause granuloma, phlebitis, and thrombosis;

[0053] c) Avoid large particles from entering other organs of the body, such as the liver and kidneys, through blood circulation, causing damage;

[0054] d) The present invention provides a micellar injection solution that is clear and easier to observe for visible foreign matter and insoluble particles than an emulsion, allowing for early prediction of medication use and reducing medication risks.

[0055] e) The particle size of the clevidipine butyrate micelle injection prepared by the present invention is very small, 2-10 nm, and can penetrate biological membranes and be retained in tissues, thereby enhancing its aggregation in tissues and improving drug efficacy.

[0056] (3) Improved compliance: The clevidipine butyrate micellar injection prepared by the present invention can be directly diluted with sterile water for injection, 0.9% sodium chloride injection, and 5% glucose injection to obtain a drug concentration of 0.01-1 mg / mL during clinical use. The drug concentration prepared by the compatible injection and the controlled drug delivery rate do not require a Y-shaped infusion set or a "special infusion device with precise dosage adjustment." The drug delivery rate is controlled within the range of 1-32 mg / h, making the clinical delivery method more flexible and convenient, and the dosage more accurate and controllable.

[0057] (4) Increase drug loading:

[0058] In the system of the present invention, clevidipine butyrate is dissolved in phospholipid-bile salt mixed micelles, and a fat-soluble nano-micelle solubilizer is creatively added to the formulation to further improve the drug loading capacity of the mixed micelles;

[0059] The clevidipine butyrate micellar injection prepared by this invention has a concentration of up to 10 mg / mL, significantly improving drug solubility compared to the commercially available injection (0.5 mg / mL). During clinical administration, the total infusion volume of this preparation is 1 / 2-1 / 20 of that of Cleviprex®, eliminating or reducing the risk of extravasation of hypertonic fat emulsion injections and significantly reducing the risk of adverse reactions such as phlebitis, thus offering greater clinical value.

[0060] (5) Improve stability:

[0061] A large number of experiments have shown that the addition of nanomicelle stabilizers to the system of the present invention can achieve unexpected results. The addition of stabilizers can form a hydrophilic layer on the surface of the micelles, reducing the aggregation between particles, or the addition of sodium oleate can increase the Zeta potential of the micelle particles, both of which can achieve the purpose of improving the stability of the micelle injection, especially significantly improving the compatibility stability of the injection.

[0062] Intravenous emulsions fall into the category of submicroemulsions in terms of pharmaceutics, with average particle sizes generally ranging from 100 to 500 nm, making them thermodynamically unstable systems. While Cleviprex® has a shelf life of no more than two months at room temperature, the clevidipine butyrate micellar injection prepared by the present invention has been tested for freeze-thaw cycles, low-temperature cycles, and compatibility stability, and after three months at room temperature, the solution remains clear and free of precipitated crystals. This overcomes the disadvantage of fat emulsions that they cannot be frozen for long periods or even temporarily, and improves ease of transportation, storage, and use. The clevidipine butyrate micellar injection prepared by the present invention creatively incorporates a nano-micelle stabilizer, significantly improving the stability of the formulation. Terminal sterilization at 121°C / 15 minutes produces a stable, clear solution, effectively preventing droplet aggregation, particle size enlargement, and localized demulsification that can occur after sterilization and long-term storage of intravenous emulsions.

[0063] (6) The preparation process is simple and easy to scale up: The existing fat emulsion preparation process requires complex processes such as high-pressure homogenizers to obtain emulsion particles, and process-related quality control is difficult and costly. However, the system of the present invention does not require special equipment and is easy to control the process. In particular, the blank micelle method can be used to select the production route of ordinary water injections. Therefore, the process provided by the present invention is simple, the production cost is low, and it is easy to implement. Furthermore, the existing published patent literature uses organic solvents such as dichloromethane, chloroform, and propylene glycol with large toxic side effects, which are not environmentally friendly and hinder scale-up production. The present invention does not require or use a small amount of methanol or ethanol, which is conducive to scale-up production. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 This is a diagram showing the appearance of the clevidipine butyrate mixed micelle injection prepared by the thin film dispersion method in Example 1;

[0065] Figure 2 The particle size distribution diagram of clevidipine butyrate micellar injection prepared by thin film dispersion method in Example 1 is shown in FIG. Figure 2 a is Figure 2 b shows the average particle size, aggregation index and intercept of the particle size distribution, Figure 2 b is the particle size distribution diagram;

[0066] Figure 3 This is the Zeta potential diagram of the clevidipine butyrate micelle injection prepared by the blank micelle method in Example 7, wherein Figure 3 a is Figure 3 b Zeta potential, Zeta potential standard deviation and conductivity of the Zeta potential distribution, Figure 3 b is the Zeta potential distribution diagram;

[0067] Figure 4 This is a high performance liquid chromatogram of the determination of related substances of the clevidipine butyrate micellar injection prepared by the blank micelle method in Example 3 of Experimental Example 1;

[0068] Figure 5 This is the content change curve (0.25 mg / mL and 0.1 mg / mL) of the clevidipine butyrate micellar injection prepared by the blank micelle method in Example 11 of Experimental Example 2 when investigating the compatibility stability (0.9% sodium chloride injection);

[0069] Figure 6 This is the content change curve (0.25 mg / mL and 0.1 mg / mL) of the clevidipine butyrate micellar injection prepared by the blank micelle method in Example 11 of Experimental Example 2 when conducting a compatibility stability study (5% glucose injection);

[0070] Figure 7This is the Zeta potential diagram of the freeze-thaw cycle test of the clevidipine butyrate micelle injection prepared by the blank micelle method in Example 11 of Experimental Example 5, wherein Figure 7 a is Figure 7 b Zeta potential, Zeta potential standard deviation and conductivity of the Zeta potential distribution, Figure 7 b is the Zeta potential distribution diagram;

[0071] Figure 8 This is the particle size distribution diagram of the accelerated / 3-month clevidipine butyrate micellar injection prepared by the blank micelle method in Example 9 of Experimental Example 6, wherein Figure 8 a is Figure 8 b shows the average particle size, aggregation index and intercept of the particle size distribution, Figure 8 b is the particle size distribution diagram. DETAILED DESCRIPTION

[0072] The following examples are used to illustrate the present invention but are not intended to limit the scope of the present invention. Where specific techniques or conditions are not specified in the examples, the techniques or conditions described in the literature in the art or the product instructions were used.

[0073] The water for injection and the liquid medicine after volume adjustment used in the examples were filled with protective gas, and the dissolved oxygen was controlled within the range of 0-5 mg / L. The residual oxygen content in the headspace after packaging and before sealing was controlled within the range of 0-5%.

[0074] Example 1 A clevidipine butyrate micellar injection, the raw material formula of which is as follows:

[0075] Clevidipine butyrate 500 mg, soybean lecithin 5.33 g, glycocholic acid 6.67 g, sodium hydroxide 0.57 g, add water for injection to 100 mL.

[0076] The thin film dispersion method is adopted, and the preparation method includes the following steps:

[0077] (1) Weigh the prescribed amount of clevidipine butyrate, soybean lecithin, and glycocholic acid, add 5 mL of methanol and 2 mL of anhydrous ethanol to dissolve them completely, and then rotary evaporate them in a 40°C water bath for 4 h to obtain a loose film.

[0078] (2) Take the prescribed amount of sodium hydroxide, add 80 mL of nitrogen-filled water for injection, stir to dissolve, transfer to the loose film obtained in step (1), stir at 300 rpm and 40°C in a water bath for 30 minutes to completely hydrate it, and adjust the pH value to 6.5 with 1 mol / L sodium hydroxide solution and 0.5 mol / L citric acid solution.

[0079] (3) The solution obtained in (2) was diluted to 100 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0080] (4) Fill the ampoule with nitrogen (20 s / ampule), divide the volume into 5 mL / ampule, seal the ampule, and sterilize by autoclave at 121°C for 15 min.

[0081] like Figure 1 As shown, the clevidipine butyrate micellar injection obtained in Example 1 is clear and transparent, light yellow-green in color, and has a pH of 6.6.

[0082] like Figure 2 As shown, the Z-Average of the mixed micelle solution is 2.655 nm, the PDI is 0.296, and the Zeta potential is -36.8 mV.

[0083] Example 2 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0084] Clevidipine butyrate 2 g, soybean lecithin 25.66 g, deoxycholic acid 25.66 g, sodium hydroxide 2.32 g, add water for injection to 400 mL.

[0085] The thin film dispersion method is adopted, and the preparation method includes the following steps:

[0086] (1) Weigh the prescribed amount of clevidipine butyrate, soybean lecithin, and deoxycholic acid, add 20 mL of methanol and 10 mL of anhydrous ethanol, dissolve completely, place on a rotary evaporator, and evaporate in a 40°C water bath for 4 h to obtain a loose film.

[0087] (2) Weigh the prescribed amount of sodium hydroxide, add 350 mL of nitrogen-deoxygenated water for injection, stir until completely dissolved, transfer to the loose film obtained in (1), stir in a water bath at 300 rpm and 35°C for 30 min to completely hydrate it, and adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0088] (3) The solution obtained in (2) was diluted to 400 mL, filtered through a 0.22 μm pore size filter membrane, and filled with nitrogen.

[0089] Step (4) is the same as in Example 1.

[0090] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.560 nm, a PDI of 0.264, a Zeta potential of -36.5 mV, and a pH of 6.7.

[0091] Example 3 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0092] Clevidipine butyrate 1.5 g, soybean lecithin 23.98 g, sodium glycocholate 17.38 g, add water for injection to 300 mL.

[0093] The blank micelle method is adopted, and the preparation method includes the following steps:

[0094] (1) Weigh the prescribed amount of sodium glycocholate, add 160 mL of nitrogen-deoxygenated water for injection, stir for 15 minutes to dissolve, and adjust the pH to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0095] (2) Weigh the soybean lecithin in the prescription and add it to the solution in step (1). Heat it in an oil bath at 100°C and stir it at 400 rpm for 1 hour. The solution will become clear to obtain a blank micelle solution.

[0096] (3) Weigh the prescribed amount of clevidipine butyrate and add it to the blank micellar solution obtained in step (2). Heat and stir in an oil bath at 100°C until the API is completely dissolved and the solution is clear. Cool to room temperature and adjust the pH to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0097] (4) The volume of the solution prepared in step (3) was adjusted to 300 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0098] (5) Aliquot (5 mL / ampoule), fill the headspace of the ampoule with nitrogen (10 s / ampoule), seal it, and sterilize it by autoclave at 121°C for 15 min.

[0099] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.763 nm, a PDI of 0.261, a Zeta potential of -39.0 mV, and a pH of 7.0.

[0100] Example 4 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0101] Clevidipine butyrate 1.25 g, soybean lecithin 17.96 g, glycocholic acid 22.45 g, sodium hydroxide 2.02 g, add water for injection to 250 mL.

[0102] The blank micelle method is adopted, and the preparation method includes the following steps:

[0103] (1) Weigh the prescribed amount of glycocholic acid and sodium hydroxide, add 160 mL of nitrogen-deoxygenated water for injection, stir for 15 minutes to dissolve, and adjust the pH to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0104] Steps (2) and (3) were carried out in the same manner as described in Example 3.

[0105] (4) The solution prepared in step (3) was diluted to 250 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0106] (5) Aliquot (5 mL / ampoule), fill the headspace of the ampoule with nitrogen (10 s / ampoule), seal it, and sterilize it by autoclave at 121°C for 15 min.

[0107] The injection is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.725 nm, a PDI of 0.293, a Zeta potential of -38.3 mV, and a pH of 7.0.

[0108] Example 5 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0109] Clevidipine butyrate 500 mg, soybean lecithin 6.34 g, glycocholic acid 5.07 g, sodium hydroxide 0.46 g, add water for injection to 100 mL.

[0110] The preparation method is a thin film dispersion method, and the specific implementation steps are the same as those in Example 1.

[0111] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.065 nm, a PDI of 0.203, a Zeta potential of -39.6 mV, and a pH of 6.5.

[0112] Example 6 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0113] Clevidipine butyrate 500 mg, soybean lecithin 5.71 g, glycocholic acid 5.71 g, sodium hydroxide 0.52 g, add water for injection to 100 mL.

[0114] The blank micelle method is adopted, and the preparation method includes the following steps:

[0115] (1) Weigh the prescribed amount of glycocholic acid and sodium hydroxide, add 80 mL of nitrogen-deoxygenated water for injection, stir to dissolve, and adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0116] (2) Weigh the soybean lecithin in the prescription and add it to the solution in step (1). Heat and stir in an oil bath at 100°C until the solution becomes clear to obtain a blank micelle solution.

[0117] (3) Weigh the prescribed amount of clevidipine butyrate and add it to the blank micellar solution in step (2). Heat and stir in an oil bath at 90°C until the API is completely dissolved and the solution is clear. Cool to room temperature and adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0118] (4) The volume of the solution prepared in step (3) was adjusted to 100 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0119] (5) Aliquot (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it, and sterilize it by autoclave at 121°C for 15 min.

[0120] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 1.985 nm, a PDI of 0.209, a Zeta potential of -40.8 mV, and a pH of 6.5.

[0121] Example 7 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0122] Clevidipine butyrate 1 g, soybean lecithin 11.79 g, sodium glycocholate 9.43 g, vitamin E 200 mg, add water for injection to 200 mL.

[0123] The blank micelle method is adopted, and the preparation method includes the following steps:

[0124] (1) Weigh the prescribed amount of sodium glycocholate, add 160 mL of nitrogen-deoxygenated water for injection, stir for 20 minutes to dissolve, and adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0125] (2) Weigh the soybean lecithin 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 300 rpm until the solution becomes clear to obtain a blank micelle solution.

[0126] (3) Weigh the prescribed amount of clevidipine butyrate and add it to the blank micelle solution prepared in step (2). Heat and stir in an oil bath at 100°C until the solution becomes clear and translucent. Cool to room temperature and adjust the pH to 6.5.

[0127] (4) The volume of the solution prepared in step (3) was adjusted to 200 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0128] (5) Aliquot (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it, and sterilize it by autoclave at 121°C for 15 min.

[0129] The injection solution is clear, transparent, and light yellow-green. The Z-Average of the mixed micelle solution is 2.766 nm, the PDI is 0.239, and the pH is 6.5; Figure 3 As shown, the zeta potential is -48.6 mV.

[0130] Example 8 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0131] Clevidipine butyrate 1 g, soybean lecithin 9.5 g, sodium glycocholate 9.5 g, oleic acid 200 mg, add water for injection to 200 mL.

[0132] The thin film dispersion method is adopted, and the preparation method includes the following steps:

[0133] (1) Weigh the prescribed amount of clevidipine butyrate, soybean lecithin, sodium glycocholate, and oleic acid, add 5 mL of methanol and 2 mL of anhydrous ethanol to dissolve completely, place on a rotary evaporator, and evaporate in a 50°C water bath for 3 h to obtain a loose film.

[0134] (2) Add 160 mL of nitrogen-filled water for injection to the obtained loose film and stir to completely hydrate it. Adjust the pH value to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0135] (3) After pH adjustment, the solution was diluted to 200 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0136] (4) Aliquot (10 mL / vial), fill the vial with nitrogen, then cap and seal, and sterilize by autoclaving at 121°C for 15 min.

[0137] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.986 nm, a PDI of 0.293, a Zeta potential of -45.6 mV, and a pH of 6.4.

[0138] Example 9 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0139] Clevidipine butyrate 1 g, egg yolk lecithin 10.31 g, glycocholic acid 10.31 g, sodium hydroxide 0.93 g, vitamin E 200 mg, 15-hydroxystearate polyethylene glycol 400 mg, add water for injection to 200 mL.

[0140] The blank micelle method is adopted, and the preparation method includes the following steps:

[0141] (1) Weigh the prescribed amount of glycocholic acid and sodium hydroxide, add 160 mL of nitrogen-deoxygenated water for injection, stir for 20 minutes to dissolve, and adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0142] (2) Weigh the egg yolk phospholipids, vitamin E, and 15-hydroxystearic acid polyethylene glycol ester in the prescription, add the solution prepared in step (1), heat in an oil bath at 100°C, and stir at 300 rpm until the solution becomes clear to obtain a blank micelle solution.

[0143] The remaining steps are the same as those in Example 7.

[0144] The resulting injection solution was clear, transparent, and pale yellow-green. The mixed micelle solution had a Z-Average of 2.726 nm, a PDI of 0.287, a Zeta potential of -37.6 mV, and a pH of 6.5.

[0145] Example 10 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0146] Clevidipine butyrate 1 g, soybean lecithin 9.5 g, glycocholic acid 9.5 g, sodium hydroxide 0.87 g, vitamin E 200 mg, sodium oleate 200 mg, add water for injection to 200 mL.

[0147] The thin film dispersion method is adopted, and the preparation method includes the following steps:

[0148] (1) According to the prescription, clevidipine butyrate, soybean lecithin, glycocholic acid, vitamin E and sodium oleate were weighed and added with 5 mL of methanol and 2 mL of anhydrous ethanol to dissolve completely. The mixture was placed on a rotary evaporator and evaporated in a water bath at 50°C for 4 h to obtain a loose film.

[0149] (2) Take the prescribed amount of sodium hydroxide, add 160 mL of nitrogen-filled water for injection, stir to dissolve, transfer to the loose film obtained in step (1), stir at 30°C for 1 hour 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.

[0150] The remaining steps are the same as in Example 8.

[0151] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.853 nm, a PDI of 0.275, a Zeta potential of -36.7 mV, and a pH of 6.5.

[0152] Example 11 A clevidipine butyrate micellar injection, the raw material formula of which is as follows:

[0153] Clevidipine butyrate 5 g, soybean lecithin 51.53 g, glycocholic acid 41.22 g, sodium hydroxide 3.72 g, vitamin E 1.0 g, 15-hydroxystearate polyethylene glycol ester 1.25 g, add water for injection to 1000 mL.

[0154] The blank micelle method is adopted, and the preparation method includes the following steps:

[0155] (1) Weigh the glycocholic acid and sodium hydroxide in the prescription, add 800 mL of nitrogen-deoxygenated water for injection, stir for 20 minutes 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.

[0156] (2) Weigh the soybean lecithin, 15-hydroxystearic acid polyethylene glycol ester, and vitamin E in the prescription, add the solution prepared in step (1), heat in an oil bath at 100°C, and stir at 400 rpm until the solution becomes clear to obtain a blank micelle solution.

[0157] (3) Weigh the prescribed amount of clevidipine butyrate and add it to the blank micelle solution prepared in step (2). Heat and stir in an oil bath at 100°C until the solution becomes clear and translucent. Cool to room temperature and adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0158] (4) The volume of the solution prepared in step (3) was adjusted to 1000 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0159] (5) Aliquot (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it, and sterilize it by autoclave at 121°C for 15 min.

[0160] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.906 nm, a PDI of 0.330, a Zeta potential of -38.6 mV, and a pH of 6.6.

[0161] Example 12 A clevidipine butyrate micellar injection, the raw material formula of which is as follows:

[0162] Clevidipine butyrate 1 g, soybean lecithin 11.79 g, sodium deoxycholate 9.43 g, poloxamer 300 mg, vitamin E 200 mg, add water for injection to 200 mL.

[0163] The thin film dispersion method is adopted, and the preparation method includes the following steps:

[0164] (1) Weigh the prescribed amount of clevidipine butyrate, soybean lecithin, sodium deoxycholate, poloxamer, and vitamin E, add 5 mL of methanol and 2 mL of anhydrous ethanol to dissolve completely, place on a rotary evaporator, and evaporate in a 50°C water bath for 5 h to obtain a loose film.

[0165] (2) Add 160 mL of nitrogen-filled water for injection to the resulting loose film and stir at 25°C for 1 h to completely hydrate it. Adjust the pH to 6.5 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0166] (3) After pH adjustment, the solution was diluted to 200 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0167] (4) Repackaging (5 mL / vial), fill the headspace of the ampoule with nitrogen and seal it, then sterilize by autoclaving at 121°C for 15 min.

[0168] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 3.065 nm, a PDI of 0.196, a Zeta potential of -48.5 mV, and a pH of 6.5.

[0169] Example 13 A clevidipine butyrate micellar injection, the raw material formula of which is as follows:

[0170] Clevidipine butyrate 1 g, soybean lecithin 9.5 g, deoxycholic acid 9.5 g, sodium hydroxide 0.87 g, polyoxyethylene castor oil 500 mg, ethyl oleate 200 mg, add water for injection to 200 mL.

[0171] The blank micelle method is adopted, and the preparation method includes the following steps:

[0172] (1) Weigh deoxycholic acid and sodium hydroxide in the prescription, add 160 mL of nitrogen-deoxygenated water for injection, stir for 20 minutes to dissolve, and adjust the pH to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0173] (2) Weigh the soybean lecithin, polyoxyethylene castor oil, and ethyl oleate in the prescription, add the solution prepared in step (1), heat in an oil bath at 100°C, and stir at 300 rpm until the solution becomes clear to obtain a blank micelle solution.

[0174] (3) Weigh the prescribed amount of clevidipine butyrate and add it to the blank micelle solution prepared in step (2). Heat and stir in an oil bath at 100°C until the solution becomes clear and translucent. Cool to room temperature and adjust the pH to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0175] (4) The volume of the solution prepared in step (3) was adjusted to 200 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0176] (5) Aliquot (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it, and sterilize it by autoclave at 121°C for 15 min.

[0177] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 3.012 nm, a PDI of 0.129, a Zeta potential of -45.2 mV, and a pH of 7.0.

[0178] Example 14 A micellar injection of clevidipine butyrate, the raw material formula of which is as follows:

[0179] Clevidipine butyrate 2.5 g, hydrogenated soybean lecithin 27.95 g, sodium glycocholate 18.63 g, vitamin E 0.300 g, polysorbate 80 0.625 g, add water for injection to 333 mL.

[0180] The blank micelle method is adopted, and the preparation method includes the following steps:

[0181] (1) Weigh sodium glycocholate in the prescription, add 280 mL of nitrogen-deoxygenated water for injection, 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.

[0182] (2) Weigh the hydrogenated soybean lecithin, polysorbate 80, and vitamin E in the prescription, add the solution prepared in step (1), heat in an oil bath at 100°C, and stir at 300 rpm until the solution becomes clear, thereby obtaining a blank micelle solution.

[0183] (3) Weigh the prescribed amount of clevidipine butyrate and add it to the blank micelle solution prepared in step (2). Heat and stir in an oil bath at 100°C until the solution becomes clear and translucent. Cool to room temperature and adjust the pH to 7.0 with 0.5 mol / L citric acid solution and 1 mol / L sodium hydroxide solution.

[0184] (4) The solution prepared in step (3) was diluted to 333 mL, filtered through a 0.22 μm filter membrane, and filled with nitrogen.

[0185] (5) Aliquot (5 mL / vial), fill the headspace of the ampoule with nitrogen, seal it, and sterilize it by autoclave at 121°C for 15 min.

[0186] The injection solution is clear, transparent, and pale yellow-green. The mixed micelle solution has a Z-Average of 2.506 nm, a PDI of 0.189, a Zeta potential of -40.6 mV, and a pH of 7.0.

[0187] Experimental Example 1 Determination of drug loading, content, related substances, and residual solvents:

[0188] The drug loading, content, related substances, and residual solvents were determined by the following methods.

[0189] 1. Drug loading capacity:

[0190] Clevidipine butyrate / (the sum of the mass of levidipine butyrate and excipients) × 100%.

[0191] 2. Content determination method (the ratios of the mobile phase, reference solution and test solution mentioned below are all by volume):

[0192] Chromatographic conditions: octadecylsilane bonded silica gel was used as the filler (Sunniest C18, specifications: 250×4.6 mm, 5 μm); methanol-acetonitrile-10 mM NaH2PO4 (weigh approximately 1.2 g of anhydrous sodium dihydrogen phosphate, dissolve it in water and dilute to 1000 mL, and adjust the pH to 3.0 with phosphoric acid) (25:25:50) was used as the mobile phase A, and acetonitrile-water (90:10) was used as the mobile phase B (mobile phase A-mobile phase B, 20:80); the detection wavelength was 239 nm; the injection volume was 10 μL.

[0193] Reference solution: Take approximately 25 mg of clevidipine butyrate reference substance and place it in a 50 mL volumetric flask. Add methanol and shake to dissolve and dilute to the scale. Shake well. Accurately measure 2 mL and place it in a 20 mL volumetric flask. Dilute to the scale with solvent (methanol-acetonitrile-water, 40:40:20) and shake well.

[0194] Test solution: Accurately measure 2 mL of the test solution under the relevant substance, place it in a 20 mL volumetric flask, dilute to the scale with solvent (methanol-acetonitrile-water, 40:40:20), and shake well.

[0195] 3. Testing methods for related substances:

[0196] Chromatographic conditions: Octadecylsilane bonded silica gel was used as the filler (Sunniest C18, specifications: 250 × 4.6 mm, 5 μm); methanol-acetonitrile-10 mM NaH2PO4 (weigh approximately 1.2 g of anhydrous sodium dihydrogen phosphate, dissolve in water and dilute to 1000 mL, and adjust the pH to 3.0 with phosphoric acid) (25:25:50) was used as the mobile phase A, and acetonitrile-water (90:10) was used as the mobile phase B; the detection wavelength was 239 nm; the injection volume was 20 μl, and gradient elution was performed according to the method shown in Table 1.

[0197] Table 1 Gradient elution table of the test method for related substances of clevidipine butyrate micellar injection

[0198]

[0199] Test solution: Accurately measure 1 mL of the product and place it in a 10 mL volumetric flask, dilute to the mark with methanol, and shake well.

[0200] 4. Residual solvent determination method:

[0201] (1) Chromatographic conditions: capillary column with 6% cyanopropylphenyl-94% dimethylpolysiloxane (or similar polarity) as stationary phase (Agilent DB-624, 0.53 mm × 30 m, 3 μm or equivalent performance column is recommended); starting temperature is 50°C, maintained for 10 min, then heated to 150°C at a rate of 50°C per minute and maintained for 5 min; injection port temperature is 200°C; detector is a flame ionization detector, and the detector temperature is 300°C; column flow rate is 2 mL per minute; split ratio is 20:1; headspace equilibrium temperature is 80°C; quantitative loop temperature is 90°C; equilibration time is 30 min.

[0202] System suitability requirements: In the chromatogram of the reference solution, methanol and ethanol appear in sequence, and the separation between each chromatographic peak should meet the requirements.

[0203] (2) Solution preparation

[0204] Test solution: Accurately measure 2 mL of the product, place it in a 20 mL headspace vial, and seal it with a stopper.

[0205] Reference solution: Take appropriate amounts of methanol and ethanol, accurately weigh them, and quantitatively dilute them with ultrapure water to make 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 bottle, and seal it with a stopper.

[0206] The results of the tests for Examples 1-14 were shown in Table 2 according to the above-mentioned methods for determining drug loading, content, related substances and residual solvents. The chromatogram of the related substances of the micelle injection prepared in Example 3 after sterilization is shown in Table 2. Figure 4 As shown in Table 2, the drug loading of Examples 7-14 is higher than that of Examples 1-6, indicating that the addition of nano-micelle solubilizers to the micelle injection formulation can further increase the drug loading; the content of the micelle injection prepared in the examples does not change significantly before and after autoclaving.

[0207] Table 2 Test results of drug loading, content, related substances, etc. of Examples 1-14

[0208] .

[0209] Experimental Example 2 Compatibility Stability

[0210] The micellar injections prepared in Example 1 and Examples 4-11 were diluted with 0.9% sodium chloride injection and 5% glucose injection to different concentrations (e.g., 0.1 mg / mL, 0.25 mg / ml, 0.5 mg / mL, and 1 mg / mL), respectively, and placed at room temperature (25°C) for 24 hours to investigate their stability under clinical use conditions. Combined with the content and related substance analysis and detection methods in Experimental Example 1, the dilution stability was investigated. The results are shown in Tables 3-7, and the content change curve is shown in Table 3-7. Figure 5 and Figure 6 .

[0211] Table 3 Physical stability test results of sample compatibility solutions

[0212]

[0213] Note: ① “-” means “no crystal precipitation”, “+” means “crystal precipitation”, and “++” means “severe crystal precipitation”;

[0214] ②The compatible reagent is 5% glucose injection, and the concentration of the compatible solution is 0.5 mg / mL.

[0215] Table 4 Particle size (Z-Average) test results of the mixed solution of Example 11

[0216] .

[0217] Table 5 Particle size (Z-Average) test results of the mixed solution of Example 7

[0218] .

[0219] Table 6 Content determination results of the compatible solution of Example 11

[0220] .

[0221] Table 7 Determination results of related substances in the compatible solution of Example 11

[0222]

[0223] As shown in Table 3, the micelle injection prepared in the examples shown in the table and the 5% glucose injection solution were placed at room temperature (25°C) for 24 hours. Examples 1 and 4 were stable after 12 hours, which can meet clinical needs. Therefore, when the nano-micelle solubilizer and stabilizer are not added, when the drug loading is up to 4.0%, the compatibility stability is stable for 12 hours without precipitation, and crystals are precipitated after 20 hours. Examples 5 and 6 precipitated after 8 hours. It can be seen that when the nano-micelle stabilizer and nano-micelle solubilizer are not added, the compatibility stability of the injection solution is greatly reduced when the drug loading is increased to 4.2%. Examples 7-8 precipitated crystals after 20 hours, and Examples 9-11 precipitated crystals after 24 hours. No crystals were precipitated during the experiment. It can be seen that the addition of nanomicelle solubilizers (vitamin E and oleic acid were added in Examples 7-8, with drug loadings of 4.5% and 5%, respectively) to the formulation increased the drug loading and also improved the stability to a certain extent. However, the addition of nanomicelle stabilizers (sodium oleate and 15-hydroxystearate polyethylene glycol were added in Examples 9-11, with drug loadings of 4.5%, 4.9%, and 5.0%, respectively) to the formulation further improved the stability. The formulation was stable at room temperature of 25°C for 24 hours without precipitation.

[0224] From the results in Tables 4 to 7, it can be seen that when Examples 7 and 11 were respectively mixed with 5% glucose injection and placed at room temperature (25°C) for 24 hours, there was no significant change in the particle size, content and related substances in Example 11 compared with 0 hour, while the particle size of Example 7 was significantly increased. It can be seen that the addition of the nanomicelle stabilizer in Example 11 can further improve the compatibility stability of the injection.

[0225] Conclusion: Compared with the sample without nanomicelle stabilizer in the formulation, the sample prepared by adding nanomicelle stabilizer in the formulation did not precipitate crystals after being placed at room temperature for 24 h, and the content did not change significantly. It can further improve the stability of micelle injection while improving the drug loading capacity.

[0226] Experimental Example 3: Investigating the effect of adding nanomicelle stabilizer

[0227] The clevidipine butyrate micelle injection prepared by the present invention has a nano-micelle stabilizer such as 15-hydroxystearate polyethylene glycol added to the formulation, which can shorten the dissolution time of clevidipine butyrate in the blank micelle preparation process and improve the physical and chemical stability of the mixed micelles.

[0228] Comparisons were made with Example 4 (no addition), Example 5 (no addition), Example 7 (addition of a nanomicelle solubilizer), and Example 11 (addition of a nanomicelle stabilizer in addition to Example 7). After filtration, the drug solutions were left at room temperature to observe for crystal precipitation and assess physical stability. The dissolution time of clevidipine butyrate was also compared. The results are shown in Tables 8 and 9 below. The drug solution in Example 4 precipitated after 8 hours at room temperature, while that in Example 5 did after 4 hours. A small amount of precipitation occurred in Example 7 after 14 hours, while no precipitation occurred in Example 11 after 24 hours. Adding a nanomicelle solubilizer to the formulation increases both drug loading and stability. However, adding a nanomicelle stabilizer can further enhance the physical stability of the micelle injection. Compared to Example 4, the dissolution time of clevidipine butyrate in Examples 7 and 11 was shorter, reducing the time required for the blank micelle method preparation process.

[0229] Table 8 Physical stability test results

[0230]

[0231] Note: “-” means “no crystals precipitated”, “+” means “crystals precipitated”, and “N / A” means “not applicable”.

[0232] Table 9 Results of investigation on dissolution time of clevidipine butyrate

[0233] .

[0234] Experimental Example 4 Low Temperature Cycle Test

[0235] The clevidipine butyrate micellar injection prepared in Example 11 was subjected to a low-temperature cycle test. The test should include 3 cycles, each cycle of placing at 2-8°C for 2 days and then placing at 25°C for 2 days. Samples were taken and tested after each cycle. The results are shown in Table 10.

[0236] As shown in Table 10, no precipitation occurred after the third cycle. Compared with day 0, there were no significant changes in pH, clarity, color, particle size, potential, content, and related substances. This indicates that the prepared clevidipine butyrate micellar injection can withstand the low-temperature cycle test and has good low-temperature tolerance.

[0237] Note: Information on the colorimetric solution used under the color item: Name: 2020 edition Pharmacopoeia standard colorimetric solution, number: TM-2020-0901, batch number: C2011013, manufacturer: Tanmo Quality Inspection Technology Co., Ltd.

[0238] The turbidity standard solution used for clarity was prepared with reference to the 2020 edition of the Chinese Pharmacopoeia.

[0239] Table 10 Low temperature cycle test results of clevidipine butyrate micellar injection of Example 11

[0240] .

[0241] Experimental Example 5 Freeze-thaw test

[0242] The clevidipine butyrate micellar injections prepared in Example 1, Example 6 (without the addition of nanomicelle stabilizer and solubilizer) and Examples 7-11 were subjected to freeze-thaw tests. The test should include 3 cycles, each cycle of placing at -10 to -20°C for 2 days and then placing at 25°C for 2 days. Samples were taken and tested after each cycle. The results are shown in Tables 11 and 12.

[0243] After the completion of the three cycles, it can be seen from the results in Table 11 that in Example 1, no nanomicelle stabilizer and nanomicelle solubilizer were added, and crystals were precipitated in the third round of freeze-thaw cycles; in Example 6, the drug loading was increased to 4.2% based on Example 1, and crystals were precipitated in the second round of freeze-thaw cycles; while in Examples 7-8, only nanomicelle solubilizers were added but no nanomicelle stabilizers were added, and crystals were precipitated in the third round of freeze-thaw cycles, which can significantly increase the drug loading but have limited improvement in stability; in the formulation composition, a certain amount of nanomicelle stabilizer was added on the basis of the nanomicelle solubilizer (Examples 9-11), and no crystals were precipitated in the three rounds, which shows that the nanomicelle stabilizer can further improve the freeze-thaw stability of the injection.

[0244] As shown in Table 12, the sample prepared in Example 11 had no precipitated crystals. Compared with day 0, the pH value, clarity, color, particle size, potential, content, and related substances did not change significantly. The Zeta potential diagram of the three rounds of Example 11 is shown in FIG. Figure 7 As shown, the above results indicate that clevidipine benzyl alcohol micelle injection can withstand freeze-thaw test.

[0245] Table 11 Freeze-thaw test results

[0246]

[0247] Note: “-” means “no crystals precipitated”, and “+” means “crystals precipitated”.

[0248] Table 12 Freeze-thaw test results of Example 11

[0249] .

[0250] Experimental Example 6 Accelerated test and long-term test

[0251] The stability of the clevidipine butyrate mixed micellar injections in Examples 9-14 was studied under the following conditions. The changes in appearance, properties, pH, and Zeta potential were recorded. The clevidipine butyrate content and related substances were determined according to Experimental Example 1 above. The results are shown in Table 13.

[0252] Long-term test: 5℃±3℃; accelerated test: 25℃±2℃ / 60%RH±5%RH.

[0253] Table 13 Stability results of accelerated test and long-term test

[0254]

[0255]

[0256] Figure 8 The figure shows the accelerated / 3-month particle size distribution of the clevidipine butyrate micelle injection prepared in Example 9, indicating that the particle size of the prepared micelle injection after being placed under accelerated conditions for 3 months did not change significantly compared with that at 0 days.

[0257] Conclusion: As shown in the table above, the clevidipine butyrate micellar injections of Examples 9-14 showed no significant changes in appearance, pH, potential, content, or related substances after six months of storage under long-term and accelerated stability conditions. These results demonstrate that the clevidipine butyrate micellar injections of Examples 9-14 exhibit good stability. The designed storage conditions for clevidipine butyrate micellar injections are 2-8°C. Based on the results of the accelerated stability tests, the shelf life of clevidipine butyrate micellar injections is tentatively set at two years.

[0258] For ordinary technicians in this field, the specific embodiments are only illustrative descriptions of the present invention. It is obvious that the specific implementation of the present invention is not limited to the above-mentioned methods. As long as various non-substantial improvements are made using the method concepts and technical solutions of the present invention, or the concepts and technical solutions of the present invention are directly applied to other occasions without improvement, they are all within the scope of protection of the present invention.

Claims

1. A clevidipine butyrate micellar injection, characterized in that: It contains clevidipine butyrate, phospholipid, bile acid or its salt, and pH regulator; The injection solution also contains a nanomicelle stabilizer and a nanomicelle solubilizer; 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); The nanomicelle stabilizer is selected from one or more of 15-hydroxystearate polyethylene glycol, 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; The injection contains 1-10 mg / mL of clevidipine butyrate, 30-100 mg / mL of bile acid or its salt, a mass ratio of phospholipid to bile acid or its salt is 1:(0.5-1.5), a pH regulator is used to adjust the pH of the injection to 6.0-7.5, and the solvent is water for injection. 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.

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

3. 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 includes 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 evaporate by rotary evaporation to obtain a loose film; (2) Preparation of drug solution: When the bile acid or its salt added in step (1) is one or both of deoxycholic acid and glycocholic acid, an alkaline pH regulator is added at a molar ratio of 1:(0.8-1.2) to the bile acid or its salt added in step (1), and the alkaline pH regulator is completely dissolved in water for injection filled with protective gas accounting for 50-85% of the total volume of the injection preparation, and the resulting solution is transferred to the loose film of step (1). After complete hydration, the pH is adjusted to 6.0-7.5 with a pH regulator to obtain a drug solution; When the bile acid or salt thereof used in step (1) is one or both of sodium deoxycholate and sodium glycocholate, 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; (3) Volume or weight determination: The liquid 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: Pack the contents separately, fill the headspace with protective gas, and then seal the contents. Sterilize by 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).

4. The method for preparing the clevidipine butyrate micellar injection according to claim 3, characterized in that: The rotary evaporation parameters in step (1) are: water bath temperature 40-60°C, time 2-6 hours; 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 0.5-2 hours, the dissolved oxygen residual range is 0-5 mg / L, and the headspace residual oxygen content is controlled at 0-5%.

5. 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, an alkaline pH regulator is added at a molar ratio of 1:(0.8-1.2) to bile acid, and the mixture is completely dissolved in water for injection filled with protective gas accounting for 50-85% of the total volume of the injection solution, and the pH of the solution is adjusted to 6.0-7.5 with the pH regulator to obtain a bile salt solution; When the bile acid or its salt is one or both of sodium deoxycholate and sodium glycocholate, the bile acid or its salt is directly dissolved in water for injection filled with protective gas accounting for 50-85% of the total volume of the injection preparation, and then the pH is adjusted to 6.0-7.5 with a 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 to obtain a blank micelle solution; (3) Preparation of drug solution: Weigh clevidipine butyrate according to the prescription and 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 translucent. After cooling to room temperature, adjust the pH to 6.0-7.5 with a pH adjuster. (4) Volume or weight determination: The liquid obtained in step (3) is volume or weight determined, filtered, and then filled with protective gas. The filtration is performed using a 0.22 μm filter membrane; (5) Sealing and sterilization: Pack the contents separately, fill the headspace with protective gas, and then seal the contents. Autoclave sterilize 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).

6. The method for preparing the clevidipine butyrate micellar injection according to claim 5, characterized in that: 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 dissolved oxygen range is 0-5 mg / L, and the residual oxygen content in the headspace is controlled at 0-5%.

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