WO2023193694A1 - 一种安全、稳定的注射用尼莫地平及其制备方法 - Google Patents

一种安全、稳定的注射用尼莫地平及其制备方法 Download PDF

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WO2023193694A1
WO2023193694A1 PCT/CN2023/086085 CN2023086085W WO2023193694A1 WO 2023193694 A1 WO2023193694 A1 WO 2023193694A1 CN 2023086085 W CN2023086085 W CN 2023086085W WO 2023193694 A1 WO2023193694 A1 WO 2023193694A1
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nimodipine
injection
cyclodextrin
sulfobutyl ether
add
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PCT/CN2023/086085
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English (en)
French (fr)
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武鑫
许幼发
林志浙
黄永洁
陈建明
张元声
傅志勤
陈新美
陈行
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上海维洱生物医药科技有限公司
上海宝龙药业股份有限公司
宝龙药业有限公司
上海宝龙安庆药业有限公司
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Publication of WO2023193694A1 publication Critical patent/WO2023193694A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4422—1,4-Dihydropyridines, e.g. nifedipine, nicardipine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/16—Otologicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of medical technology, specifically, it is a safe and stable nimodipine for injection and its preparation method.
  • Nimodipine (structural formula shown in formula I) is a yellow crystalline powder with a melting point of 125°C. It is almost insoluble in water, soluble in ethanol and chloroform, and is easily degraded when exposed to light. Nimodipine easily penetrates the blood-brain barrier, acts on cerebral vascular smooth muscle, dilates cerebral blood vessels, increases cerebral blood flow, and can effectively prevent or reverse ischemic damage to brain tissue caused by cerebral vasospasm caused by subarachnoid hemorrhage. It is clinically used to prevent and treat ischemic cerebrovascular diseases, such as ischemic nerve damage caused by cerebral vasospasm after subarachnoid hemorrhage, migraine, sudden deafness, etc. It has great potential in the treatment of cerebrovascular diseases. Potential (Carlson AP, et al. Nimodipine Reappraised: An Old Drug With a Future. Curr Neuropharmacol. 2020; 18(1):65-82).
  • nimodipine is commercially available in tablets, capsules, oral liquids and injections. After oral administration of nimodipine, the liver first-pass effect is significant, and the bioavailability is only 5-15%; and the half-life is short, and patients need frequent administration to maintain effective blood concentration, resulting in great restrictions on oral administration. (Teng Z, et al. Preparation and characterization of nimodipine-loaded nanostructured lipid systems for enhanced solubility and bioavailability. Int J Nanomedicine. 2018; 14:119-133.)
  • Nimotong injection has no liver first-pass effect, high bioavailability, and rapid onset of action. It has unparalleled advantages over other dosage forms in clinical use.
  • this injection has the following problems: 1 Due to the level of nimodipine Due to poor solubility, Nimodipine injection adds a large amount of organic solvents to increase the solubility of nimodipine, including 23.7% (V/V) ethanol and 17% (V/V) polyethylene glycol 400.
  • nano-formulation technologies such as lipid emulsion, liposomes, and lipid nanoparticles to encapsulate nimodipine, such as Chinese patent documents CN107019682A, CN102552156A, CN101199522A, CN1554340A, and CN105434355A.
  • nano-preparations have problems such as easy aggregation of particles, low drug loading capacity, low encapsulation rate and obvious burst release effect, and are easily captured by the liver and spleen reticuloendothelial system, affecting the efficacy and even Increase toxicity.
  • the preparation process of nano-preparations is complex, the production line requirements are high, the cost is high, and product quality is difficult to control.
  • Some researchers use surfactants, polymers, etc. to form micelles with nimodipine to increase its water solubility.
  • Chinese patent documents CN101129366A and CN1771950A use extremely toxic surfactants such as Tween 80 in their formulas, which can easily lead to hemolysis and allergic reactions and are highly irritating;
  • Chinese patent documents CN113694031A, CN102525917A, and CN103315948A use polymers to encapsulate nimodipine.
  • the in vivo process of polymer micelles is difficult to predict, which affects the efficacy.
  • nimodipine cyclodextrin inclusion complex is one of the current research hotspots, and domestic and foreign scholars have conducted a large number of studies.
  • Chinese patent document CN1634050A discloses a new nimodipine composition for injection, which combines The material consists of nimodipine, polyethylene glycol 400, Tween 80 and hydroxypropyl- ⁇ -cyclodextrin.
  • This technology has some obvious shortcomings: 1 In addition to hydroxypropyl- ⁇ -cyclodextrin, the formula also adds a large amount of Tween 80 and polyethylene glycol 400 to help dissolve, and the prescription process is complicated. 2 Adding polyethylene glycol 400 to freeze-dried products causes the product to shrink, collapse and become less plump in appearance; 3 The formula contains Tween 80, which can easily lead to hemolysis and allergic reactions and is highly irritating.
  • Chinese patent document CN1424035A discloses a nimodipine freeze-dried composition.
  • the composition contains phospholipids, cyclodextrin and its derivatives or surfactants, wherein the mass ratio of nimodipine to cyclodextrin and its derivatives is 1: 1 to 1:20.
  • Chinese patent document CN1653089A discloses a complex of organic drugs and ⁇ -cyclodextrin derivatives and a preparation method thereof, in which the mass ratio of nimodipine/hydroxypropyl- ⁇ -cyclodextrin is 1:136.
  • nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex was prepared by mixing with ether- ⁇ -cyclodextrin aqueous solution and shaking on a shaking table at 37°C for 72 hours.
  • Nimodipine Ophthalmic Formulations for Management of Glaucoma. Pharm Res. 2017; 34(4):809-824. The above documents all use the shaking method to prepare inclusion compounds.
  • the cyclodextrin inclusion complex prepared by this method has poor inclusion effect.
  • these documents do not include the dosage and inclusion temperature of sulfobutyl ether- ⁇ -cyclodextrin. , inclusion time, ethanol dosage, sulfobutyl ether- ⁇ -cyclodextrin and nimodipine mass ratio (adjuvant ratio), and stirring speed were systematically studied, resulting in long preparation time and low nimodipine solubility. and the problem of high adjuvant ratio.
  • the existing technology generally has poor cyclodextrin inclusion effect, long preparation time, and high auxiliary drug ratio; in addition to cyclodextrin, a large amount of organic solvent needs to be added to the formula to help dissolve, which is not conducive to cyclodextrin inclusion. At the same time, it increases the irritation and toxicity of the preparation and has low safety. Therefore, it is of great significance to develop a safe and stable nimodipine injection, which can lay a solid foundation for improving the safety and convenience of clinical use of nimodipine.
  • the purpose of the present invention is to provide a safe and stable nimodipine for injection and its preparation method.
  • the present invention increases the solubility of nimodipine through sulfobutyl ether- ⁇ -cyclodextrin.
  • sulfobutyl ether- ⁇ -cyclodextrin is an ideal carrier for nimodipine.
  • nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex There are few studies, and a nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion compound product suitable for industrial production and clinical application has not yet been developed.
  • the inventor prepared nimodipine freeze-dried powder injection according to Chinese patent document CN1634050A.
  • the formula contains Tween 80, which can easily cause hemolysis and allergic reactions and is highly irritating.
  • the prepared freeze-dried powder has loose voids, shrinks and collapses (see Example 1), and the appearance of the unqualified freeze-dried powder affects product quality.
  • the inventor prepared nimodipine powder injection containing 2-hydroxypropyl- ⁇ -cyclodextrin according to Example 2 in Chinese patent document CN1424035A. Multiple experiments have found that the drug precipitates during the preparation process, even if the maximum proportion of excipients in the patent (drug/2-hydroxypropyl- ⁇ -cyclodextrin mass ratio 1:20) is used to prepare nimodipine cyclopaste Even with the fine inclusion compound, the drug could not be completely included, and the actual measured auxiliary drug ratio was 571 (see Example 2).
  • Example 28 The inventor operated according to Example 28 in the Chinese patent document CN1653089A. After many experimental verifications, the drugs were all precipitated during the preparation process. The actual measured auxiliary drug ratio was 1904 (see Example 3), which is much higher than the auxiliary drug ratio of the present invention. Drug comparison.
  • the inventor also adopted the shaking method used in the literature to prepare nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex, shook it at 37°C for 72 hours, added an appropriate amount of ethanol to the formula to help dissolve, and prepared the nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex.
  • Dipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex has poor inclusion effect and long preparation time, which is not conducive to the industrial production of injections (see Example 4).
  • the present invention found that the dosage of sulfobutyl ether- ⁇ -cyclodextrin, inclusion temperature, inclusion time, ethanol dosage, auxiliary drug ratio and stirring speed significantly affect the effect of sulfobutyl ether- ⁇ -cyclodextrin on nitric acid.
  • the inclusion effect of Modipine The present invention can significantly reduce the auxiliary drug ratio and achieve an ideal drug loading concentration by adjusting the dosage of sulfobutyl ether- ⁇ -cyclodextrin, inclusion temperature, inclusion time, ethanol dosage and stirring speed, and the preparation is safe and The dilution stability is good.
  • the formula of the present invention can contain a small amount of ethanol.
  • Dissolving nimodipine in a small amount of ethanol can improve the solubility of the drug in the aqueous solution of sulfobutyl ether- ⁇ -cyclodextrin, increase the convenience of the preparation process, and ethanol can Removed during preparation, less residue remains in the product (see Examples 33 and 65).
  • the first aspect of the present invention provides a safe and stable nimodipine for injection, which is made of sulfobutyl ether- ⁇ -cyclodextrin and nimodipine.
  • the sulfobutyl ether- ⁇ -cyclodextrin The mass ratio of semen and nimodipine is 200:1-700:1.
  • the mass ratio of the sulfobutyl ether- ⁇ -cyclodextrin and nimodipine is 350:1-700:1.
  • the content of nimodipine for injection before freeze-drying is 0.01-0.14% g/ml
  • the content of sulfobutyl ether- ⁇ -cyclodextrin is 10-50% g/ml.
  • the mass ratio of the sulfobutyl ether- ⁇ -cyclodextrin and nimodipine is 400:1-600:1, the nimodipine content is 0.02-0.10% g/ml, and the sulfobutyl ether- ⁇ - Cyclodextrin concentration 10-40% g/ml.
  • the mass ratio of the sulfobutyl ether- ⁇ -cyclodextrin and nimodipine is 450:1-550:1
  • the nimodipine content is 0.04-0.06% g/ml
  • the sulfobutyl ether- ⁇ -cyclodextrin and nimodipine are 450:1-550:1.
  • ⁇ -Cyclodextrin concentration is 20-30% g/ml.
  • a safe and stable nimodipine injection of the present invention is made of the following ingredients:
  • nimodipine injection is made of the following ingredients:
  • nimodipine injection is made of the following ingredients:
  • nimodipine injection is made of the following ingredients:
  • the above-mentioned nimodipine injection does not contain ethanol.
  • the above-mentioned nimodipine injection may also contain a buffer to adjust the pH value to 4.5-7.5; the buffer is selected from the group consisting of sodium citrate-citric acid, disodium hydrogen phosphate-citric acid, and phosphoric acid.
  • the buffer is selected from the group consisting of sodium citrate-citric acid, disodium hydrogen phosphate-citric acid, and phosphoric acid.
  • disodium hydrogen phosphate-sodium dihydrogen phosphate disodium hydrogen phosphate-potassium dihydrogen phosphate and potassium dihydrogen phosphate-sodium hydroxide.
  • a second aspect of the present invention provides a method for preparing safe and stable nimodipine injection as described above, which includes the following steps:
  • step (c) Add the solution of step (b) or nimodipine drug powder to the solution of step (a) under certain stirring temperature and speed conditions, stir for a certain period of time, add water for injection to the full amount, and use a 0.22 ⁇ m microporous filter membrane without Bacterial filtration, filling, freeze-drying and packaging are carried out to obtain the safe and stable nimodipine injection.
  • stirring temperature in steps (a) and (c) is 20-100°C, more preferably 40-95°C, and more preferably 60-90°C.
  • stirring time in step (c) is 10-300 min, further preferably 30-240 min, more preferably 60-180 min.
  • the stirring linear speed in step (c) is 0.5-10.0m/s, further preferably 0.8-6.0m/s, more preferably 1.0-3.0m/s.
  • steps (a), (b) and (c) clean and dry nitrogen (N 2 ) can be introduced to saturation, including freeze-drying, nitrogen filling, and packaging.
  • the ethanol residual amount of nimodipine for injection described in step (c) is less than 0.3%, more preferably less than 0.1%, even more preferably less than 0.05%.
  • the present invention found that the dosage of sulfobutyl ether- ⁇ -cyclodextrin, inclusion temperature, inclusion time, ethanol dosage, auxiliary drug ratio and stirring speed significantly affect the effect of sulfobutyl ether- ⁇ -cyclodextrin on Nemo.
  • the inclusion effect of the flat earth Through the control conditions of the present invention, the relative proportion of sulfobutyl ether- ⁇ -cyclodextrin and nimodipine can be significantly reduced, the safety and dilution stability of the preparation can be improved, and the defects of commercially available nimodipine injection can be overcome. Therefore, the core technology of the present invention lies in the control of sulfobutyl ether- ⁇ -cyclodextrin dosage, inclusion temperature, inclusion time, ethanol dosage, auxiliary drug ratio and stirring speed.
  • nimodipine for injection of the present invention is significantly improved, the toxicity problems caused by using a large amount of organic solvents are avoided, the irritation of the preparation is reduced, and the patient's compliance is improved.
  • nimodipine for injection can be directly dispersed into 0.9% sodium chloride injection or 5% glucose injection without the need for a three-way valve, improving convenience of use.
  • the relative proportion of sulfobutyl ether- ⁇ -cyclodextrin and nimodipine in nimodipine for injection of the present invention is smaller, which can not only reduce production costs, but also reduce potential safety hazards caused by cyclodextrin.
  • this patent can greatly shorten the preparation time by optimizing the preparation process; at the same time, the use of a small amount of ethanol not only shortens the inclusion time, but also the small amount of ethanol in the formula does not require special procedures to be removed, and can The ethanol residue in the final product is very low, which increases production convenience and shortens the production cycle.
  • the present invention provides a nimodipine cyclodextrin freeze-dried powder injection containing sulfobutyl ether- ⁇ -cyclodextrin, which can significantly improve the solubility, medication safety and dilution stability of nimodipine , increase patient use compliance and ease of clinical use.
  • the present invention Compared with the commercially available nimodipine injection, the present invention has significantly lower toxicity, significantly improved safety, dilution stability and clinical use convenience, and has obvious advantages; it is compared with the existing nimodipine cyclodextrin inclusion complex Compared with other technologies, it avoids the use of a large amount of organic solvents, surfactants, etc., greatly reduces the auxiliary drug ratio, shortens the production cycle, and has very good application prospects.
  • Figure 4 Stability chart of dilution of nimodipine and nimodipine for injection with injection;
  • Example 1 Effect of polyethylene glycol 400 on nimodipine freeze-dried powder injection
  • nimodipine to the mixture of polyethylene glycol 400 and Tween-80, heat and stir to dissolve.
  • hydroxypropyl- ⁇ -cyclodextrin to water for injection, heat and stir to dissolve, add the above solution while stirring, continue stirring, and bring to room temperature.
  • Add an appropriate amount of activated carbon and keep at 60°C for 20 minutes. and stir. Filter, fill, and freeze-dry to get it.
  • freeze-dried powder prepared by this formula has loose voids and a shrunken, collapsed and unfull appearance.
  • the appearance of unqualified freeze-dried powder affects product quality.
  • nimodipine powder for injection Take 2 mg of nimodipine and stir with 2 mL of absolute ethanol until completely dissolved. Take 20 mg of hydroxypropyl- ⁇ -cyclodextrin and add 7 mL of water for injection to dissolve. Add the nimodipine ethanol solution to the hydroxypropyl- ⁇ -cyclodextrin while stirring. In the cyclodextrin aqueous solution, a uniform cyclodextrin inclusion compound is prepared. Dry under reduced pressure on a rotary evaporator to remove ethanol, add 300 mg of mannitol, filter the solution with a sterile funnel, put the filtrate into a sterile vial, determine the content by high-performance liquid chromatography, freeze-dry, and seal. , that is, nimodipine powder for injection.
  • the nimodipine content determination method in the present invention is determined according to the high performance liquid chromatography method (2020 edition of the Chinese Pharmacopoeia, Part 4, General Chapter 0512), and the operation is protected from light.
  • Chromatographic conditions and system suitability test Use octadecylsilane bonded silica gel as the filler (C 18 column 250 ⁇ 4.6mm, 5 ⁇ m); use methanol-acetonitrile-water (35:38:27) as the mobile phase; detection The wavelength is 235nm; the injection volume is 10 ⁇ L.
  • the number of theoretical plates calculated based on the nimodipine peak should not be less than 8000, and the separation between the nimodipine peak and adjacent impurity peaks should meet the requirements.
  • test solution Precisely measure 1 mL of the prepared solution, place it in a 50 mL volumetric flask, dilute to the mark with mobile phase, and shake well.
  • HPLC chromatogram for content determination of the test solution is shown in Figure 1.
  • Preparation of reference substance solution Take nimodipine reference substance, weigh it accurately, add mobile phase to dissolve and quantitatively dilute it to make a solution containing approximately 20 ⁇ g per 1 mL.
  • Determination method Precisely measure the test solution and reference solution, inject them into the liquid chromatograph respectively, and record Chromatogram. Calculated by peak area according to external standard method.
  • the drug precipitated After the sample was filtered, the drug content in the solution was determined to be 0.005 mg/mL, and the adjuvant ratio was calculated to be 571.
  • This patented technology uses 22% ethanol, which is not conducive to the inclusion of nimodipine by cyclodextrin, and a large amount of ethanol poses certain safety risks; in addition, the need to remove ethanol through rotary evaporation increases the complexity of the process.
  • the drug precipitated during the mixing and stirring process of nimodipine ethanol solution and hydroxypropyl- ⁇ -cyclodextrin aqueous solution a large amount of drug also precipitated during the process of drying under reduced pressure to remove ethanol.
  • the drug content in the solution was measured to be 0.21 mg/mL, and the calculated auxiliary drug ratio was 1904, which is much higher than the auxiliary drug ratio of the present invention.
  • Example 4 Preparation of nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex by shaking method
  • Nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex was prepared using the shaking method with reference to the literature (Maria DN, et al. Nimodipine Ophthalmic Formulations for Management of Glaucoma. Pharm Res. 2017; 34(4):809- 824.), in addition, nimodipine sulfobutyl ether- ⁇ -cyclodextrin inclusion complex was prepared using the same formula with a small amount of ethanol shaking method for comparison.
  • Example 5 Effect of different solvents on the solubility of nimodipine in sulfobutyl ether- ⁇ -cyclodextrin
  • Example 6 Effect of ethanol dosage on the solubility of nimodipine in sulfobutyl ether- ⁇ -cyclodextrin
  • sulfobutyl ether- ⁇ -cyclodextrin Take 6g of sulfobutyl ether- ⁇ -cyclodextrin, make 7 parallel groups, add an appropriate amount of water for injection, stir at 30°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, add 0.6 mL, 0.9mL, Dissolve 1.2mL, 1.5mL, and 1.8mL ethanol. Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions. Stir for 2 hours. The resulting solution is filtered through a 0.45 ⁇ m polyethersulfone membrane. Filter, and detect the nimodipine content by HPLC.
  • the content of the prepared nimodipine cyclodextrin was determined and the results are as follows:
  • Example 7 Effect of the dosage of sulfobutyl ether- ⁇ -cyclodextrin on the solubility of nimodipine
  • ⁇ -cyclodextrin aqueous solution take 45 mg of nimodipine, add 0.9 mL of ethanol to dissolve it, add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 2 hours, and the resulting solution Filter through a 0.45 ⁇ m polyethersulfone filter membrane, and detect the nimodipine content by HPLC.
  • the content of the prepared nimodipine cyclodextrin was determined and the results are as follows:
  • Example 8 Effect of dissolution temperature and time on the solubility of nimodipine
  • nimodipine ethanol solution to sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, respectively at 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C , 90°C, and 100°C for 5 hours, and take points at 1h, 2h, 3h, 4h, and 5h.
  • the resulting solution was filtered through a 0.45 ⁇ m polyethersulfone filter membrane, and the nimodipine content was detected by HPLC.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 6g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 40°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, add 0.3 mL of ethanol, and dissolve in Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 30 minutes, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. , freeze-drying, packaging, and it is ready.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 10.8g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 60°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 24 mg of nimodipine, add 0.3 mL of ethanol to dissolve, Add nimodipine ethanol solution to sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 1 hour, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, and filled Install, freeze-dry, and encapsulate, and you have it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 9.9g of sulfobutyl ether- ⁇ -cyclodextrin, 520 mg of disodium hydrogen phosphate and 281 mg of sodium dihydrogen phosphate, add an appropriate amount of water for injection, stir at 60°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; Take 30 mg of nimodipine, add 0.3 mL of ethanol to dissolve it, add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 1 hour, add water for injection to make up to 30 mL, and the resulting solution Sterile filter through 0.22 ⁇ m microporous filter membrane, fill, freeze-dry, and package.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 9g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 100°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 30 mg of nimodipine, add 0.72 mL of ethanol, and dissolve in Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 2 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. , freeze-drying, packaging, and it is ready.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 10.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 50°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 30 mg of nimodipine, add 0.48 mL of ethanol to dissolve, Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 5 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, and filled Install, freeze-dry, and encapsulate, and you have it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 10.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 60°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 30 mg of nimodipine, add 0.72 mL of ethanol to dissolve, Stirring Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under the conditions, stir for 1 hour, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, and filled. Freeze-dry, package and get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 13.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 50°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 30 mg of nimodipine, add 0.48 mL of ethanol to dissolve, Stirring Under the conditions, add nimodipine ethanol solution to sulfobutyl ether- ⁇ -cyclodextrin aqueous solution and stir for 3 hours. Add water for injection to make up to 30 mL. The resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and frozen. Dry, package, and it's ready.
  • nimodipine Take 36 mg of nimodipine, add 0.72 mL of ethanol to dissolve it, add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 2 hours, add water for injection to make up to 30 mL, the result The solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, freeze-dried, and packaged.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 13.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 80°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 45 mg of nimodipine, add 0.9 mL of ethanol to dissolve, Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under stirring conditions, stir for 4 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, and filled Install, freeze-dry, and encapsulate, and you have it.
  • Example 33 Determination of ethanol residue in nimodipine for injection
  • ethanol content in nimodipine for injection is determined according to the residual solvent determination method (Chinese Pharmacopoeia 2020 Edition Four General Chapters 0861 Second Method), as follows:
  • DB-624 capillary column as the chromatographic column; programmed temperature rise, the starting temperature is 70°C, maintained for 2 minutes, first heated to 120°C at a rate of 10°C per minute, and then heated to 220°C at a rate of 20°C per minute. Maintain for 3 minutes; split ratio is 20:1; column flow rate is 1mL/min; inlet temperature is 200°C; detector temperature is 250°C; headspace bottle equilibrium temperature is 80°C, and equilibrium time is 30min.
  • Example 34 Dilution stability study of nimodipine for injection
  • nimodipine for injection and the nimodipine injection prepared in Example 21, and dilute them to clinical dosage concentrations with 0.9% sodium chloride injection and 5% glucose respectively. Observe and take points at 0, 3, 6, 9, 12, and 24 hours. After filtration with a 0.45 ⁇ m polyethersulfone filter, determine the nimodipine content according to the above content determination method.
  • Example 35 Hemolytic study of nimodipine for injection
  • nimodipine for injection prepared in Example 21 and the commercially available nimodipine injection were respectively diluted with 0.9% sodium chloride injection to a clinical dosage concentration of 0.04 mg/mL. Take 8 10mL clean glass test tubes and number them. Tubes 1 to 5 are nimodipine for injection with different concentrations, tube 6 is a negative control tube, tube 7 is a positive control tube, and tube 8 is a commercially available nimodipine for injection. liquid. As shown in the table below, add 2% red blood cell suspension, 0.9% sodium chloride injection, distilled water, and medicinal solution in sequence. After mixing, immediately place it in a 37 ⁇ 0.5°C water bath and incubate it. Observe and record the hemolysis of each tube. . Start recording every 15 minutes. After 1 hour, observe every 1 hour for 3 hours.
  • Example 36 Study on high temperature stability of nimodipine for injection
  • Nimodipine for injection prepared in Example 21 was selected and placed at 60°C for one month to detect changes in drug content and related impurities.
  • the determination method of nimodipine related substances in the present invention is determined according to the high performance liquid chromatography method (2020 version of the Chinese Pharmacopoeia, Part 4, General Chapter 0512), and the operation is protected from light.
  • Chromatographic conditions and system suitability test Use octadecylsilane bonded silica gel as the filler (C 18 column 250 ⁇ 4.6mm, 5 ⁇ m); use methanol-acetonitrile-water (35:38:27) as the mobile phase; detection The wavelength is 235nm; the injection volume is 10 ⁇ L.
  • the separation between the nimodipine peak and the peaks of impurities B, C, and I should be greater than 3.0.
  • test solution Precisely measure an appropriate amount of this product and quantitatively dilute it with mobile phase to prepare a solution containing approximately 0.2 mg of nimodipine per 1 mL.
  • HPLC chromatogram for determination of related substances in the test solution is shown in Figure 3.
  • Preparation of the control solution Precisely measure 1 mL of the test solution, place it in a 100 mL volumetric flask, quantitatively dilute it to the mark with mobile phase, and shake well.
  • Impurity I reference substance solution Precisely weigh the impurity I reference substance and quantitatively dilute it with mobile phase to prepare a solution containing approximately 1 ⁇ g per 1 mL.
  • Preparation of system suitability solution Take appropriate amounts of nimodipine and impurities B, C, and I reference standards, add mobile phase to dissolve and dilute to make a mixed solution containing approximately 200 ⁇ g and 1 ⁇ g each in 1 mL.
  • Determination method Precisely measure the test solution, impurity I reference solution and control solution, and inject them into the liquid chromatograph respectively. If there are impurity peaks in the chromatogram of the test solution, except for the chromatographic peaks with a relative retention time less than 0.45, the chromatographic peaks with the same retention time as the impurity peak I will be calculated based on the peak area according to the external standard method; other single impurities will be calculated as the main component Calculated by self-contrast method.
  • nimodipine for injection was stored in a 60°C stability test chamber for one month, the indicated percentage content of nimodipine showed almost no change, and the relevant substances complied with the prescribed limit requirements.
  • the above results show that nimodipine for injection of the present invention has good high-temperature stability and its quality complies with relevant regulations.
  • Example 37 Effects of preparation temperature and time on the solubility of nimodipine in sulfobutyl ether- ⁇ -cyclodextrin. Using nimodipine content and related substances as indicators, the effects of preparation temperature and time on the solubility of nimodipine in sulfonbutyl ether- ⁇ -cyclodextrin were investigated. Solubility of ether- ⁇ -cyclodextrin and the influence of related substances.
  • Example 38 Effect of stirring speed on the solubility of nimodipine in sulfobutyl ether- ⁇ -cyclodextrin
  • sulfobutyl ether- ⁇ -cyclodextrin Take 25g of sulfobutyl ether- ⁇ -cyclodextrin, make 8 parallel groups, add an appropriate amount of water for injection, stir at 75°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 50 mg of nimodipine, and add them to Add nimodipine under the conditions of stirring linear speed of 0.25m/s, 0.5m/s, 1.0m/s, 1.5m/s, 2.0m/s, 3.0m/s, 6.0m/s, and 10.0m/s.
  • stirring speed significantly affects the inclusion effect of sulfobutyl ether- ⁇ -cyclodextrin on nimodipine.
  • Stirring speed is one of the important core technologies of the present invention, so we have added the protection scope of stirring speed in the claims.
  • Example 39 Effect of auxiliary drug ratio on dilution stability of nimodipine cyclodextrin inclusion complex
  • sulfobutyl ether- ⁇ -cyclodextrin Take 15g, 17.5g, 20g, 22.5g, 25g, 30g, and 35g of sulfobutyl ether- ⁇ -cyclodextrin respectively, add an appropriate amount of water for injection, stir at 75°C to dissolve, and obtain sulfobutyl ether- ⁇ -cyclodextrin.
  • Dextrin aqueous solution take 50 mg of nimodipine, add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution at a stirring linear speed of 1.8m/s, stir for 1.5h, and add water for injection to make up to 100mL.
  • the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, freeze-dried, and packaged.
  • the prepared nimodipine for injection was diluted to a drug concentration of 0.2 mg/mL with 0.9% sodium chloride injection. Observe and take points at 0, 12, 24, 36, 48, and 60 hours. After filtration with a 0.45 ⁇ m polyethersulfone filter, determine the nimodipine content according to the above content determination method.
  • the dilution stability results are shown in Table 24.
  • the adjuvant ratio is ⁇ 350, the dilution stability time of nimodipine for injection can reach more than 24 hours; when the adjuvant ratio is ⁇ 400, the dilution stability time of nimodipine for injection can reach more than 36 hours;
  • the adjuvant ratio is ⁇ 450, the dilution and stability time of nimodipine for injection can reach more than 48 hours; when the adjuvant ratio is ⁇ 500, the dilution and stability time of nimodipine for injection can reach more than 60 hours.
  • the results show that the excipient ratio significantly affects the dilution stability of nimodipine cyclodextrin inclusion complex.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 3g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 70°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 3 mg of nimodipine, add 0.07 mL of ethanol, and dissolve in Add nimodipine ethanol solution to sulfobutyl ether- ⁇ -cyclodextrin aqueous solution at a stirring speed of 3.0 m/s, stir for 10 minutes, add water for injection to make up to 30 mL, and the resulting solution is filtered through a 0.22 ⁇ m microporous membrane. Bacteria passed Filter, fill, freeze-dry, and package.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 3g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 60°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 6 mg of nimodipine, stir at a stirring speed of 2.0m/ Add nimodipine ethanol solution to sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under s conditions, stir for 30 minutes, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. Freeze-dry, package and get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 3g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 20°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 6 mg of nimodipine, and stir at a stirring speed of 10.0 m/ Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under s conditions, stir for 4 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and freeze-dried. , encapsulate, and you get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 6g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 85°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 12 mg of nimodipine, stir at a stirring speed of 0.8m/ Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under s conditions, stir for 2 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and freeze-dried. , encapsulate, and you get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 75°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, stir at a stirring speed of 1.8 m /s condition will Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution, stir for 1.5 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, freeze-dried, and packaged. That’s it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 80°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, and stir at a stirring speed of 1.7 Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under m/s conditions, stir for 2 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. Freeze-dry, package and get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 90°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, stir at a stirring speed of 1.5 m Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under /s conditions, stir for 1 hour, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and frozen Dry, package, and it's ready.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 65°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, stir at a stirring speed of 2.0 m Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under /s conditions, stir for 2 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. Freeze-dry, package and get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 75°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, stir at a stirring speed of 2.5 m Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under /s conditions, stir for 3 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and frozen Dry, package, and it's ready.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 60°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, stir at a stirring speed of 3.0 m Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under /s conditions, stir for 3 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and frozen Dry, package, and it's ready.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 7.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 70°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 15 mg of nimodipine, add 0.45 mL of ethanol to dissolve, Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution at a stirring speed of 2.0 m/s, stir for 1 hour, and add water for injection to make up to 30 mL. The resulting solution is sterile through a 0.22 ⁇ m microporous filter. Filter, fill, freeze-dry, encapsulate and you're ready.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 10.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 40°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 19.10 mg of nimodipine, stir at a stirring speed of 6.0 Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under m/s conditions, stir for 5 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. Freeze-dry, package and get it.
  • ether- ⁇ -cyclodextrin aqueous solution stir for 2 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, freeze-dried, and packaged.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 12g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 95°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 30 mg of nimodipine, stir at a stirring speed of 0.8m/ Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under s conditions, stir for 4 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane, filled, and freeze-dried. , encapsulate, and you get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 13.5g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, stir at 75°C to dissolve, and obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 22.5 mg of nimodipine, stir at a stirring speed of 3.0 Add nimodipine to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution under m/s conditions, stir for 2 hours, add water for injection to make up to 30 mL, and the resulting solution is sterile filtered through a 0.22 ⁇ m microporous filter membrane and filled. Freeze-dry, package and get it.
  • sulfobutyl ether- ⁇ -cyclodextrin Take 15g of sulfobutyl ether- ⁇ -cyclodextrin, add an appropriate amount of water for injection, and stir to dissolve at 100°C to obtain a sulfobutyl ether- ⁇ -cyclodextrin aqueous solution; take 42 mg of nimodipine, add 1.05 mL of ethanol, and dissolve in Add the nimodipine ethanol solution to the sulfobutyl ether- ⁇ -cyclodextrin aqueous solution at a stirring speed of 1.7m/s, stir for 1.5h, add water for injection to make up to 30mL, and the resulting solution is filtered through a 0.22 ⁇ m microporous membrane. Sterile filtration, filling, freeze-drying, packaging, ready to go.
  • Example 65 Determination of ethanol residue in nimodipine for injection
  • Nimodipine for injection prepared in Examples 40, 41, 47, 55, 56, and 63 was selected to detect ethanol residue.
  • the ethanol content in nimodipine for injection is determined according to the residual solvent determination method (Chinese Pharmacopoeia 2020 Edition Four General Chapters 0861 Second Method).
  • Example 66 Dilution stability study of nimodipine for injection
  • the nimodipine for injection prepared in Example 48 was reconstituted with water for injection and the nimodipine injection was diluted to a clinical dosage concentration of 0.04 mg/mL with 0.9% sodium chloride injection and 5% glucose respectively. Observe and take points at 0, 3, 6, 9, 12, and 24 hours. After filtration with a 0.45 ⁇ m polyethersulfone filter, determine the nimodipine content according to the above content determination method.
  • Nimodipine injection is diluted with 0.9% sodium chloride injection or 5% glucose, a large amount of the drug precipitates in 3 hours, and the drug content drops significantly in 24 hours, and the dilution stability is extremely poor;
  • Nimodipine for injection of the present invention is diluted with 0.9% chloride injection After dilution with sodium chloride injection or 5% glucose, the content does not change significantly within 24 hours, the appearance is clear, and the dilution stability is good.
  • the above results show that compared with nimodipine injection, the dilution stability of nimodipine for injection of the present invention is significantly improved and has obvious advantages.
  • Nimodipine for injection prepared in Example 48 was selected, and nimodipine for injection was investigated in accordance with the "9001 Stability Test Guidelines for Raw Materials and Preparations" in the 2020 edition of the "Pharmacopoeia of the People's Republic of China" High temperature, high humidity, light and accelerated test stability, detect drug content and changes in related substances.
  • Nimodipine for injection has almost no change in the content of nimodipine and related substances under high temperature, high humidity and accelerated test conditions.
  • the content of nimodipine for injection was only 96.59%, and related substances increased by 0.11%. This may be due to the fact that nimodipine is relatively unstable and easy to decompose under light conditions. Even after being included in cyclodextrin, it still degrades under light conditions, indicating that nimodipine for injection should be stored away from light.
  • the above results show that the nimodipine for injection of the present invention has good stability and the quality complies with relevant regulations.
  • Example 68 Study on vascular irritation of nimodipine for injection
  • Example 48 Nimodipine for injection prepared in Example 48 was added to water for injection to reconstitute and commercially available Nimodipine was treated with 0.9% chloride for injection.
  • the sodium injection was diluted to a clinical dosage concentration of 0.04 mg/mL, and the solution was injected through the rabbit's right ear margin vein at a dosage of 0.4 mg/kg and a bolus speed of 1 mL/min.
  • an equal volume of 0.9% sodium chloride injection was given to the left ear through the marginal ear vein as a control, once a day for three consecutive days. Animal behavior and injection site changes were visually observed during daily dosing.
  • Euthanasia was performed 24-48 hours after the last dose, and the bilateral distance from the injection site was removed.
  • the auricular vein tissue located about 0.5-3.0cm from the heart end was fixed in 10% paraformaldehyde solution, dehydrated using ethanol gradient, embedded in paraffin, and stained with hematoxylin-eosin solution to evaluate pathological changes.
  • Nimotong injection showed obvious collagen swelling, red blood cell leakage and spot hemorrhage, endothelial cell swelling, and infiltration of a large number of inflammatory cells, indicating that the body had a strong inflammatory response after administration of Nimotong injection group.
  • the rabbit struggled violently and felt severe pain, which may be due to the strong vascular irritation and pain caused by high concentration of ethanol entering the blood vessels.
  • nimodipine and 0.9% sodium chloride injection for injection of the present invention is intact, the sebaceous glands and sweat gland tissues have no special lesions, the central part of the cartilage has normal shape, no obvious lesions and injuries are seen, and the rabbit has no symptoms during the administration process. Visibly unwell.
  • nimodi injection had only a small amount of inflammatory cell infiltration and no other abnormalities.
  • Example 69 Hemolytic study of nimodipine for injection
  • Nimodipine for injection prepared in Example 48 was reconstituted with water for injection and commercially available Nimotop injection, and diluted to a clinical dosage concentration of 0.04 with 0.9% sodium chloride injection. mg/mL. Take 8 10mL clean glass test tubes and number them. Tubes 1 to 5 are NIMO-CD of different concentrations, tube 6 is a negative control tube, tube 7 is a positive control tube, and tube 8 is Nimotop injection. As shown in the table below, add 2% red blood cell suspension, 0.9% sodium chloride injection, distilled water, and medicinal solution in sequence. After mixing, immediately place it in a 37 ⁇ 0.5°C water bath and incubate it. Observe and record the hemolysis of each tube. . Start recording every 15 minutes. After 1 hour, observe every 1 hour for 3 hours.
  • NIMO-CD No. 1-5 red blood cells were deposited at the bottom of the test tube. The supernatant was colorless and clear, and could be evenly dispersed after shaking, indicating that NIMO-CD would not cause hemolysis and red blood cell aggregation. .
  • the above results show that the nimodipine for injection of the present invention has no hemolytic property and can be used for injection.
  • Example 70 Pharmacodynamic study of nimodipine for injection
  • Example 48 32 male SD rats were randomly divided into 4 groups: sham operation group (Sham group), model control group (Model group), Nimotop injection group (Nimotop group), and Nimodipine injection group (NIMO-CD group). , Example 48). After adding nimodipine for injection prepared in Example 48 to water for injection to reconstitute, it was diluted to 0.2 mg/mL with 0.9% sodium chloride injection. The Nimotop group and the NIMO-CD group were administered via tail vein injection at a dose of 1 mg/kg, once a day from the day of modeling to 72 hours after the operation, and immediately after the operation.
  • the brain stereotaxic instrument was used in the left striatum area (centered on the bregma, 2.0mm on the posterior side, 2.0mm on the right side, The needle was held at a depth of 4.0 mm for 2 min, and 2 ⁇ L of 2 mg/mL collagenase solution was injected at a rate of 1 ⁇ L/5 min. The needle was slowly removed after being held for 3 min to establish a cerebral hemorrhage model. After the operation is completed, the wound is sutured and iodophor is used to disinfect the wound.
  • Gentamicin 40 mg/kg was injected intramuscularly into the hind limbs to prevent infection, and 10 mg/kg Tonlidine was injected intramuscularly twice a day to relieve pain.
  • the experimental procedures for animals in the Sham group were the same as above, except that the same volume of PBS solution was used instead of collagenase injection.
  • CT modified neurological severity score
  • Rat brain tissue sections were fixed with 4% paraformaldehyde solution, embedded in paraffin, and stained with HE, and the lesions in the sections were observed under an optical microscope.
  • the degree of brain edema, inflammatory infiltration and neuronal damage in the slices was evaluated according to the histopathological scoring standards. The scoring standards are shown in Table 30.
  • the mNSS score is shown in Table 31 and Figure 7A.
  • the international standard definition of mNSS score is: 0 points for normal SD rats, mild injury between 1 and 6 points, moderate injury between 7 and 12 points, and moderate injury between 13 and 18 points. severe injuries.
  • the mNSS score of the Model group was the highest on the first day, and the neurological damage was the most severe. On the second and third days, the score gradually decreased, and the neurological function gradually recovered, but all were greater than 7 points, indicating moderate damage.
  • the neurological function scores of the NIMO-CD group and the Nimotop group also decreased over time. The scores were both greater than 7 points on the two days after treatment, indicating moderate injury, and were lower than 7 points on the third day, indicating mild injury.
  • the mNSS scores of experimental animals in the NIMO-CD group and Nimotop group were significantly reduced.
  • the CT score results are shown in Table 32 and Figure 7B.
  • the CT scores decreased with time, and the hemiplegia of experimental animals in each group gradually improved.
  • the final CT scores of the Sham group and Model group were 48.75% and 65.00% respectively, and the final CT scores of the NIMO-CD group and Nimotop group were 58.75% and 60.00% respectively.
  • the CT scores of experimental animals in the NIMO-CD group and Nimotop group were both reduced.
  • nimodipine injection has a significant improvement effect on neurobehavioral dysfunction caused by brain injury in the rat cerebral hemorrhage model, and there is no significant difference with the nimodipine injection group (P>0.05 ).
  • the brain tissues of the test animals were collected, and the differences in the total hematoma area of each group were compared. The results are shown in Table 33 and Figure 7C. There was no hematoma in the Sham group, the total hematoma area accounted for 4.45% in the Model group, and the total hematoma area accounted for both the NIMO-CD group and Nimotop group was 0.98%. Compared with the Model group, the proportion of total hematoma area in experimental animals in the NIMO-CD group and Nimotop group was significantly reduced.
  • nimodipine injection can significantly reduce hematoma caused by cerebral hemorrhage in rats and significantly improve cerebral hemorrhage damage, and there is no significant difference with nimodipine injection (P>0.05).
  • the pathological sections are shown in Figure 7E. Based on the pathological sections, the degree of brain edema, inflammation and neuronal damage are comprehensively scored. The scoring results are shown in Table 34 and Figure 7D.
  • the histopathological evaluation scores of the Sham group and Model group were 1.38 and 5.00 respectively.
  • the histopathological evaluation scores of the Nimotop group and NIMO-CD group were 2.63 and 2.25 respectively. Compared with the Model group, the total score of histopathological evaluation of experimental animals in the NIMO-CD group was reduced. Histopathological results showed that nimodipine injection could significantly improve the degree of cerebral edema, inflammation and neuronal damage, and there was no significant difference with nimodipine injection (P>0.05).
  • nimodipine for injection has a significant therapeutic effect on the prognosis of cerebral hemorrhage, which greatly improves the neurological damage, cerebral hematoma, cerebral edema, inflammation and neuronal damage caused by cerebral hemorrhage, and the treatment
  • the effect is basically the same as that of Nyima Tong Injection.
  • Example 71 Pharmacokinetic study of nimodipine for injection
  • NIMO-CD nimodipine for injection
  • Example 48 16 male SD rats were randomly divided into two groups, with 8 rats in each group. They were fasted for 12 hours before the experiment. water.
  • the nimodipine for injection (NIMO-CD) prepared in Example 48 was added to water for injection and reconstituted, it was diluted to 0.2 mg/mL with 0.9% sodium chloride injection.
  • Nimotop injection (Nimotop) and NIMO-CD at 1.6 mg/kg were administered through the tail vein respectively, and 0.5 mL of blood was taken from the orbit at 5, 15, 30, 45, 60, 120, 240, 360 and 480 min after administration, 4000 rpm. Centrifuge for 5 minutes, take 300 ⁇ L of supernatant, and store in a -80°C refrigerator.
  • Chromatographic column Zorbax SB C 18 column (250mm ⁇ 4.6mm, 5 ⁇ m)
  • NIMO-CD plasma concentration-time curves of SD rats after intravenous injection of Nimotop injection and NIMO-CD are shown in Figure 8.
  • the learning elimination curves are almost the same.
  • NIMO-CD and Nimotop injection NIMO can be rapidly eliminated within a short period of time after entering the body. HPLC cannot detect the drug in the plasma 240 minutes after administration.
  • the main pharmacokinetic parameters of NIMO-CD and Nimotop injection are shown in Table 35.
  • the results show that the pharmacokinetic parameters of NIMO-CD and Nimotop injection are basically consistent, including the area under the plasma concentration-time curve (AUC). , mean residence time (MRT), half-life (t 1/2 ), clearance (CL) and maximum plasma concentration (C max ), and there was no statistically significant difference between the groups (P>0.05).
  • the above results show that the pharmacokinetics of nimodipine and nimotong injection after tail vein injection in SD rats The properties are basically the same.

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Abstract

本发明涉及医药技术领域，具体是一种安全、稳定的注射用尼莫地平及其制备方法。本发明提供了一种含磺丁基醚-β-环糊精的尼莫地平环糊精冻干粉针，可显著提高尼莫地平溶解性、用药安全性和稀释稳定性，增加患者使用的顺应性及临床使用的便捷性。本发明与市售尼膜同注射液相比，安全性和稀释稳定性均有显著提升，具有明显优势。

Description

一种安全、稳定的注射用尼莫地平及其制备方法 技术领域
本发明涉及医药技术领域，具体地说，是一种安全、稳定的注射用尼莫地平及其制备方法。
背景技术
尼莫地平(结构式如式I所示)为黄色结晶粉末，熔点125℃，在水中几乎不溶，溶于乙醇和氯仿，遇光极易降解。尼莫地平易透过血脑屏障，作用于脑血管平滑肌，扩张脑血管，增加脑血流量，可有效防止或逆转蛛网膜下腔出血引起的脑血管痉挛造成的脑组织缺血性损害。临床上用于预防和治疗缺血性脑血管疾病，如蛛网膜下腔出血后脑血管痉挛引起的缺血性神经损伤、偏头痛、突发性耳聋等，在治疗脑血管疾病上具有极大的潜力(Carlson AP,et al.Nimodipine Reappraised:An Old Drug With a Future.Curr Neuropharmacol.2020；18(1):65-82)。
目前尼莫地平市售剂型有片剂、胶囊剂、口服液和注射剂。尼莫地平口服后，肝脏首过效应显著，生物利用度仅为5-15％；且半衰期短，患者需频繁给药以维持有效血药浓度，导致口服给药受到极大的限制。(Teng Z,et al.Preparation and characterization of nimodipine-loaded nanostructured lipid systems for enhanced solubility and bioavailability.Int J Nanomedicine.2018；14:119-133.)
为了改善尼莫地平口服制剂的缺陷，德国拜耳公司开发了一款尼膜同注射液。尼膜同注射液无肝脏首过效应，生物利用度高，起效迅速，临床使用时具有其它剂型无可比拟的优势。然而该注射液存在以下问题：①由于尼莫地平水 溶性差，尼膜同注射液添加大量有机溶媒以增加尼莫地平溶解度，其中包括23.7％(V/V)乙醇和17％(V/V)聚乙二醇400。大量有机溶媒对血管刺激性大，造成给药部位疼痛、红肿等，易引发静脉炎，导致患者依从性较差(Schneider AC,et al.Chronic exposure to ethanol causes steatosis and inflammation in zebrafish liver.World J Hepatol.2017；9(8):418-426.)。②尼膜同注射液临床使用时直接稀释药物易析出。因此，给药时需借助三通阀与5％葡萄糖、0.9％氯化钠等液体以1：4(V/V)的比例混合输注，每日给药时间为10h，治疗时间为10-14天。该输注方式存在较大安全隐患，滴注速度和混合比例不易控制，操作繁琐、时间较长，临床使用十分不便，患者顺应性差。
鉴于现有尼莫地平制剂的不足，行业内的研究人员对尼莫地平开展了广泛的研究。
为了提高尼莫地平溶解度，研究人员利用脂肪乳、脂质体、脂质纳米粒等纳米制剂技术包裹尼莫地平，如中国专利文献CN107019682A、CN102552156A、CN101199522A、CN1554340A、CN105434355A。虽然改善了尼莫地平的水溶性，但纳米制剂存在微粒易聚集、载药量低、包封率低和突释效应明显等问题，并且易被肝脾网状内皮系统捕获，影响疗效，甚至增加毒性。此外，纳米制剂制备工艺复杂，生产线要求高，成本高昂，产品质量难以控制。
还有研究人员采用表面活性剂、聚合物等与尼莫地平形成胶束，从而增加其水溶性。如中国专利文献CN101129366A、CN1771950A，其配方中采用吐温80等毒性极大的表面活性剂，易导致溶血和过敏反应，刺激性大；中国专利文献CN113694031A、CN102525917A、CN103315948A利用聚合物包裹尼莫地平，聚合物胶束体内过程难以预测，影响疗效。
鉴于以上纳米制剂的不足，研究人员开始寻找新的制剂技术改善尼莫地平的水溶性，其中尼莫地平环糊精包合物是目前研究热点之一，国内外学者进行了大量的研究。
文献报道尼莫地平由于侧链的空间位阻作用较大，难于同β-环糊精形成包合物(武雪芬等.β-环糊精与二氢吡啶类药物的相互作用[J].中国药学杂志,2005(08):599-601.)。鉴于β-环糊精的不足，研究人员尝试寻找新的β-环糊精衍生物包载尼莫地平。
中国专利文献CN1634050A公开了一种新的注射用尼莫地平组合物，组合 物由尼莫地平、聚乙二醇400、吐温80和羟丙基-β-环糊精。该技术存在一些明显缺陷：①配方中除含羟丙基-β-环糊精外，还添加大量的吐温80和聚乙二醇400助溶，处方工艺较复杂。②冻干产品中添加聚乙二醇400导致产品外观出现萎缩、塌陷和不饱满；③配方中含吐温80，易导致溶血和过敏反应，刺激性强。
中国专利文献CN1424035A公开了一种尼莫地平冻干组合物，组合物含有磷脂、环糊精及其衍生物或表面活性剂，其中尼莫地平和环糊精及其衍生物质量比为1:1至1:20。中国专利文献CN1653089A公开了一种有机药物与β-环糊精衍生物配合物及其制备方法，其中尼莫地平/羟丙基-β-环糊精质量比为1：136。
以上专利普遍存在以下问题：①羟丙基-β环糊精和药物的相对比例过高，包合效果差。②配方中添加大量有机溶媒助溶，刺激性强。③大量有机溶媒的存在不利于环糊精包合。综上所述，解决以上环糊精包合物的技术缺陷仍是目前研究的热点。近年来，含磺丁基醚-β-环糊精的注射剂陆续上市，磺丁基醚-β-环糊精在药物制剂领域的应用受到研究人员的广泛关注。文献报道磺丁基醚-β-环糊精对尼莫地平的增溶效果显著优于羟丙基-β-环糊精，相较于羟丙基-β-环糊精，磺丁基醚-β-环糊精是尼莫地平更为理想的载体。(Semcheddine F,et al.Effects of the Preparation Method on the Formation of True Nimodipine SBE-β-CD/HP-β-CD Inclusion Complexes and Their Dissolution Rates Enhancement.AAPS PharmSciTech.2015；16(3):704-715.)。
经文献检索发现，有学者采用研磨法制备尼莫地平磺丁基醚-β-环糊精包合物，该法制备的包合物载药量低，制备完成后还需抽滤和洗涤除去未溶解的药物，重复性差，产品质量难以保证，不适合工业化生产(杨星昊,任勇.尼莫地平/磺丁醚-β-环糊精固体包合物的性质特征研究[J].中国现代应用药学,2003(05):380-383.)。许多研究人员采用振摇法制备尼莫地平磺丁基醚-β-环糊精包合物。Serena等将过量尼莫地平加入到50mM磺丁基醚-β-环糊精水溶液中混合，室温摇床振摇48h，制备尼莫地平磺丁基醚-β-环糊精包合物。(Tongiani S,et al.Sulfobutyl ether-alkyl ether mixed cyclodextrin derivatives with enhanced inclusion ability.J Pharm Sci.2009；98(12):4769-4780.)；Farouk等将过量尼莫地平加入到16mM磺丁基醚-β-环糊精水溶液中混合，37℃摇床振摇72h，制备尼莫地平磺丁基醚-β-环糊精包合物。(Semcheddine F,et al.Effects of the  Preparation Method on the Formation of True Nimodipine SBE-β-CD/HP-β-CD Inclusion Complexes and Their Dissolution Rates Enhancement.AAPS PharmSciTech.2015；16(3):704-715.)；Doaa Nabih等将过量尼莫地平加入到磺丁基醚-β-环糊精水溶液中混合，形成的悬浮液涡旋5min后，超声15min，最后37℃摇床振摇72h，制备尼莫地平磺丁基醚-β-环糊精包合物。(Maria DN,et al.Nimodipine Ophthalmic Formulations for Management of Glaucoma.Pharm Res.2017；34(4):809-824.)。以上文献均采用振摇法制备包合物，该方法制备的环糊精包合物的包合效果不佳，此外由于这些文献没有对磺丁基醚-β-环糊精用量、包合温度、包合时间、乙醇用量、磺丁基醚-β-环糊精和尼莫地平质量比(辅药比)以及搅拌速度等进行系统地研究，导致普遍存在制备时间长、尼莫地平溶解度低和辅药比高的问题。
综上所述，现有技术普遍存在环糊精包合效果差，制备时间长，辅药比高；配方中除含环糊精外还需添加大量有机溶媒助溶，不利于环糊精包合的同时，增加制剂的刺激性和毒性，安全性低。因此研发一种安全、稳定的尼莫地平注射剂具有重要意义，可为提高尼莫地平临床用药的安全性和便利性奠定坚实的基础。
发明内容
基于上述现有技术并未从根本上改变尼膜同注射液稀释稳定性差、安全性低的缺陷，本发明目的在于提供一种安全、稳定的注射用尼莫地平及其制备方法。
本发明通过磺丁基醚-β-环糊精增加尼莫地平的溶解性。在前期文献调研及实验验证的基础上发现，磺丁基醚-β-环糊精是尼莫地平较为理想的载体，然而目前对尼莫地平磺丁基醚-β-环糊精包合物的研究较少，尚未开发出一款适合工业化生产、可应用于临床的尼莫地平磺丁基醚-β-环糊精包合物产品。
发明人按照中国专利文献CN1634050A制备尼莫地平冻干粉针。配方含吐温80，易导致溶血和过敏反应，刺激性强。且制备所得的冻干粉针空隙疏松，萎缩塌陷(见实施例1)，不合格的冻干粉针外观影响产品质量。
发明人按照中国专利文献CN1424035A中实施例2制备了含2-羟丙基-β-环糊精的尼莫地平粉针剂。多次实验均发现制备过程中药物析出，即使按该专利中最大辅料占比(药物/2-羟丙基-β-环糊精质量比1:20)制备尼莫地平环糊 精包合物，药物也无法被完全包合，实际测得辅药比为571(见实施例2)。
发明人按照中国专利文献CN1653089A中的实施例28操作，经过多次实验验证，药物在制备过程中均析出，实际测得辅药比为1904(见实施例3)，远高于本发明的辅药比。
发明人还采用了文献中采用的振摇法制备尼莫地平磺丁基醚-β-环糊精包合物，37℃条件下振摇72h，配方中添加适量乙醇助溶，制备的尼莫地平磺丁基醚-β-环糊精包合物包合效果较差，制备时间较长，不利于注射剂的工业化生产(见实施例4)。
本发明通过大量实验研究发现磺丁基醚-β-环糊精用量、包合温度、包合时间、乙醇用量、辅药比和搅拌速度显著影响磺丁基醚-β-环糊精对尼莫地平的包合效果。本发明通过磺丁基醚-β-环糊精用量、包合温度、包合时间、乙醇用量和搅拌速度的调整可显著降低辅药比，达到理想的载药浓度，且制剂的安全性和稀释稳定性均良好。此外，本发明配方中可含少量乙醇，将尼莫地平溶解在少量乙醇中可提高药物在磺丁基醚-β-环糊精中水溶液中的溶解度，增加制备过程的便利性，且乙醇可在制备过程中除去，在产品残留较少(见实施例33和65)。
本发明的第一方面，提供一种安全、稳定的注射用尼莫地平，由磺丁基醚-β-环糊精和尼莫地平制成，所述的磺丁基醚-β-环糊精和尼莫地平质量比为200:1-700:1。
进一步的，所述的磺丁基醚-β-环糊精和尼莫地平质量比为350:1-700:1。
进一步的，所述的注射用尼莫地平冻干前尼莫地平含量为0.01-0.14％克/毫升，磺丁基醚-β-环糊精含量为10-50％克/毫升。
进一步的，所述的磺丁基醚-β-环糊精和尼莫地平质量比为400:1-600:1，尼莫地平含量为0.02-0.10％克/毫升，磺丁基醚-β-环糊精浓度为10-40％克/毫升。
进一步优选的，所述的磺丁基醚-β-环糊精和尼莫地平质量比为450:1-550:1，尼莫地平含量为0.04-0.06％克/毫升，磺丁基醚-β-环糊精浓度为20-30％克/毫升。
本发明的一种安全、稳定的尼莫地平注射剂，由以下成分制成：

进一步的，所述的尼莫地平注射剂，由以下成分制成：
进一步的，所述的尼莫地平注射剂，由以下成分制成：
更进一步的，所述的尼莫地平注射剂，由以下成分制成：
更进一步的，上述的尼莫地平注射剂中不含乙醇。
进一步的，上述的尼莫地平注射剂中还可含有缓冲剂，调节pH值至4.5-7.5；所述的缓冲剂选自枸橼酸钠-枸橼酸、磷酸氢二钠-枸橼酸、磷酸氢二钠-磷酸二氢钠、磷酸氢二钠-磷酸二氢钾和磷酸二氢钾-氢氧化钠其中一种。
本发明的第二方面，提供一种如上所述的安全、稳定的尼莫地平注射剂的制备方法，包括以下步骤：
(a)称取处方量的磺丁基醚-β-环糊精(和缓冲剂)，加入适量的注射用水，在一定温度下搅拌使溶解，得磺丁基醚-β-环糊精水溶液；
(b)称取处方量的尼莫地平，加入处方量的乙醇溶解，即得尼莫地平乙醇溶液；
(c)在一定的搅拌温度和速度条件下将步骤(b)溶液或尼莫地平药物粉末加入到步骤(a)溶液中，搅拌一定时间，加注射用水至全量，0.22μm微孔滤膜无 菌过滤，灌装，冷冻干燥，封装，即得所述的安全、稳定的尼莫地平注射剂。
进一步的，步骤(a)和(c)所述的搅拌温度为20-100℃，进一步优选为40-95℃，更优选为60-90℃。
进一步的，步骤(c)所述的搅拌时间为10-300min，进一步优选为30-240min，更优选为60-180min。
进一步的，步骤(c)所述的搅拌线速度为0.5-10.0m/s，进一步优选为0.8-6.0m/s，更优选为1.0-3.0m/s。
进一步的，步骤(a)、(b)和(c)中可通入洁净干燥的氮气(N2)至饱和，包括冷冻干燥后充氮，封装。
进一步的，步骤(c)所述的注射用尼莫地平乙醇残留量低于0.3％，进一步优选低于0.1％，更进一步优选低于0.05％。
本发明通过大量实验研究发现磺丁基醚-β-环糊精用量、包合温度、包合时间、乙醇用量、辅药比和搅拌速度显著影响磺丁基醚-β环糊精对尼莫地平的包合效果。通过本发明的控制条件可显著降低磺丁基醚-β-环糊精和尼莫地平相对比例，提高制剂的安全性和稀释稳定性，克服市售尼膜同注射液的缺陷。故本发明对磺丁基醚-β-环糊精用量、包合温度、包合时间、乙醇用量、辅药比和搅拌速度的控制，是本发明的核心技术所在。
本发明优点在于：
1、本发明的注射用尼莫地平的安全性显著提高，避免使用大量有机溶媒带来的毒性问题，降低制剂刺激性，提高患者使用顺应性。
2、稀释稳定性提高。注射用尼莫地平重建后可直接分散至0.9％氯化钠注射液或5％葡萄糖注射液中，不需要使用三通阀，提高使用便利性。
3、本发明的注射用尼莫地平中磺丁基醚-β-环糊精与尼莫地平相对比例更小，不仅可减少生产成本，并可降低由环糊精可能带来的安全隐患。
4、增加生产的便利性。与普遍采用的振摇法相比，本专利通过优化制备工艺，可大大缩短制备时间；同时少量乙醇的使用不仅缩短了包合时间，而且配方中少量的乙醇也不需要特殊的工序去除，并能做到终产品乙醇残留非常低，增加了生产便利性，缩短了生产周期。
综上所述，本发明提供了一种含磺丁基醚-β-环糊精的尼莫地平环糊精冻干粉针，可显著提高尼莫地平溶解性、用药安全性和稀释稳定性，增加患者使用 的顺应性及临床使用的便捷性。本发明与市售尼膜同注射液相比，毒性明显降低，在安全性、稀释稳定性以及临床使用便利性都得到显著提高，具有明显优势；与尼莫地平环糊精包合物现有技术相比，避免了大量有机溶剂、表面活性剂等使用，极大地降低辅药比，缩短生产周期，具有非常好的应用前景。
附图说明
图1.含量测定供试品溶液HPLC色谱图；
图2.注射用尼莫地平溶血作用图；
图3.有关物质测定供试品溶液HPLC色谱图；
图4.注射用尼莫地平及尼膜同注射液稀释稳定性图；
图5.注射用尼莫地平血管刺激性病理切片图；
图6.注射用尼莫地平溶血作用图；
图7.注射用尼莫地平药效学评价图；
图8.注射用尼莫地平血药浓度-时间曲线图。
具体实施方式
下面结合实施例对本发明提供的具体实施方式作详细说明。应理解，以下实施例仅用于说明本发明而非限定本发明的范围。
实施例1：聚乙二醇400对尼莫地平冻干粉针的影响
参照专利文献CN1634050A实施例1进行操作实验，制备尼莫地平冻干粉针。
1.处方
2.制备工艺
将处方量的尼莫地平加入到聚乙二醇400和吐温-80的混合物中，加热并搅拌使溶解。将羟丙基-β-环糊精加入到注射用水中，加热并搅拌使溶解，边搅拌边加入上述溶液，继续搅拌，至室温。加入适量活性炭，60℃保温20min 并搅拌。过滤，灌装，冷冻干燥，即得。
3.实验结果
该配方制备所得的冻干粉针空隙疏松，外观呈萎缩、塌陷和不饱满的状态，不合格的冻干粉针外观影响产品质量。
实施例2：尼莫地平羟丙基-β-环糊精包合物的制备
参照中国专利文献CN1424035A实施例2进行操作实验，制备尼莫地平羟丙基-β-环糊精包合物冻干粉针。
1.处方
2.制备工艺
取尼莫地平2mg，用2mL无水乙醇搅拌至完全溶解，取羟丙基-β-环糊精20mg，加7mL注射用水溶解，搅拌下将尼莫地平乙醇溶液加入到羟丙基-β-环糊精水溶液中，制成均匀的环糊精包合物。在旋转蒸发仪上减压干燥除去乙醇，加入甘露醇300mg，溶液用事先经灭菌处理过的垂熔漏斗过滤，滤液置无菌西林瓶中，高效液相色谱法测定含量，冷冻干燥，封口，即得尼莫地平粉针剂。
3.本发明中的尼莫地平含量测定方法照高效液相色谱法(2020年版中国药典第四部通则0512)测定，避光操作。
色谱条件与系统适用性试验：用十八烷基硅烷键合硅胶为填充剂(C18柱250×4.6mm，5μm)；以甲醇-乙腈-水(35:38:27)为流动相；检测波长为235nm；进样体积10μL。理论板数按尼莫地平峰计算不低于8000，尼莫地平峰与相邻杂质峰的分离度应符合要求。
供试品溶液的制备：精密量取制备的溶液1mL，置50mL量瓶中，用流动相稀释至刻度，摇匀。供试品溶液含量测定HPLC色谱图如图1所示。
对照品溶液的制备：取尼莫地平对照品，精密称定，加流动相溶解并定量稀释制成每1mL中约含20μg的溶液。
测定法：精密量取供试品溶液与对照品溶液，分别注入液相色谱仪，记录 色谱图。按外标法以峰面积计算。
4.实验结果
尼莫地平乙醇溶液和羟丙基-β-环糊精水溶液混合搅拌过程中，药物析出。样品过滤后，测定溶液中药物含量为0.005mg/mL，计算得辅药比为571。
5.结果分析
该专利技术使用了22％乙醇不利于环糊精对尼莫地平包合，而且大量的乙醇存在一定的安全隐患；此外，需要通过旋转蒸发除去乙醇增加了工艺的复杂性。
实施例3：尼莫地平羟丙基-β-环糊精包合物的制备
参照中国专利文献CN1653089A实施例28进行操作实验，制备尼莫地平羟丙基-β-环糊精包合物。
1.处方
2.制备工艺
将0.5g尼莫地平溶于处方量乙醇中，称取68g羟丙基-β-环糊精溶于适量水中，上述溶液混合，60℃搅拌加热，加活性炭脱热源，过滤，滤液输入1立升旋转罐中，保温70-80℃，减压浓缩，回收溶剂，并转化为水系统，继续保温，减压使物料膨化，干燥。
3.实验结果
经过多次实验重复验证，尼莫地平乙醇溶液和羟丙基-β-环糊精水溶液混合搅拌过程中，药物即析出；在减压干燥除去乙醇过程中药物也大量析出。样品经过滤后，测定溶液中药物含量为0.21mg/mL,计算得辅药比为1904，远高于本发明的辅药比。
实施例4：振摇法制备尼莫地平磺丁基醚-β-环糊精包合物
参照文献采用振摇法制备尼莫地平磺丁基醚-β-环糊精包合物(Maria DN,et al.Nimodipine Ophthalmic Formulations for Management of Glaucoma.Pharm Res.2017；34(4):809-824.)，另外，采用同样的配方含少量乙醇振摇法制备尼莫地平磺丁基醚-β-环糊精包合物，进行对比。
1.处方
表1处方设计
2.制备工艺
分别取磺丁基醚-β-环糊精1.3g，平行两组，加入适量注射用水，搅拌使溶解，得磺丁基醚-β-环糊精水溶液；一组不添加乙醇，另一组取处方量的乙醇加入到磺丁基醚-β-环糊精水溶液中混合，两组分别加注射用水补足至30mL，置于西林瓶中，加入处方量的尼莫地平，加塞压盖，悬浮液涡旋5min后，超声15min，37℃条件下电热恒温水浴锅振荡72h。所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量。
3.实验结果
表2振摇法制备的尼莫地平磺丁基醚-β-环糊精包合物溶解性
结果表明,即使处方中添加少量的乙醇助溶,振摇72h制备的尼莫地平磺丁基醚-β-环糊精包合物包合效果较差，制备时间较长，不利于注射剂的工业化生产。
实施例5：不同溶剂对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响
1.处方
表3处方设计

2.制备工艺
取磺丁基醚-β-环糊精9g，平行5组，加入适量注射用水，30℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平45mg，加入0.9mL的溶剂溶解，在搅拌条件下将尼莫地平溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量。
3.实验结果
表4不同溶剂对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响
结果表明，添加适量的有机溶剂有利于环糊精对尼莫地平的增溶作用，其中效果最佳为乙醇。
实施例6：乙醇用量对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响
1.处方
表5处方设计
2.制备工艺
取磺丁基醚-β-环糊精6g，平行7组，加入适量注射用水，30℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，分别加入0.6mL、0.9mL、 1.2mL、1.5mL、1.8mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量。
3.实验结果
对制备的尼莫地平环糊精中进行含量测定，结果如下：
表6不同乙醇用量对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响
实验结果表明，随着乙醇用量的增加，尼莫地平在磺丁基醚-β-环糊精水溶液中溶解度降低，辅药比升高。
实施例7：磺丁基醚-β-环糊精用量对尼莫地平溶解性的影响
1.处方
表7处方设计
2.制备工艺
分别取3g、6g、7.5g、9g、10.5g、12g、13.5g、15g的磺丁基醚-β-环糊精，加入适量的注射用水，30℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平45mg，加入0.9mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量。
3.实验结果
对制备的尼莫地平环糊精中进行含量测定，结果如下：
表8不同磺丁基醚-β-环糊精用量对尼莫地平溶解性的影响
实验结果表明，当磺丁基醚-β-环糊精用量小于45％时，随着磺丁基醚-β-环糊精用量的增加，尼莫地平溶解度增加，辅药比显著降低；而当磺丁基醚-β-环糊精用量大于45％时，尼莫地平溶解度未明显增加，辅药比增大。
实施例8：溶解温度和时间对尼莫地平溶解性的影响
1.处方
表9处方设计
2.制备工艺
取磺丁基醚-β-环糊精12g，平行9组，加入适量注射用水，搅拌使溶解，得磺丁基醚-β-环糊精水溶液；分别取尼莫地平60mg，加入1.2mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，分别在20℃、30℃、40℃、50℃、60℃、70℃、80℃、90℃、100℃搅拌5h，于1h、2h、3h、4h、5h取点，所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量。
3.结果分析
对不同时间所取的尼莫地平环糊精中进行含量测定，结果如下
表10不同温度尼莫地平搅拌5h的溶解性
表11不同温度搅拌5h磺丁基醚-β-环糊精和尼莫地平比例
实验表明随着温度的升高，尼莫地平溶解度增加，辅药比降低；在温度较低的情况下，随着搅拌时间的增加，尼莫地平溶解度降低，辅药比升高。这表明提高温度有利于磺丁基醚-β-环糊精对尼莫地平的包合，但温度过高可能导致有关物质的产生。
实施例9：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精3g，加入适量注射用水，20℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平3mg，加入0.06mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌10 min，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例10：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精6g，加入适量注射用水，40℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，加入0.3mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌30min，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例11：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精10.5g、枸橼酸钠60mg和枸橼酸9mg，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例12：注射用尼莫地平的制备
1.处方

2.制备工艺
取磺丁基醚-β-环糊精9g，加入适量注射用水，50℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平24mg，加入0.48mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例13：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精10.8g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平24mg，加入0.3mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例14：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精12g、磷酸氢二钠709mg和枸橼酸102mg，加入适 量注射用水，70℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平24mg，在搅拌条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例15：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精9g，加入适量注射用水，50℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.3mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例16：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精9.9g、磷酸氢二钠520mg和磷酸二氢钠281mg，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.3mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例17：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精11.1g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.48mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例18：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精9g、枸橼酸钠60mg和枸橼酸9mg，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.48mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例19：注射用尼莫地平的制备
1.处方

2.制备工艺
取磺丁基醚-β-环糊精9g，加入适量注射用水，100℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.72mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例20：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精10.5g，加入适量注射用水，50℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.48mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例21：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精10.5g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.72mL乙醇溶解，在搅拌 条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例22：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精10.5g，加入适量注射用水，70℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.9mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例23：注射用尼莫地平的制备
1.处方
尼莫地平            30mg
磺丁基醚-β-环糊精  10.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精10.5g，加入适量注射用水，80℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，在搅拌条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例24：注射用尼莫地平的制备
1.处方

2.制备工艺
取磺丁基醚-β-环糊精12g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.9mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例25：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精12g、磷酸氢二钠520mg、磷酸二氢钠281mg，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，在搅拌条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例26：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精13.5g，加入适量注射用水，50℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，加入0.48mL乙醇溶解，在搅拌 条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例27：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精12g，加入适量注射用水，70℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平36mg，加入0.72mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例28：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精12.6g、磷酸氢二钠59mg和磷酸二氢钾6.7mg，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平36mg，加入0.72mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例29：注射用尼莫地平的制备
1.处方
尼莫地平            36mg
磺丁基醚-β-环糊精  13.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精13.5g，加入适量注射用水，100℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平36mg，在搅拌条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例30：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精13.5g，加入适量注射用水，80℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平45mg，加入0.9mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌4h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例31：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精15g，加入适量注射用水，100℃搅拌使溶解，得磺 丁基醚-β-环糊精水溶液；取尼莫地平60mg，加入1.5mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例32：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精12g、磷酸二氢钾204mg和氢氧化钠3.5mg，加入适量注射用水，100℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平60mg，加入0.9mL乙醇溶解，在搅拌条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。。
实施例33：注射用尼莫地平的乙醇残留测定
选取实施例9、10、12、13、15、16、17、18、19、20、21、22、24、26、27、28、30、31、32制备的注射用尼莫地平，检测乙醇残留。
1.注射用尼莫地平中乙醇含量照残留溶剂测定法(中国药典2020年版四部通则0861第二法)测定，如下：
色谱条件：
以DB-624毛细管柱为色谱柱；程序升温，起始温度为70℃，维持2min，先以每分钟10℃的速率升温至120℃，然后再以每分钟20℃的速率升温至220℃，维持3min；分流比为20：1；柱流量1mL/min；进样口温度为200℃；检测器温度为250℃；顶空瓶平衡温度为80℃，平衡时间为30min。
空白溶液的制备：
分别精密称取制备的空白辅料冻干粉2g，置于3个20mL容量瓶中，用 DMF稀释至刻度，摇匀，精密量取4mL，置顶空瓶中，密封，即得。
对照品溶液的制备：
取乙醇适量，精密称定，用DMF稀释制成每1mL中含5mg的溶液，作为对照品储备液。精密量取对照品储备液2mL，置20mL容量瓶中，用DMF稀释至刻度，摇匀，精密量取4mL，置顶空瓶中，密封，即得对照品溶液。
供试品溶液的制备：
精密称取注射用尼莫地平2g，置于3个20mL容量瓶中，用DMF稀释至刻度，摇匀，精密量取4mL，置顶空瓶中，密封，即得供试品溶液。
测定法：
取空白溶液及对照品溶液顶空进样，记录色谱图。再取供试品溶液与对照品溶液分别顶空进样，记录色谱图，按外标法以峰面积计算。
2.实验结果
表12注射用尼莫地平乙醇残留结果
结果表明，通过温度和时间的控制，本发明的注射用尼莫地平乙醇残留均满足质量标准要求。
实施例34：注射用尼莫地平的稀释稳定性研究
1.实验方法
取实施例21制备的注射用尼莫地平重建后以及尼膜同注射液，分别用0.9％氯化钠注射液和5％葡萄糖稀释至临床给药浓度。于0、3、6、9、12、24h观察并取点，0.45μm聚醚砜滤膜过滤后，按上述含量测定方法测定尼莫地平含量。
2.实验结果
表13注射用尼莫地平及尼膜同注射液0.9％氯化钠注射液稀释后的含量变化

注：-表示未析出，+表示析出。
表14注射用尼莫地平及尼膜同注射液5％葡萄糖稀释后的含量变化


注：-表示未析出，+表示析出。
尼膜同注射液经0.9％氯化钠注射液或5％葡萄糖稀释后，3h药物即大量析出，含量显著下降，24h后含量分别仅剩2.30％和6.24％，稀释稳定性极差；本发明的注射用尼莫地平经0.9％氯化钠注射液或5％葡萄糖稀释后，24h内含量未发生明显变化，外观澄明，稀释稳定性良好。以上结果表明，相较于尼膜同注射液，本发明的注射用尼莫地平稀释稳定性显著提高，具有明显优势。
实施例35：注射用尼莫地平溶血性研究
1.实验方法
分别用0.9％氯化钠注射液稀释实施例21制备的注射用尼莫地平及市售尼膜同注射液至临床给药浓度0.04mg/mL。取10mL干净玻璃试管8支，编号，1至5号管为不同浓度的注射用尼莫地平，6号管为阴性对照管，7号管为阳性对照管，8号为市售尼膜同注射液。按下表所示，依次加入2％红细胞悬液、0.9％氯化钠注射液、蒸馏水、药液，混匀后，立即置于37±0.5℃水浴中温育，观察并记录各管的溶血情况。开始每隔15分钟记录一次，1小时后，每隔1小时观察一次，观察3小时。
表15溶血实验设计表
2.实验结果
溶血情况如图2所示，本发明的注射用尼莫地平(1-5号)均未发生溶血现象，表明本发明的注射用尼莫地平安全性良好。
实施例36：注射用尼莫地平高温稳定性研究
选取实施例21制备的注射用尼莫地平于60℃条件下放置1月，检测药物含量及有关杂质变化。
1.本发明中的尼莫地平有关物质测定方法照高效液相色谱法(2020年版中国药典第四部通则0512)测定，避光操作。
色谱条件与系统适用性试验：用十八烷基硅烷键合硅胶为填充剂(C18柱250×4.6mm，5μm)；以甲醇-乙腈-水(35:38:27)为流动相；检测波长为235nm；进样体积10μL。系统适用性溶液色谱图中，尼莫地平峰与杂质B、C、I峰的分离度应大于3.0。
供试品溶液的制备：精密量取本品适量，用流动相定量稀释制成每1mL中约含尼莫地平0.2mg的溶液。供试品溶液的有关物质测定HPLC色谱图如图3所示。
对照溶液的制备：精密量取供试品溶液1mL，置100mL量瓶中，用流动相定量稀释至刻度，摇匀。
杂质I对照品溶液：精密称取杂质I对照品，用流动相定量稀释制成每1mL中约含1μg的溶液。
系统适用性溶液的制备：取尼莫地平与杂质B、C、I对照品适量，加流动相溶解并稀释制成每1mL中各约含200μg与1μg的混合溶液。
测定法：精密量取供试品溶液、杂质I对照品溶液和对照溶液，分别注入液相色谱仪。供试品溶液色谱图中如有杂质峰，除相对保留时间小于0.45的色谱峰不计外，与杂质I峰保留时间一致的色谱峰，按外标法以峰面积计算；其他单个杂质以主成分自身对照法计算，。
2.实验结果
表16注射用尼莫地平高温稳定性实验结果

2.结果分析
注射用尼莫地平于60℃稳定性试验箱储存1个月后，尼莫地平标示百分含量几乎无变化，有关物质符合规定的限度要求。以上结果表明，本发明的注射用尼莫地平高温稳定性良好，质量符合相关规定。
实施例37：制备温度和时间对尼莫地平在磺丁基醚-β-环糊精溶解性的影响以尼莫地平含量和有关物质为指标，考察制备温度和时间对尼莫地平在磺丁基醚-β-环糊精溶解性以及有关物质的影响。
1.处方
表17处方设计
2.制备工艺
取磺丁基醚-β-环糊精12g，平行9组，加入适量注射用水，搅拌使溶解，得磺丁基醚-β-环糊精水溶液；分别取尼莫地平60mg，在搅拌线速度为2.0m/s条件下，将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，分别在20℃、30℃、40℃、50℃、60℃、70℃、80℃、90℃、100℃搅拌5h，于1h、2h、3h、4h、5h取点，所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量及有关物质。
3.实验结果
对不同时间所取的尼莫地平环糊精包合物中进行含量及有关物质测定，结果如下。
表18不同温度尼莫地平搅拌5h在磺丁基醚-β-环糊精中溶解性

表19不同温度搅拌5h磺丁基醚-β环糊精和尼莫地平比例
表20不同温度下搅拌5h尼莫地平环糊精的有关物质

实验表明，在不添加乙醇的情况下，尼莫地平溶解度随着温度的升高和搅拌时间的增加而增大，辅药比降低；尼莫地平有关物质随温度的升高和搅拌时间的增加而增大。因此制备尼莫地平磺丁基醚-β-环糊精包合物需选择适当的制备温度和时间。
实施例38：搅拌速度对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响
1.处方
表21处方设计
2.制备工艺
取磺丁基醚-β-环糊精25g，平行8组，加入适量的注射用水，75℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平50mg，分别在搅拌线速度为0.25m/s、0.5m/s、1.0m/s、1.5m/s、2.0m/s、3.0m/s、6.0m/s、10.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌1.5h，加注射用水补足至100mL，所得溶液经0.45μm聚醚砜滤膜过滤，HPLC法检测尼莫地平含量。
3.实验结果
搅拌速度对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响如表22所示。结果表明，药物含量随着搅拌速度增大而增加，搅拌速度过低无法达到理想的载药浓度。
表22搅拌速度对尼莫地平在磺丁基醚-β-环糊精中溶解性的影响
4.讨论
研究表明，搅拌速度显著影响磺丁基醚-β-环糊精对尼莫地平的包合效果，搅拌速度越大，包合效果越好，药物含量越高。我们通过实验意外地发现，在不添加乙醇作为助溶剂的情况下，通过调整搅拌速度也可以达到较为理想的载药浓度。搅拌速度是本发明重要的核心技术之一，因此我们在权利要求中添加了搅拌速度的保护范围。
实施例39：辅药比对尼莫地平环糊精包合物稀释稳定性的影响
1.处方
表23处方设计
2.制备工艺
分别取15g、17.5g、20g、22.5g、25g、30g、35g的磺丁基醚-β-环糊精，加入适量的注射用水，75℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平50mg，在搅拌线速度为1.8m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌1.5h，加注射用水补足至100mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
将制备的注射用尼莫地平分别用0.9％氯化钠注射液稀释至0.2mg/mL药物浓度。于0、12、24、36、48、60h观察并取点，0.45μm聚醚砜滤膜过滤后，按上述含量测定方法测定尼莫地平含量。
3.实验结果
稀释稳定性结果如表24所示，当辅药比≥350，注射用尼莫地平稀释稳定时间可达24h以上；当辅药比≥400，注射用尼莫地平稀释稳定时间可达36h以上；当辅药比≥450，注射用尼莫地平稀释稳定时间可达48h以上；当辅药比≥500，注射用尼莫地平稀释稳定时间可达60h以上。结果表明，辅药比显著影响尼莫地平环糊精包合物稀释稳定性。
表24不同辅药比的尼莫地平环糊精包合物稀释稳定性
4.讨论
研究结果表明，尼莫地平环糊精包合物稀释稳定时间随辅药比增大而增加。按照说明书推荐的用药方案，最大规格的尼膜同注射液(250mL:50mg)最长持续给药时间为51h。研究发现，当辅药比≥500时尼莫地平环糊精包合物时，稀释稳定时间可达60h以上，满足临床使用需求。基于上述原因，我们提高了权利要求中磺丁基醚-β-环糊精和尼莫地平质量比的范围。
实施例40：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精3g，加入适量注射用水，70℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平3mg，加入0.07mL乙醇溶解，在搅拌速度为3.0m/s条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液中，搅拌10min，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过 滤，灌装，冷冻干燥，封装，即得。
实施例41：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精3g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平6mg，在搅拌速度为2.0m/s条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液中，搅拌30min，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例42：注射用尼莫地平的制备
1.处方
尼莫地平            6mg
磺丁基醚-β-环糊精  3g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精3g，加入适量注射用水，20℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平6mg，在搅拌速度为10.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌4h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例43：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精4.5g、枸橼酸钠60mg和枸橼酸9mg，加入适量注射用水，90℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平11.25mg，在搅拌速度为0.5m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，
实施例44：注射用尼莫地平的制备
1.处方
尼莫地平            12mg
磺丁基醚-β-环糊精  6g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精6g，加入适量注射用水，70℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平12mg，在搅拌速度为2.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例45：注射用尼莫地平的制备
1.处方
尼莫地平            12mg
磺丁基醚-β-环糊精  6g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精6g，加入适量注射用水，85℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平12mg，在搅拌速度为0.8m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例46：注射用尼莫地平的制备
1.处方

2.制备工艺
取磺丁基醚-β-环糊精6.3g、磷酸氢二钠709mg和枸橼酸102mg，加入适量注射用水，40℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平14mg，在搅拌速度为6.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例47：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精6.6g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平14.67mg，加入0.3mL乙醇溶解，在搅拌速度为2.0m/s条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例48：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，75℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌速度为1.8m/s条件下将 尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌1.5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例49：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，80℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，，在搅拌速度为1.7m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例50：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，90℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌速度为1.5m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例51：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，75℃搅拌使溶解，得磺 丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌速度为1.9m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌1.5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例52：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，65℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌速度为2.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例53：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，75℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌速度为2.5m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例54：注射用尼莫地平的制备
1.处方
尼莫地平            15mg
磺丁基醚-β-环糊精  7.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，60℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，在搅拌速度为3.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例55：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精7.5g，加入适量注射用水，70℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平15mg，加入0.45mL乙醇溶解，在搅拌速度为2.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌1h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例56：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精9g，加入适量注射用水，90℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平18mg，加入0.75mL乙醇溶解，在搅拌速度为1.0m/s条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例57：注射用尼莫地平的制备
1.处方
尼莫地平            18mg
磺丁基醚-β-环糊精  9g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精9g，加入适量注射用水，75℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平18mg，在搅拌速度为3.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例58：注射用尼莫地平的制备
1.处方
尼莫地平            19.10mg
磺丁基醚-β-环糊精  10.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精10.5g，加入适量注射用水，40℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平19.10mg，在搅拌速度为6.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例59：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精11.4g、磷酸二氢钾204mg和氢氧化钠3.5mg，加 入适量注射用水，90℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平20.72mg，在搅拌速度为1.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例60：注射用尼莫地平的制备
1.处方
尼莫地平            30mg
磺丁基醚-β-环糊精  12g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精12g，加入适量注射用水，95℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，在搅拌速度为0.8m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌4h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例61：注射用尼莫地平的制备
1.处方
尼莫地平            30mg
磺丁基醚-β-环糊精  12g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精12g，加入适量注射用水，80℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平30mg，在搅拌速度为2.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌3h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例62：注射用尼莫地平的制备
1.处方
尼莫地平            22.5mg
磺丁基醚-β-环糊精  13.5g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精13.5g，加入适量注射用水，75℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平22.5mg，在搅拌速度为3.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例63：注射用尼莫地平的制备
1.处方
2.制备工艺
取磺丁基醚-β-环糊精15g，加入适量注射用水，100℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平42mg，加入1.05mL乙醇溶解，在搅拌速度为1.7m/s条件下将尼莫地平乙醇溶液加入到磺丁基醚-β-环糊精水溶液中，搅拌1.5h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得。
实施例64：注射用尼莫地平的制备
1.处方
尼莫地平            21.43mg
磺丁基醚-β-环糊精  15g
注射用水            补足至30mL
2.制备工艺
取磺丁基醚-β-环糊精15g，加入适量注射用水，95℃搅拌使溶解，得磺丁基醚-β-环糊精水溶液；取尼莫地平21.43mg，在搅拌速度为2.0m/s条件下将尼莫地平加入到磺丁基醚-β-环糊精水溶液中，搅拌2h，加注射用水补足至30mL，所得溶液经0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，充氮，封装，即得。整个溶解及过滤过程通入洁净干燥N2。
实施例65：注射用尼莫地平的乙醇残留测定
1.实验方法
选取实施例40、41、47、55、56、63制备的注射用尼莫地平，检测乙醇残留。注射用尼莫地平中乙醇含量照残留溶剂测定法(中国药典2020年版四部通则0861第二法)测定。
2.实验结果
表25注射用尼莫地平乙醇残留结果
结果表明，通过温度和时间的控制，本发明的注射用尼莫地平乙醇残留均较低。
实施例66：注射用尼莫地平的稀释稳定性研究
1.实验方法
取实施例48制备的注射用尼莫地平加入注射用水复溶后以及尼膜同注射液，分别用0.9％氯化钠注射液和5％葡萄糖稀释至临床给药浓度0.04mg/mL。于0、3、6、9、12、24h观察并取点，0.45μm聚醚砜滤膜过滤后，按上述含量测定方法测定尼莫地平含量。
2.实验结果
注射用尼莫地平及尼膜同注射液经0.9％氯化钠注射液稀释稳定性结果如表26和27所示，溶液外观如图4所示。
表26注射用尼莫地平及尼膜同注射液0.9％氯化钠注射液稀释后的含量变化


注：“-”表示药物无析出，“+”表示药物析出。
表27注射用尼莫地平及尼膜同注射液5％葡萄糖稀释后的含量变化

注：“-”表示药物无析出，“+”表示药物析出。
尼膜同注射液经0.9％氯化钠注射液或5％葡萄糖稀释后，3h药物即大量析出，24h药物含量显著下降，稀释稳定性极差；本发明的注射用尼莫地平经0.9％氯化钠注射液或5％葡萄糖稀释后，24h内含量未发生明显变化，外观澄明，稀释稳定性良好。以上结果表明相较于尼膜同注射液，本发明的注射用尼莫地平稀释稳定性显著提高，具有明显优势。
实施例67：注射用尼莫地平影响因素及加速稳定性考察
1.实验方法
选取实施例48制备的注射用尼莫地平，按照2020年版《中华人民共和国药典》中《9001原料药物与制剂稳定性试验指导原则》，考察注射用尼莫地平 高温、高湿、光照及加速试验稳定性，检测药物含量及有关物质变化。
2.实验结果
表28注射用尼莫地平稳定性考察结果
3.结果分析
注射用尼莫地平在高温、高湿、加速试验条件下，尼莫地平含量和有关物质几乎无变化。但在光照强度条件下放置10天，注射用尼莫地平含量仅剩96.59％，有关物质增加了0.11％。可能是由于尼莫地平光照条件下较不稳定，易分解，即使被环糊精包合后，仍然在光照条件下降解，表明注射用尼莫地平应避光储存。以上结果表明，本发明的注射用尼莫地平稳定性良好，质量符合相关规定。
实施例68：注射用尼莫地平血管刺激性研究
1.实验方法
取6只家兔(体重1.8-2.0kg)随机分为2组，取实施例48制备的注射用尼莫地平加入注射用水复溶后及市售尼膜同注射液，分别经0.9％氯化钠注射液稀释至临床给药浓度0.04mg/mL，经兔右耳缘静脉注射药液，给药剂量为0.4mg/kg，推注速度为1mL/min。同时左耳经耳缘静脉给予等体积的0.9％氯化钠注射液作为对照，连续三天，每天给药一次。每天给药期间肉眼观察动物行为和注射部位变化。最后一次给药后24-48h实施安乐死，取双侧距注射部 位向心端约0.5-3.0cm处耳缘静脉组织，置于10％多聚甲醛溶液中固定，使用乙醇梯度脱水，嵌入石蜡中，苏木素-伊红溶液染色，对病理变化进行评估。
2.实验结果
血管刺激性兔耳病理切片结果如图5所示。
尼膜同注射液出现明显的胶原肿胀，红细胞渗漏及点状出血，内皮细胞肿胀，有大量炎症细胞浸润，说明尼膜同注射液组给药后机体有较强的炎症反应。给药过程中兔子剧烈挣扎，疼痛感强烈，可能是由于高浓度的乙醇进入血管后产生强烈的血管刺激性及疼痛感。
本发明注射用尼莫地平与0.9％氯化钠注射液耳廓皮肤表面上皮结构完整，皮脂腺及汗腺组织无特殊病变，中心部位软骨形态正常，未见明显病变损伤情况，且给药过程兔子无明显不适。与0.9％氯化钠注射液组相比，注射用尼莫地仅有少量炎症细胞浸润，未见其它异常情况。
上述结果表明本发明的注射用尼莫地平的血管刺激性明显低于尼膜同注射液，安全性提高。
实施例69：注射用尼莫地平溶血性研究
1.实验方法
取实施例48制备的注射用尼莫地平(NIMO-CD)加入注射用水复溶后及市售尼膜同注射液(Nimotop)，分别经0.9％氯化钠注射液稀释至临床给药浓度0.04mg/mL。取10mL干净玻璃试管8支，编号，1至5号管为不同浓度的NIMO-CD，6号管为阴性对照管，7号管为阳性对照管，8号为Nimotop注射液。按下表所示，依次加入2％红细胞悬液、0.9％氯化钠注射液、蒸馏水、药液，混匀后，立即置于37±0.5℃水浴中温育，观察并记录各管的溶血情况。开始每隔15分钟记录一次，1小时后，每隔1小时观察一次，观察3小时。
表29溶血实验设计表
2.实验结果
溶血情况如图6所示，NIMO-CD(1-5号)红细胞均在试管底部沉积，上清液无色澄明，振摇后均可均匀分散，表明NIMO-CD不会引起溶血及红细胞凝聚。上述结果表明，本发明注射用尼莫地平无溶血性，可注射使用。
实施例70：注射用尼莫地平药效学研究
1.实验方法
1.1试验动物分组及给药
32只雄性SD大鼠随机分为4组：假手术组(Sham组)、模型对照组(Model组)、尼膜同注射液组(Nimotop组)、注射用尼莫地平组(NIMO-CD组，实施例48)。取实施例48制备的注射用尼莫地平加入注射用水复溶后，经0.9％氯化钠注射液稀释至0.2mg/mL。Nimotop组及NIMO-CD组经尾静脉注射给药，给药剂量为1mg/kg，造模当日至术后72h，每日给药一次，术后立即给药。
1.2脑出血模型建立
Model组、Nimotop组及NIMO-CD组大鼠经1～4％异氟烷麻醉后，脑立体定位仪于左侧纹状体区域(以前囟为中心，后侧2.0mm，右侧2.0mm，深度4.0mm)驻针2min，以1μL/5min速率注射2mg/mL胶原酶溶液2μL，驻针3min后缓慢移出，建立脑出血模型。手术完成后缝合伤口，碘伏对伤口进行消毒。后肢肌肉注射40mg/kg庆大霉素防止感染，每天两次肌肉注射10mg/kg痛立定缓解疼痛。Sham组动物实验操作同上，使用同等体积的PBS溶液替代胶原酶注射。
1.3检测指标
1.3.1行为学评价
术前及脑出血模型建立后24h、48h和72h，观察动物行为，改良神经功能缺损评分(Modified neurological severity score，mNSS)分别评价实验动物的运动、感觉、平衡和反射能力。采用转角实验(Corner test，CT)测量实验动物感觉运动障碍。将大鼠放入一个两块木板所形成的30°夹角内，使其转身，观察并记录大鼠向右侧转身的次数，测试10次，计算CT分数公式如下所示。
1.3.2脑出血损伤面积
实验终点时，腹腔注射25-50mg/kg舒泰将所有实验动物麻醉安乐死。取大脑，4％多聚甲醛溶液固定24h后将脑置于脑模具中，以进针位置为中心，按冠状方式从前到后，切成6片。将6片脑切片按序排放整齐，切片旁附标尺，用数码相机成像。通过图像分析软件ImageJ(版本：1.4.3.67)计算每一切片的血肿区域面积占比，比较各组之间的总血肿面积差异。若实验过程中出现动物死亡，立即取大脑，按上述操作进行。
1.3.3组织病理学评价
大鼠脑组织切片经4％多聚甲醛溶液固定、石蜡包埋、HE染色后，光学显微镜观察切片病变情况。按照组织病理学评分标准评估切片的脑水肿、炎性浸润和神经元损伤程度，评分标准如表30所示。
表30组织病理学评分标准
2.实验结果
2.1行为学评价
mNSS评分如表31和图7A所示，mNSS评分国际标准定义：正常SD大鼠为0分，1-6分之间为轻度损伤，7-12分之间为中度损伤，13-18分之间为重度损伤。Model组mNSS评分在第1天最高，神经功能损伤最严重，第2、3天评分逐步下降，神经功能逐步恢复，但均大于7分，呈中度损伤。NIMO-CD组和Nimotop组神经功能评分同样的也随时间递减，治疗后两天评分均大于7分，呈中度损伤，第三天均低于7分，呈轻度损伤。与Model组相比，NIMO-CD组和Nimotop组实验动物mNSS评分均显著降低。
表31 mNSS评分表(Mean±SD，n＝8)

注：与Model组比较，*P＜0.05，**P＜0.01，***P＜0.001。
CT分数结果评分如表32和图7B所示，CT分数随时间递减，各组实验动物偏瘫情况逐渐改善。Sham组和Model组CT最终分数分别为48.75％和65.00％，NIMO-CD组和Nimotop组CT分数最终分别为58.75％和60.00％。与Model组相比，NIMO-CD组和Nimotop组实验动物CT分数均降低。
表32 CT评分表(Mean±SD，n＝8)

注：与Model组比较*P＜0.05，**P＜0.01，***P＜0.001。
行为学评价结果表明，注射用尼莫地平对大鼠脑出血模型产生的脑损伤引起的神经行为功能异常，具有明显的改善作用，且与尼膜同注射液组无显著性差异(P>0.05)。
2.2脑出血损伤面积
收集试验动物脑组织，比较各组的总血肿面积差异，结果如表33和图7C所示。Sham组未出现血肿，Model组总血肿面积占比为4.45％，NIMO-CD组和Nimotop组总血肿面积占比均为0.98％。与Model组相比，NIMO-CD组和Nimotop组实验动物总血肿面积占比显著降低。脑出血损伤面积测定实验结果表明，注射用尼莫地平可显著降低大鼠脑出血导致的血肿，显著改善脑出血损伤，且与尼膜同注射液无显著性差异(P>0.05)。
表33脑出血损伤面积表(Mean±SD，n＝8)

注：与Model组比较，*P＜0.05，**P＜0.01，***P＜0.001。
2.3组织病理学评价
显微镜观察并分析脑组织切片情况，病理学切片如图7E所示，根据病理学切片对脑水肿程度、炎症程度和神经元损伤进行综合评分，评分结果如表34和图7D所示。Sham组和Model组组织病理学评价分数分别为1.38和5.00。Nimotop组和NIMO-CD组组织病理学评价分数分别为2.63和2.25。与Model组相比，NIMO-CD组实验动物组织病理学评价总分降低。组织病理学结果表明，注射用尼莫地平可显著改善脑水肿程度、炎症程度和神经元损伤，且与尼膜同注射液无显著性差异(P>0.05)。
表34组织病理学评分表(Mean±SD，n＝8)

注：与Model组比较，*P＜0.05，**P＜0.01，***P＜0.001。
综上所述，注射用尼莫地平对脑出血预后有显著的治疗作用，极大地改善了脑出血所引起神经功能损伤、脑血肿程度、脑水肿程度、炎症程度以及神经元损伤程度，且治疗效果与尼膜同注射液基本相同。
实施例71：注射用尼莫地平药物代谢动力学研究
1.实验方法
1.1试验动物分组及给药
雄性SD大鼠16只，随机分为两组，每组8只，实验前禁食12h，不禁 水。取实施例48制备的注射用尼莫地平(NIMO-CD)加入注射用水复溶后，经0.9％氯化钠注射液稀释至0.2mg/mL。尾静脉分别给予1.6mg/kg的尼膜同注射液(Nimotop)和NIMO-CD，于给药后5、15、30、45、60、120、240、360和480min眼眶取血0.5mL，4000rpm离心5min，取上清液300μL，-80℃冰箱冷冻保存。
1.2血浆样品处理方法
将冻存的血浆样品室温下放置回温后，4000rpm离心5min，取血浆200μL置于2.0mL EP管中，加入5μg/mL的尼群地平内标液40μL，涡旋5min。加入1mol/L的NaOH 100μL，定量加入1.5mL无水乙醚：正己烷(1:1)提取液，涡旋5min，超声5min，10000rpm离心10min，取所有上清液于1.5mL EP管中，45℃吹干，残留物用100μL流动相复溶，涡旋混合5min，10000rpm离心10min，HPLC进样测定。
1.3色谱条件
色谱柱：Zorbax SB C18柱(250mm×4.6mm,5μm)
流动相：乙腈：水(60:40,v/v)
流速：1.0mL/min
检测波长：358nm
柱温：30℃
进样量：20μL
2.实验结果
SD大鼠尾静脉分别注射Nimotop注射液及NIMO-CD后的血药浓度-时间曲线，如图8所示，NIMO-CD和Nimotop注射液静脉给药后，在SD大鼠体内的药代动力学消除曲线几乎一致。给予NIMO-CD和Nimotop注射液后，NIMO进入体内短时间内可快速消除，给药240min后HPLC无法检测到血浆中的药物。
NIMO-CD及Nimotop注射液主要药代动力学参数如表35所示，结果表明，NIMO-CD和Nimotop注射液的药代动力学参数基本一致，包括血药浓度-时间曲线下面积(AUC)，平均滞留时间(MRT)，半衰期(t1/2)、清除率(CL)和最大血药浓度(Cmax)，且组间差异无统计学意义(P>0.05)。上述结果表明，注射用尼莫地平和尼膜同注射液在SD大鼠体内尾静脉注射后药物代谢动力学 性质基本一致。
表35 NIMO-CD及Nimotop注射液主要药物代谢动力学参数(Mean±SD，n＝8)
以上已对本发明创造的较佳实施例进行了具体说明，但本发明创造并不限于所述实施例，熟悉本领域的技术人员在不违背本发明创造精神的前提下还可做出种种的等同的变型或替换，这些等同的变型或替换均包含在本申请权利要求所限定的范围内。

Claims (11)

  1. 一种安全、稳定的注射用尼莫地平，其特征在于，由磺丁基醚-β-环糊精和尼莫地平制成，所述的磺丁基醚-β-环糊精和尼莫地平质量比为200:1-700:1。
  2. 根据权利要求1所述的注射用尼莫地平，其特征在于，所述的磺丁基醚-β-环糊精和尼莫地平质量比为350:1-700:1。
  3. 根据权利要求2所述的注射用尼莫地平，其特征在于，所述的注射用尼莫地平冻干前尼莫地平含量为0.01-0.14％克/毫升，磺丁基醚-β-环糊精含量为10-50％克/毫升。
  4. 根据权利要求3所述的注射用尼莫地平，其特征在于，所述的磺丁基醚-β-环糊精和尼莫地平质量比为400:1-600:1，所述的注射用尼莫地平冻干前尼莫地平含量为0.02-0.10％克/毫升，磺丁基醚-β-环糊精含量为10-40％克/毫升。
  5. 根据权利要求3所述的注射用尼莫地平，其特征在于，所述的磺丁基醚-β-环糊精和尼莫地平质量比为450:1-550:1，所述的注射用尼莫地平冻干前尼莫地平含量为0.04-0.06％克/毫升，磺丁基醚-β-环糊精含量为20-30％克/毫升。
  6. 一种安全、稳定的尼莫地平注射剂，其特征在于，由以下成分制成：
  7. 根据权利要求6所述的安全、稳定的尼莫地平注射剂，其特征在于，由以下成分制成：
  8. 一种如权利要求6或7所述的安全、稳定的尼莫地平注射剂的制备方法，其特征在于，包括以下步骤：
    (a)称取处方量的磺丁基醚-β-环糊精，加入适量的注射用水，在一定温度下搅拌使溶解，得磺丁基醚-β-环糊精水溶液；
    (b)称取处方量的尼莫地平，加入处方量的乙醇溶解，即得尼莫地平乙醇溶液；
    (c)在一定的搅拌温度和速度条件下将步骤(b)溶液或尼莫地平药物粉末加入到步骤(a)溶液中，搅拌一定时间，加注射用水至全量，0.22μm微孔滤膜无菌过滤，灌装，冷冻干燥，封装，即得所述的安全、稳定的尼莫地平注射剂。
  9. 根据权利要求8所述的制备方法，其特征在于，步骤(a)和(c)所述的搅拌温度为20-100℃。
  10. 根据权利要求8所述的制备方法，其特征在于，步骤(c)所述的搅拌时间为10-300min。
  11. 根据权利要求8所述的制备方法，其特征在于，步骤(c)所述的搅拌线速度为0.5-10.0m/s。
PCT/CN2023/086085 2022-04-06 2023-04-04 一种安全、稳定的注射用尼莫地平及其制备方法 WO2023193694A1 (zh)

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