WO2020048533A1 - 一种尼莫地平注射液组合物及其制备方法 - Google Patents

一种尼莫地平注射液组合物及其制备方法 Download PDF

Info

Publication number
WO2020048533A1
WO2020048533A1 PCT/CN2019/104760 CN2019104760W WO2020048533A1 WO 2020048533 A1 WO2020048533 A1 WO 2020048533A1 CN 2019104760 W CN2019104760 W CN 2019104760W WO 2020048533 A1 WO2020048533 A1 WO 2020048533A1
Authority
WO
WIPO (PCT)
Prior art keywords
injection
nimodipine
composition
oil
group
Prior art date
Application number
PCT/CN2019/104760
Other languages
English (en)
French (fr)
Inventor
李宏
高山
Original Assignee
睢宁九旭神州医药科技有限公司
李宏
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 睢宁九旭神州医药科技有限公司, 李宏 filed Critical 睢宁九旭神州医药科技有限公司
Priority to EP19857267.9A priority Critical patent/EP3848021A4/en
Priority to CN201980063913.9A priority patent/CN112912066B/zh
Priority to JP2021537460A priority patent/JP2021535932A/ja
Priority to US17/274,299 priority patent/US20210338649A1/en
Publication of WO2020048533A1 publication Critical patent/WO2020048533A1/zh

Links

Images

Classifications

    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • A61K47/51—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 non-active ingredient being a modifying agent
    • A61K47/54—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 non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/547—Chelates, e.g. Gd-DOTA or Zinc-amino acid chelates; Chelate-forming compounds, e.g. DOTA or ethylenediamine being covalently linked or complexed to the pharmacologically- or therapeutically-active agent
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • 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
    • A61K9/10—Dispersions; Emulsions
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the invention belongs to the technical field of medicine, and relates to an injection solution of the composition of the invention, a preparation method thereof and non-clinical safety.
  • Nimododipine is a second-generation pyridine calcium antagonist. Nimodipine injection was developed and marketed by the German company Bayer in April 1985. There are more than 100 manufacturers in our country. Due to its high lipophilicity, it can easily penetrate the blood-brain barrier, selectively expand cerebral blood vessels, and significantly reverse the basilar and anterior spinal artery spasm. It is clinically used to treat hypertension, stroke, migraine, and cobwebs. Sub-membrane hemorrhage and other cerebral hemorrhage diseases are currently the drugs of choice for cerebrovascular treatment, and have high clinical application value for senile dementia.
  • nimodipine preparations commonly used in clinical practice include tablets, capsules, and injections.
  • Nimodipine is a poorly water-soluble drug with low solubility and strong first-pass effect in the liver, resulting in low oral bioavailability.
  • the bioavailability of healthy subjects and patients with subarachnoid hemorrhage is 5-13% and 3-28%.
  • the biological half-life of the drug is short (about 1.5-2h), and frequent medication is required 3-4 times a day. Not only is it inconvenient to use, but it can also cause the peak concentration of the blood drug to cause toxic and side effects; because nimodipine is water Insolubility.
  • nimodipine infusion is a water-insoluble preparation containing ethanol.
  • crystals can be precipitated, the content of which is reduced, which reduces the efficacy of the drug and also brings patients Come a certain degree of danger.
  • Nimodipine exists in different crystalline forms, namely alpha and beta. Different crystal forms have different solubility in different solvents. According to literature reports, the melting point of ⁇ crystal form is 124-125 ° C, which has good solubility in water (3.6ug / ml); the melting point of ⁇ crystal form is 114-116 ° C, and the solubility in water is 2.5ug / ml, the solubility in the oil phase has not been reported; the present invention compared the solubility of commercially available nimodipine ( ⁇ crystal form, Xinhua Pharmaceutical) and homemade nimodipine ( ⁇ crystal form) in different oils, and found nimodipine The solubility in oil is much greater than the solubility in water, and the solubility of the ⁇ crystal form in medium chain fatty oils and the solubility of soybean oil and medium chain fatty oil mixtures is much greater than the ⁇ crystal form (see the table below). Type nimodipine dissolved in oil and made
  • Injectable emulsions containing soybean oil, medium-chain fatty oil, and a mixture of soybean oil and medium-chain fatty oil as the main components have both solvent properties and are almost non-toxic.
  • Fat-soluble drugs are introduced and dissolved in the core lipid portion of the emulsion particles.
  • Lipid oil droplets are metabolized and slowly released to maintain effective blood drug concentration, reduce the toxic and side effects that the drug may cause, and have many advantages of the composition injection of the present invention; the composition injection of the present invention can increase nimodipine drug
  • the solubility can increase drug loading, reduce drug hydrolysis, increase stability, reduce toxicity, and has good clinical application prospects.
  • Benzyl alcohol has the functions of disinfection, antiseptic and local anesthesia. It is used as a bacteriostatic agent and analgesic in injections and has hemolytic effect. Children's injections for intramuscular injection can cause gluteal muscle contracture when it contains benzyl alcohol. The 2015 edition of the "Pharmacopoeia of the People's Republic of China" has strict restrictions on the addition of bacteriostatic agents to intravenous injections and is not recommended. At the same time, all nimodipine emulsions do not consider the crystal form. The other crystal forms have low solubility, small drug loading, easy to crystallize during storage, and poor product stability. Considering the potential safety and security hazards of the above nimodipine composition, it is necessary to develop a new nimodipine emulsion composition to meet clinical needs.
  • One object of the present invention is to provide a nimodipine injection composition, which is characterized in that the injection solution composition includes the following components: nimodipine, oil for injection, emulsifier, complexing agent, stability Agent, osmotic pressure regulator.
  • the injection solution composition includes nimodipine 0.02-0.23%, injection oil 2-30%, emulsifier 0.8-3%, complexing agent 0-0.1%, stabilizer 0-0.3%, osmotic pressure regulator 1-3%.
  • the preferred ratios are: nimodipine 0.04-0.20%, injection oil 3-20%, emulsifier 1-2.5%, complexing agent 0-0.05%, stabilizer 0-0.2%, osmotic pressure regulator 2-3 %; More preferred ratios are: nimodipine 0.08-0.18%, injection oil 8-12%, emulsifier 1-2%, complexing agent 0-0.01%, stabilizer 0-0.1%, osmotic pressure regulator 2-2.5%.
  • the prepared nimodipine injection composition has a pH of 6.0-8.5, a preferred pH of 6.5-8.0, and a more preferred pH of 7.0-7.5.
  • the prepared nimodipine injection composition has an average particle diameter of 100-600 nm, preferably an average particle diameter of 120-400 nm, and more preferably an average particle diameter of 140-300 nm.
  • the nimodipine is selected from an alpha crystal form or a beta crystal form, preferably an alpha crystal form having a melting point of 114-116 ° C.
  • the emulsifier in the injection solution composition is selected from soybean phospholipids or egg yolk phospholipids.
  • the content of phosphatidylcholine (PC) in soybean phospholipid or egg yolk phospholipid is 60-90%, and the content of phosphatidylethanolamine (PE) is 2-20%, and the preferred range is the soybean phospholipid or
  • the content of phosphatidylcholine (PC) in egg yolk phospholipids is 70-85%, and the content of phosphatidylethanolamine (PE) is 5-18%.
  • the complexing agent in the injection solution composition may be selected from one or more compositions of calcium sodium edetate, disodium edetate, sodium edetate, and edetic acid, and a preferred complexing agent Disodium edetate.
  • the injection oil in the injection solution composition is soybean oil for injection or medium chain triglyceride for injection, or a mixture of the two, preferably soybean oil for injection, medium chain triglyceride for injection, and a preferred mass ratio. It is 2: 8-5: 5, a more preferred mass ratio is 4: 6-5: 5, and a most preferred mass ratio is 4: 6.
  • the preferred stabilizer in the injection solution composition is oleic acid or sodium oleate.
  • the osmotic pressure regulator in the injection solution composition is glycerin.
  • the present invention provides a method for preparing a nimodipine injection composition.
  • the advantage of the present invention is that the injection of the composition of the present invention does not contain solubilizing ethanol, thereby avoiding irritation to the skin and blood vessels.
  • auxiliary emulsifiers such as Tween-80 and co-solvents such as benzyl alcohol, which solves the emulsification and solubilization problems of nimodipine injection.
  • the emulsifier By adding the emulsifier to the oil phase and the water phase separately, it is preferred that part of the emulsifier is added to the oil phase and part is added to the water phase.
  • the emulsification effect is better, the milk particles are more uniform, and the emulsion is more stable.
  • the oil phase is easier to emulsify and disperse, which can reduce the number of homogenization times and is more conducive to industrial production.
  • the prior art nimodipine emulsion has hemolysis and agglutination, and the composition injection of the present invention has no hemolysis and agglutination.
  • composition injection of the present invention and the prior art nimodipine emulsion have no obvious stimulating effect on rabbit ear marginal veins (vessels).
  • nimodipine API nimodipine water injection
  • prior art nimodipine emulsion nimodipine emulsion
  • composition injection of the present invention can induce zebrafish heart rate reduction. Slow, atrial beating stopped, pericardial edema, and circulatory defects, all four drugs have cardiovascular toxicity.
  • the maximum non-lethal concentration of nimodipine drug substance and nimodipine water injection is 2.5 ⁇ g / ml
  • the maximum non-lethal concentration of nimodipine emulsion in the prior art is 5.0 ⁇ g / ml
  • the maximum non-lethal concentration of the injection of the composition of the present invention The concentration is 10 ⁇ g / mL, which is four times that of nimodipine raw materials and nimodipine water injection, and twice that of the prior art nimodipine emulsion, indicating that the composition injection of the present invention is far less toxic to zebrafish than nimodipine.
  • Dipine APIs, nimodipine injections, and prior art nimodipine emulsions are examples of the composition injection of the present invention.
  • a nimodipine injection composition in terms of mass concentration, includes the following components: nimodipine 0.02-0.23%, injection oil 2-30%, emulsifier 0.8-3%, complexing agent 0-0.1% , Stabilizer 0-0.3%, osmotic pressure regulator 1-3%.
  • the preferred ratios are: nimodipine 0.04-0.20%, injection oil 3-20%, emulsifier 1-2.5%, complexing agent 0-0.05%, stabilizer 0-0.2%, osmotic pressure regulator 2-3 %; More preferred ratios are: nimodipine 0.08-0.18%, injection oil 8-12%, emulsifier 1-2%, complexing agent 0-0.01%, stabilizer 0-0.1%, osmotic pressure regulator 2-2.5%.
  • the prepared nimodipine injection composition has a pH of 6.0 to 8.5, preferably a pH of 6.5 to 8.0, and more preferably a pH of 7.0 to 7.5.
  • the prepared nimodipine injection composition has an average particle diameter of 100-600 nm, preferably an average particle diameter of 120-400 nm, and more preferably an average particle diameter of 140-300 nm.
  • the nimodipine is selected from an alpha crystal form or a beta crystal form, preferably an alpha crystal form having a melting point of 114-116 ° C.
  • the emulsifier is selected from one or more compositions of soybean phospholipid, egg yolk phospholipid, hydrogenated soybean phospholipid, and hydrogenated egg yolk phospholipid.
  • the complexing agent is selected from one or more compositions of calcium sodium edetate, disodium edetate, sodium edetate, and edetic acid.
  • a preferred stabilizer is oleic acid or sodium oleate.
  • the osmotic pressure regulator is glycerin.
  • the pH of the nimodipine injection composition is 6.0-8.5, the preferred pH is 6.5-8.0, and the more preferred pH is 7.0-7.5.
  • the nimodipine injection composition has an average particle diameter of 100-600 nm, preferably an average particle diameter of 120-400 nm, and more preferably an average particle diameter of 140-300 nm.
  • the invention also provides a method for preparing a nimodipine injection composition, comprising the following steps:
  • the complexing agent is selected from one or more compositions of calcium sodium edetate, disodium edetate, sodium edetate, and edetic acid.
  • the preferred complexing agent is disodium edetate .
  • the osmotic pressure regulator is preferably glycerin.
  • nimodipine is selected from an alpha crystal form or a beta crystal form, preferably an alpha crystal form having a melting point of 114-116 ° C.
  • the oil for injection is selected from the group consisting of soybean oil for injection or medium chain triglyceride for injection, or a mixture of the two.
  • a preferred soybean oil for injection and a medium chain triglyceride mixture for injection has a preferred mass ratio of 2: 8. -5: 5, a more preferred mass ratio is 4: 6-5: 5, and a most preferred mass ratio is 4: 6.
  • the emulsifier is selected from soybean phospholipids or egg yolk phospholipids, and the content of phosphatidylcholine (PC) in soybean phospholipids or egg yolk phospholipids is 60-90%, and the content of phosphatidylethanolamine (PE) is 2-20%.
  • a preferred range is that the content of phosphatidylcholine (PC) in the soybean phospholipid or egg yolk phospholipid is 70-85%, and the content of phosphatidylethanolamine (PE) is 5-18%.
  • FIG. 1 is a diagram showing changes in cerebral blood flow of No. 9 rats in the high-dose group of the composition of the present invention.
  • the dot curve is the whole craniocerebral region, and the diamond curve is the sagittal suture region.
  • Figure 2 is a graph showing the changes in cerebral blood flow of No. 16 rats in the high-dose nimodipine acupuncture group.
  • the dot curve is the whole craniocerebral region and the diamond curve is the sagittal suture region.
  • FIG. 3 is a graph showing the time-to-dose curve of nimodipine water injection and the composition of the present invention.
  • Nimodipine 1g soybean oil 40g, medium chain triglyceride 60g, lecithin 12g, edetate disodium 0.05g, oleic acid 0.3g, glycerol 22.5g, appropriate amount of pH adjuster, water for injection to 1000ml.
  • the method is as follows:
  • the colostrum is homogenized and circulated until the emulsion particle size meets the requirements.
  • the method is as follows:
  • the colostrum is homogenized and circulated until the emulsion particle size meets the requirements.
  • Nimodipine 0.2g soybean oil 50g, lecithin 8g, sodium edetate 0g, sodium oleate 0.2g, glycerin 25g, appropriate amount of pH adjuster, water for injection to 1000ml.
  • the method is as follows:
  • the colostrum is homogenized and circulated until the emulsion particle size meets the requirements.
  • the pH was 7.4 and the average particle diameter was 150 nm.
  • Nimodipine 2.3g soybean oil 80g, medium chain triglyceride 120g, lecithin 15g, edetate disodium 0.1g, oleic acid 1g, glycerol 20g, appropriate amount of pH adjuster, water for injection to 1000ml.
  • the pH was 7.2 and the average particle diameter was 296 nm.
  • Nimodipine 0.4g soybean oil 40g, medium chain triglyceride 60g, lecithin 8g, edetate disodium 0.03g, oleic acid 0.3g, glycerol 22.5g, appropriate amount of pH adjuster, water for injection to 1000ml.
  • the pH was 7.9 and the average particle diameter was 600 nm.
  • the pH was 6.8 and the average particle size was 146 nm.
  • the pH was 6.5 and the average particle diameter was 352 nm.
  • Nimodipine 1.2g soybean oil 50g, medium chain triglyceride 50g, lecithin 30g, edetate disodium 0.0g, oleic acid 0.0g, glycerol 22.5g, appropriate amount of pH adjuster, water for injection to 1000ml.
  • the pH was 8.5 and the average particle diameter was 246 nm.
  • Soy phospholipids or egg yolk phospholipids have a phosphatidylcholine (PC) content of 70% -85% and a phosphatidylethanolamine (PE) content of 5% -18%.
  • PC phosphatidylcholine
  • PE phosphatidylethanolamine
  • the composition of the present invention has a higher ⁇ -potential and better stability.
  • Example 6 the effect of the complexing agent on the methoxyaniline value of the composition injection of the present invention was examined.
  • the metal ion complexing agent can be used to achieve anti-oxidation, which can also greatly reduce the methoxyaniline value in the preparation, improve the safety of the preparation, and the addition of other antioxidants cannot reduce the methoxyl of the injection of the composition of the present invention Aniline value.
  • Example 1 In order to determine the stability of the preparation process of the present invention, a pilot test was performed according to Example 1, and a dilution stability study and a long-term stability study were performed.
  • the measured results from the main items of the diluted sample showed that there was no significant change in the pH value, milk particle size, related substances, and content of all diluted samples within 12 hours.
  • the composition injection of the present invention is diluted with glucose injection or sodium chloride injection during clinical use, and it is safe and reliable to use within 12 hours.
  • composition injection of the present invention is placed in a light-shielded condition at a temperature of 25 ⁇ 2 ° C, and samples are taken at 0, 3, 6, 9, 12, and 18 months, respectively, and tested according to the stability of the key inspection items, compared with 0.
  • composition injection prepared in Example 1 of the present invention was subjected to special allergic, hemolytic, and local (vascular, skin, mucosal, muscle, etc.) irritation and other special
  • the safety test, zebrafish test and pharmacodynamic test on rat subarachnoid hemorrhage model the experimental results are as follows:
  • Healthy and clean SD rats were intracutaneously injected with 0.1ml of the corresponding antiserum for passive sensitization for 48 hours, and then each group was injected intravenously with a dose of 8, 4 mg / kg of the composition injection of the present invention, which was prepared using the prior art patented process.
  • Dipine emulsion 8, 4mg / kg dose (application number: 200910021091.6 prescription contains Tween 80) 100mg / kg bovine serum albumin and 10ml / kg dose of 0.9% NaCl injection, and 1% Evans Blue solution 1ml / only excitation.
  • Dose group Blue spot response (mm, ⁇ s) Composition injection of the present invention 8 mg / kg - Composition injection of the present invention 4mg / kg - Nimodipine emulsion 8mg / kg + (18.0 ⁇ 3.6) Nimodipine emulsion 4mg / kg + (11.6 ⁇ 3.3) 0.9% NaCl injection 10ml / kg - Bovine serum albumin 100mg / kg + (22.0 ⁇ 4.3)
  • Healthy white guinea pigs were injected intraperitoneally with a dosage of the composition injection of the present invention at a dose of 4, 2 mg / kg, once every other day, and sensitization was performed 3 times in a row.
  • the 13th day after the last sensitization was stimulated by a single intravenous injection of a sensitizing dose of the composition injection of the present invention.
  • the sensitization and challenge test methods of the prior art nimodipine emulsion were the same as above. Immediate (about 1 minute) similar allergic reactions such as prone motion and subsequent death occurred with a mortality rate of 100%, and no significant abnormal reaction was observed in the guinea pigs in the low dose group.
  • Bovine serum albumin positive control guinea pigs showed obvious allergic reaction symptoms after challenge, mainly manifested as restlessness, vertical hair, trembling, blowing nose, sneezing, cough, shortness of breath, urination, tearing, dyspnea, and gait Stable, wheezing, jumping, spasm, and even death, the intensity of allergic reaction is ++ ⁇ ++++, the time of death is about 5 minutes after administration, and the surviving guinea pig with allergic reaction gradually returns to normal within 30 minutes after administration, The response rate is 100% and the mortality rate is 50%.
  • the prior art nimodipine emulsion at a dose of 2 mg / kg has an allergic reaction to guinea pigs
  • the composition injection of the present invention at a dose of 2 mg / kg has no allergic reaction to guinea pigs.
  • Rabbit ear marginal veins (vessels) were injected once a day for 7 consecutive days, and the injection of the composition injection of the present invention and the prior art nimodipine emulsion 1mg (10ml) / (kg.d) were slowly injected.
  • the macroscopic observation did not show any abnormalities in the prior art nimodipine emulsion.
  • Histopathological examination revealed only two rabbits with edema of vascular endothelial cells adjacent to the local marginal vein and mild edema in the surrounding stroma. No thrombus, embolism and Large-scale bleeding, edema, necrosis and other severe irritating lesions were not significantly different from the 0.9% NaCl injection control group.
  • the left and right leg quadriceps of rabbits were injected once a day for 7 consecutive days with the injection of the composition of the present invention and the prior art nimodipine emulsion at a dose of 0.1 mg (1 ml) / (dot. D).
  • Eight rabbits received unilateral quadriceps injections with congestive focus lesions below 0.5 ⁇ 1.0cm, and the other rabbits did not see significant congestion, swelling and necrosis.
  • the sum of all quadriceps response levels was 4, Less than 6; histopathological examination showed that the localized quadriceps injection with small focal or microfocal interstitial hyperplasia and monocyte infiltration were the main lesions.
  • Rat passive allergy test The composition injection of the present invention has no passive skin allergic reaction to sensitized rats.
  • the prior art nimodipine emulsion has a passive skin allergic reaction to sensitized rats; a guinea pig whole body active allergy test
  • the guinea pigs of the high-injection composition group of the invention immediately (approximately 1 minute) experienced similar allergic reactions such as prone motion and subsequent death.
  • the mortality rate was 100%, and the guinea pigs in the low-dose group did not have obvious abnormal reactions.
  • allergic reactions such as proneness and subsequent death occurred in the high and low dose groups.
  • the mortality of the high dose group was 100%, and the death of the low dose group was 40%.
  • the prior art nimodipine emulsion has hemolysis and agglutination, and the composition injection of the present invention has no hemolysis and agglutination.
  • test situation is as follows:
  • the drugs to be tested in this project are nimodipine raw materials, nimodipine water injection (Nimotide, manufactured by Bayer), the injection of the composition of the present invention (prepared according to Example 1), and blank emulsion, using the existing patented technology
  • the prepared nimodipine emulsion (application number: 200910021091.6 contains Tween 80 in the prescription). All are provided by Zhejiang Jiuxu Pharmaceutical Co., Ltd.
  • Dissecting microscope (SMZ645, Nikon Corporation, Japan); precision electronic balance (CP214, Ohaus); six-well plate (Nest Biotech).
  • zebrafish juveniles were naturally incubated from embryos produced in fertility experiments.
  • Fish water quality RO per 1L of water was added 200mg instant salt, a conductivity of 480 ⁇ 510 ⁇ S / cm; pH 6.9 to 7.2; a hardness of 53.7 ⁇ 71.6mg / LCaCO 3.
  • zebrafish at various developmental stages were sacrificed and anesthetized with 0.25 mg / ml of tricaine mesylate.
  • the procedure of anesthesia is in accordance with the requirements of the American Veterinary Medicine Association (AVMA) for animal anesthesia.
  • AVMA American Veterinary Medicine Association
  • the wild-type AB-type zebrafish juveniles were treated with the drug to be tested for a certain period of time for 24 hours.
  • the five initial detection concentrations were: 0.1 ⁇ g / ml, 1 ⁇ g / ml, 10 ⁇ g / ml, 100 ⁇ g / ml, and 500 ⁇ g / ml.
  • 30 zebrafish were treated at each concentration. After the treatment, the number of zebrafish deaths in each experimental group was counted to provide a basis for the next experimental concentration design.
  • the detection concentration range of the drug to be tested is 1000 ⁇ g / ml (if the maximum solubility is less than 1000 ⁇ g / ml, the highest solubility shall prevail), and the lower limit is 0.001 ⁇ g / ml.
  • the maximum non-lethal concentration of nimodipine API and nimodipine injection is 2.5 ⁇ g / ml
  • the maximum non-lethal concentration of nimodipine emulsion in the prior art is 5.0 ⁇ g / ml
  • the maximum non-lethal concentration of the injection of the composition of the present invention It is 10 ⁇ g / ml, 4 times that of nimodipine raw materials and water preparations, and 2 times that of nimodipine emulsion in the prior art. It can be seen that the composition injection of the present invention is far less toxic to zebrafish than nimodipine APIs, nimodipine water injections and prior art nimodipine emulsions.
  • zebrafish heart rate As the drug concentration increased, zebrafish heart rate slowed.
  • the zebrafish heart rate induced by nimodipine API, nimodipine injection, and the prior art nimodipine emulsion slowed down with the increase of the drug concentration
  • the zebrafish heart rate induced by the injection of the composition of the present invention increased with the drug.
  • the increase in concentration slowed down slightly.
  • Nimodipine API, Nimodipine Water Injection, and prior art Nimodipine emulsion can induce atrial beating stop in some zebrafish at 2.5 ⁇ g / ml.
  • Nimodipine API, Nimodipine The incidence of atrial beating stop induced by dipine water injection and the prior art nimodipine emulsion in zebrafish reached 90%, 100%, and 70%, respectively.
  • the concentration of atrial beating stop induced by some injections of the composition injection of the present invention is At 20 ⁇ g / ml, the concentration at which atrial beating stopped at 100% was 40 ⁇ g / ml, and the occurrence concentrations were significantly higher than those of nimodipine API, nimodipine water injection, and prior art nimodipine emulsion.
  • pericardial edema induced by four drugs in zebrafish increased with increasing drug concentration.
  • the incidence of pericardial edema in zebrafish induced by nimodipine injection increased fastest with increasing concentration, and pericardial edema could be induced in all zebrafish at 2.5 ⁇ g / ml.
  • Nimodipine was the second drug at 5.0 ⁇ g / ml.
  • Pericardial edema was induced in all zebrafish at the time; pericardial edema was induced in all the zebrafish at 10.0 ⁇ g / ml in the prior art nimodipine emulsion; and the incidence of pericardial edema in zebrafish induced by the injection of the composition of the present invention varied with the drug The increase in concentration was the slowest, and pericardial edema was not induced in all zebrafish until the concentration was 20 ⁇ g / ml.
  • nimodipine API nimodipine injection
  • prior art nimodipine emulsion injection of the composition of the present invention can induce zebrafish to slow down heart rate, stop atrial beating, pericardial edema and circulation defects. All drugs have cardiovascular toxicity.
  • the maximum non-lethal concentration of nimodipine drug substance and nimodipine water injection is 2.5 ⁇ g / ml
  • the maximum non-lethal concentration of nimodipine emulsion in the prior art is 5.0 ⁇ g / ml
  • the maximum non-lethal concentration of the injection of the composition of the present invention The concentration is 10 ⁇ g / ml, which is four times that of nimodipine raw materials and nimodipine water injection, and twice that of the prior art nimodipine emulsion, indicating that the composition injection of the present invention is far less toxic to zebrafish than nimodipine.
  • Dipine APIs, nimodipine injections, and prior art nimodipine emulsions are examples of the composition injection of the present invention.
  • the wild type AB zebrafish juveniles are treated by the test drug for 24 hours.
  • the five initial detection concentrations are: 0.1 mg / ml, 1 mg / ml, 10 mg / ml. ml, 100mg / ml and 500mg / ml, blank control group was set at the same time, each concentration treated 30 zebrafish. After the treatment, the number of zebrafish deaths in each experimental group was counted to provide a basis for the next experimental concentration design.
  • the detection concentration range of the test drug is 500mg / ml (if the maximum solubility is less than 500mg / ml, the highest solubility shall prevail), and the lower limit is 0.001mg / ml.
  • Group mortality rate(%) Blank 0 (0/30) 24% ethanol 0 (0/30)
  • Positive control 0 (0/30) 10nl 0 (0/30) 20nl 0 (0/30) 40nl 0 (0/30)
  • nimodipine API 4.0mg / ml and above on zebrafish increased with increasing concentration, and it could induce pericardial edema and circulation defects.
  • Nimodipine's drug substance concentration of 2.0mg / ml (67mg / kg) induces pericardial edema in 30% of individuals, and the maximum dose of nimodipine water injection 40nl (26.7mg / kg) can induce pericardial edema in 40% of individuals.
  • Cardiovascular toxicity was not observed at the maximum administered dose of the invention composition injection (106.7 mg / kg), which indicates that the cardiovascular toxicity of the composition injection of the present invention to zebrafish is less than that of nimodipine water injection and nimodipine drug substance.
  • the experimental animals were randomly divided into 8 groups, which were a control group that only exposed the skull but did not inject blood; a high-medium-low-dose group of nimodipine injection; a high-medium and low-dose group of the injection of the composition of the present invention; More than 10 survived after each composition.
  • the administration method and dosage of each group are as follows.
  • Nimodipine injection group high dose 2mg / kg body weight, middle dose 1mg / kg body weight, low dose 0.5mg / kg body weight; drug concentration is 1mg / 5mL.
  • composition injection group of the present invention the composition injection of the present invention is diluted with physiological saline to a concentration of 1 mg / 5 mL before use. High dose 2mg / kg body weight, medium dose 1mg / kg body weight, low dose 0.5mg / kg body weight.
  • Model group The tail vein was injected with saline 1 mL / 100 g body weight.
  • Dosing volume and method high dose 1mL / 100g body weight, medium dose 0.5mL / 100g body weight, low dose 0.25mL / 100g body weight. About 30min, 48h, and 96h after modeling, respectively, the drugs were administered by tail vein injection according to the above dosage.
  • compositions of the invention are low-before administration 211.81 ⁇ 66.28 ⁇ 179.08 ⁇ 45.33 ⁇ In the composition of the present invention-before administration 189.98 ⁇ 41.52 ⁇ 177.53 ⁇ 37.70 ⁇ The composition of the invention is high-before administration 193.04
  • the blood flow was measured after the cerebral blood flow was measured, and the blood flow was measured again in the whole craniocerebral region and sagittal suture region 30 minutes after the administration.
  • One-way analysis of variance was performed using EXCEL software. The results are shown in Table 21 and Table 22 below.
  • composition of the present invention is low-poor before and after administration 13.99 ⁇ 39.85 30.94 ⁇ 25.90 In the composition of the present invention-poor before and after administration 14.11 ⁇ 63.07 25.40 ⁇ 52.50 The composition of the present invention is high-poor before and after administration 26.42 ⁇ 28.53 23.42 ⁇ 32.45
  • composition of the invention is low 225.80 ⁇ 49.14 210.02 ⁇ 41.22 In the composition of the present invention 204.09 ⁇ 44.66 202.93 ⁇ 33.18 The composition of the invention is high 217.53 ⁇ 43.15 205.22 ⁇ 39.65
  • Table 23 Cerebral blood flow table (mean ⁇ SD) of the composition of the present invention and nimodipine acupuncture high dose group after 10 days of modeling
  • Cerebral blood flow values before and after modeling and before and after administration of the high-dose nimodipine and high-dose groups of the composition of the present invention are shown in Table 24.
  • the changes in cerebral blood flow are shown in Figures 1 and 2.
  • Table 24 Cerebral blood flow table (mean ⁇ SD) of nimodipine water injection and high dose group of the composition of the present invention
  • composition of the present invention has a reduced value after high molding 122.90 133.37 After high administration of the composition of the present invention 217.53 ⁇ 43.15 205.22 ⁇ 39.65
  • the composition of the present invention is poor before and after high administration 26.42 ⁇ 28.53 23.42 ⁇ 32.45 Nimodipine Water Needle Before Modeling 306.10 ⁇ 102.35 301.90 ⁇ 86.88 Nimodipine Water Needle After Modeling 198.12 ⁇ 32.19 186.57 ⁇ 26.78 Nimodipine Water Needle Decreases Value After High Modeling 107.98 115.33 Nimodipine water after needle high dose 219.42 ⁇ 65.60 209.08 ⁇ 57.46 Nimodimodi
  • Brain tissue was dehydrated, embedded in paraffin, and brain stem tissue sections including the basilar artery were taken. After HE staining and sealing, the microscope was observed and the circumference and area of the basilar artery blood vessels were measured with an image analysis system to determine the degree of cerebral vasospasm. The results are shown in Table 25. below.
  • composition of the invention is low 567.43 ⁇ 104.38 ⁇ 18825.13 ⁇ 6587.00 In the composition of the present invention 595.82 ⁇ 164.43 ⁇ 22503.74 ⁇ 11587.18 The composition of the invention is high 603.41 ⁇ 123.66 ⁇ 24031.65 ⁇ 9632.43 ⁇
  • Nimo has a significant improvement effect with high, medium and low doses, of which Nimo has a very significant improvement effect with high doses; low, medium and high doses of fatty milk also have significant improvement effects.
  • the cross-sectional area of blood vessels in some animals changed greatly, which increased the SD in the group.
  • the cross-sectional area of the basilar artery was significantly improved only in the high-dose group and the high-dose fat milk group.
  • the curative effect ranks as follows: high dose of nimodipine water needle ⁇ high dose of the composition of the present invention > nimodipine in the composition of the present invention Among the water needles ⁇ low nimodipine water needles> the composition of the present invention is low.
  • the high-dose group of the composition of the present invention has a significant improvement. There is a quantitative effect relationship between different doses.
  • the experimental animals performed open-field autonomous activity experiments at 24h, 72h, and 5d after administration, respectively, and opened a video monitoring system to record the movement distance and activity time of rats within 5min. The results are shown in Tables 27 and 28.
  • composition of the invention is low 106.44 ⁇ 44.73 129.91 ⁇ 43.42 129.03 ⁇ 64.83 In the composition of the present invention 200.73 ⁇ 20.92 ⁇ 179.42 ⁇ 52.92 146.13 ⁇ 33.15
  • the composition of the invention is high
  • the activity distance and duration have improved, and the overall activity ability has been enhanced. It also slightly improved with the increase of the number of administrations.
  • the movement distance of the high-dose group of the composition of the invention 5 days after administration was significantly improved; the high-dose nimodipine water injection and the activity time of the dose group of the composition of the invention also significantly improved, but there was no significant difference. difference.
  • Nimodipine water injection contains high concentration of ethanol, and intravenous injection has a strong "narcotic" effect. It takes about 10-30 minutes for rats to recover slowly after injection. Among them, the nimodipine water injection high-dose group had obvious toxic and side effects, and many died during injection administration. After repeated administration of nimodipine water injection, many animals developed phlebitis and tail ulceration. In each dosage group of the composition injection of the present invention, no death and other visible adverse reactions occurred during administration.
  • the same dosage of the injection of the composition of the present invention is basically equivalent to the effect of nimodipine water injection without significant differences.
  • the injection of the composition of the present invention did not show toxic side effects and other visible adverse reactions. Therefore, the injection of the composition of the present invention is equivalent to the commercially available nimodipine injection and the prior art nimodipine emulsion injection, but has higher safety.
  • the rats were administered by tail vein injection at a dose of 2 mg / kg body weight.
  • 0.3 ml of blood was collected from the venous fundus veins of rats, placed in a heparinized blood collection tube, centrifuged at 4000 rpm for 10 minutes, and the upper plasma was stored at -80 ° C.
  • UPLC-PDA analysis was used to determine nimodipine plasma samples.
  • the sample plasma was processed one by one and analyzed by injection, and the concentration of nimodipine in the plasma was calculated.
  • the results are shown in Table 29.
  • the blood drug concentration (ng / ml) is plotted against time (min), and the blood drug concentration time curve is drawn.
  • the results are shown in FIG. 3 of the accompanying drawings.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Dermatology (AREA)
  • Dispersion Chemistry (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pain & Pain Management (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Medicinal Preparation (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

一种尼莫地平注射液组合物及其制备方法。所述尼莫地平注射液组合物，以质量浓度计，包括如下组分：尼莫地平0.02-0.23%，注射用油2-30%，乳化剂0.8-3%，络合剂0-0.1%，稳定剂0-0.3%，渗透压调节剂1-3%。该组合物不含增溶剂乙醇，无吐温-80等辅助乳化剂、苯甲醇等助溶剂。

Description

一种尼莫地平注射液组合物及其制备方法 技术领域
本发明属于医药技术领域，涉及本发明组合物注射液及其制备方法和非临床安全性。
背景技术
尼莫地平(Nimodipine，Nim)又称硝苯吡酯(Nimotop)，是第二代吡啶类钙拮抗剂，尼莫地平注射液于1985年4月由德国拜尔(Bayer)公司研制上市，目前我国有一百多个厂家生产。由于它具有很高的亲脂性特点，易透过血脑屏障，具有选择性扩张脑血管、显著逆转基底动脉和脊髓前动脉痉挛的作用，临床上用于治疗高血压、中风、偏头痛、蛛网膜下腔出血及其它脑出血疾病，是目前脑血管治疗的首选药物，特别对老年性痴呆具有较高的临床应用价值。
在现有的技术中，临床上常用的尼莫地平制剂有片剂、胶囊剂、注射剂。尼莫地平为水难溶性药物，溶解度小并且有较强的肝首过效应，致使口服生物利用度低，健康受试者及蛛网膜下腔出血患者的生物利用度分别为5-13％和3-28％。该药生物半衰期短(约1.5-2h)，每日需3-4次频繁用药，不仅使用不方便，而且可使血药浓度出现“峰谷”现象，引起毒副作用；由于尼莫地平为水不溶性，临床上所用的尼莫地平注射液均含较高浓度的乙醇以增溶，对皮肤及血管刺激性大。由于尼莫地平必须以1-2mg/h的速度慢速滴注，否则病人无法耐受其副作用，即10mg药物所需滴注时间一般需至少5小时，使用时需与葡萄糖或生理盐水混合滴注或用特殊的三通输液器与葡萄糖或生理盐水同时滴注，混合液直接输入病人体内。多数临床医生在使用时是将尼莫地平注射液加入输液瓶中，混匀后使用。由于尼莫地平在乙醇中溶解，在水中不溶，尼莫地平输液为含乙醇的非水溶性制剂，当与其它输液配伍后，均可析出结晶，含量下降，降低了药物疗效，也给病人带来一定程度的危险。
尼莫地平存在不同晶型，即α型和β型。不同晶型在不同溶剂中溶解度不同，据文献报道，β晶型熔点124-125℃，在水中溶解度较好(3.6ug/ml)；α晶型熔点114-116℃，在水中溶解度2.5ug/ml，在油相中溶解度未见报道；本发明比较了市售尼莫地平(β晶型，新华药业)及自制尼莫地平(α晶型)在不同油中的溶解度，发现尼莫地平在油中的溶解度远大于在水中的溶解度，且α晶型在中链脂肪油溶解度及大豆油和中链脂肪油混合物中的溶解度远大于β晶型(见下表)，因此，将α晶型的尼莫地平溶于油中制成脂肪乳注射液有望解决尼莫地平水针剂及现有技术尼莫地平脂肪乳注射液载药量低、安全性和稳定性差的问题。
尼莫地平在油相中的溶解度测定试验(mg/ml)
原料来源 大豆油 中链油 大豆油：中链油(4：6) 水
β晶型(常温) 6.7 11.8 11.0 0.0036
α晶型(常温) 7.5 23.22 18.1 0.0025
以大豆油、中链脂肪油及大豆油和中链脂肪油混合物等为主要成分的注射乳剂既具有溶剂特性，又几乎无毒性，将脂溶性药物引入并溶于乳剂颗粒核心脂质部分,随脂质油滴进行代谢、缓慢释放从而维持有效的血药浓度，降低了药物可能引起的毒副作用，更具有本发明组合物注射液的诸多优点；本发明组合物注射液可增加尼莫地平药物的溶解度，可以提高载药量，减少药物的水解，增加稳定性，降低毒性，具有良好的临床应用前景。
目前，有较多的有关尼莫地平脂肪乳的文献和专利申请，但都存在一定不足，如中国专利申请号为200910021091.6的已授权专利，处方中使用吐温80。目前含吐温80的药物静脉注射剂的临床不良反应报道非常多，可引起溶血反应、急性超敏反应、外周神经毒性、抑制P-糖蛋白活性、内在抗肿瘤效应、肝毒性等，因此要慎用含吐温80的静脉注射剂；中国专利申请号为200510081668.4的已授权专利，处方中加入助溶剂苯甲醇。苯甲醇具有消毒防腐、局部麻醉作用，在注射剂中用于抑菌剂、止痛剂，且具有溶血作用。儿童用于肌内注射的注射液含有苯甲醇时可引起臀肌挛缩症。《中华人民共和国药典》2015年版附录注射剂项下对静脉注射剂添加抑菌剂有严格限定，不建议使用。同时，所有尼莫地平乳剂都没有考虑晶型问题，采用其它晶型溶解度低，制剂载药量小，贮存期间易析出结晶，产品稳定性差。考虑到以上尼莫地平组合物存在的稳定性安全性隐患，因此有必要开发新的尼莫地平乳剂组合物来满足临床需求。
发明内容
本发明的目的之一是提供一种尼莫地平注射液组合物，其特征在于，所述的注射液组合物包括如下组分：尼莫地平、注射用油、乳化剂、络合剂、稳定剂、渗透压调节剂。
所述的注射液组合物包括尼莫地平0.02-0.23％，注射用油2-30％，乳化剂0.8-3％，络合剂0-0.1％，稳定剂0-0.3％，渗透压调节剂1-3％。优选的比例为：尼莫地平0.04-0.20％，注射用油3-20％，乳化剂1-2.5％，络合剂0-0.05％，稳定剂0-0.2％，渗透压调节剂2-3％；更优选的比例为：尼莫地平0.08-0.18％，注射用油8-12％，乳化剂1-2％，络合剂0-0.01％，稳定剂0-0.1％，渗透压调节剂2-2.5％。制成的尼莫地平注射液组合物pH值为6.0-8.5，优选的 pH值为6.5-8.0，更优选的pH值为7.0-7.5。制成的尼莫地平注射液组合物平均粒径为100-600nm，优选的平均粒径为120-400nm，更优选的平均粒径为140-300nm。
所述尼莫地平选自α晶型或β晶型，优选熔点为114-116℃的α晶型。
所述的注射液组合物中乳化剂选自大豆磷脂或蛋黄磷脂。
所述的注射液组合物中大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为60-90％，磷脂酰乙醇胺(PE)含量为2-20％，优选的范围为所述大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为70-85％，磷脂酰乙醇胺(PE)含量为5-18％。
所述的注射液组合物中络合剂可选自依地酸钙钠、依地酸二钠、依地酸钠、依地酸中的一种或一种以上组合物，优选的络合剂为依地酸二钠。
所述的注射液组合物中注射用油为注射用大豆油或注射用中链甘油三酸酯或二者混合物，优选注射用大豆油、注射用中链甘油三酸酯混合物，优选的质量比为2:8-5:5，更优选的质量比为4:6-5:5，最优选的质量比为4:6。
所述的注射液组合物中优选的稳定剂为油酸或油酸钠。
所述的注射液组合物中渗透压调节剂为甘油。
本发明提供了一种制备尼莫地平注射液组合物的方法，本发明的优点在于，本发明组合物注射液不含增溶剂乙醇，避免了对皮肤及血管刺激性。无需添加吐温-80等辅助乳化剂、苯甲醇等助溶剂，解决了尼莫地平注射液乳化问题和增溶性问题。通过对大豆油与中链甘油三酸酯不同比例及尼莫地平不同晶型的研究，找出既最大程度地负载尼莫地平原料药又可避免由于中链甘油三酸酯用量过大而造成的油水分离现象。具体来说解决了以下问题：
1、解决了稳定性问题。通过对乳化剂的筛选，选用一定含量范围的磷脂酰胆碱(含量在70-85％)和磷脂酰乙醇胺(含量在5-18％)的卵磷脂或豆磷脂做乳化剂，无须添加吐温-80作辅助乳化剂即可解决稳定性问题，降低了临床应用过程中致敏、变态反应、溶血反应等风险。
2、通过将乳化剂分别加入油相和水相研究，优选乳化剂一部分加入油相、一部分加入水相，乳化效果更好，乳粒更均匀，乳剂更稳定。
3、解决了增溶性问题和工业化均质次数过多问题。注射用油选用注射用大豆油、注射用中链甘油三酸酯质量比为4:6优选熔点为114-116℃的α晶型尼莫地平原料时可以达到最大载药量，同时避免造成油水分离现象。
4、无须添加苯甲醇等助溶剂，避免临床使用添加苯甲醇而造成的不良反应。
5、由于中链甘油三酸酯的加入，油相更易乳化分散，可以降低均质次数，更有利于工业 化生产。
6、使用络合剂不仅可以实现抗氧化作用，还能够大幅降低制剂中甲氧基苯胺值，提高了制剂的安全性。
7、通过过敏、刺激、溶血、大鼠等非临床安全性试验研究，实验结果证明：
(1)大鼠被动过敏试验表明，本发明组合物注射液对致敏大鼠无被动皮肤过敏反应，现有技术尼莫地平乳剂对致敏大鼠有被动皮肤过敏反应；豚鼠全身主动过敏试验本发明组合物注射液高剂量组豚鼠即刻(约1分钟)出现俯卧不动、继而死亡等类似过敏反应，死亡率100％，低剂量组豚鼠未见明显异常反应。而现有技术尼莫地平乳剂高、低剂量组均出现俯卧不动、继而死亡等类似过敏反应，高剂量组死亡率100％，低剂量组死亡40％。
(2)现有技术尼莫地平乳剂有溶血和凝集作用，本发明组合物注射液无溶血和凝集作用。
(3)兔血管刺激性试验，本发明组合物注射液和现有技术尼莫地平乳剂对兔耳缘静脉(血管)均无明显刺激作用。
(4)兔肌肉刺激性试验，本发明组合物注射液和现有技术尼莫地平乳剂对兔股四头肌均无明显刺激反应。
本发明组合物注射液诱发斑马鱼心血管毒性实验结果表明，尼莫地平原料药、尼莫地平水针剂、现有技术尼莫地平乳剂和本发明组合物注射液均可诱导斑马鱼发生心率减慢、心房跳动停止、心包水肿以及循环缺陷，四种药物均有心血管毒性。尼莫地平原料药和尼莫地平水针剂的最大非致死浓度为2.5μg/ml，现有技术尼莫地平乳剂最大非致死浓度为5.0μg/ml，而本发明组合物注射液的最大非致死浓度为10μg/mL，为尼莫地平原料药和尼莫地平水针剂的4倍，为现有技术尼莫地平乳剂的2倍，表明本发明组合物注射液对斑马鱼的毒性远小于尼莫地平原料药、尼莫地平水针剂以及现有技术尼莫地平乳剂。
大鼠注射给药表明，尼莫地平水针剂高剂量组出现明显毒副作用，在注射给药时有多只死亡。尼莫地平水针剂多次给药后多只动物出现明显静脉炎和尾巴溃烂。本发明组合物注射液各剂量组均未在给药时出现死亡和其它可见不良反应。
本发明所采用的技术方案如下：
一种尼莫地平注射液组合物，以质量浓度计，包括如下组分：尼莫地平0.02-0.23％，注射用油2-30％，乳化剂0.8-3％，络合剂0-0.1％，稳定剂0-0.3％，渗透压调节剂1-3％。优选的比例为：尼莫地平0.04-0.20％，注射用油3-20％，乳化剂1-2.5％，络合剂0-0.05％，稳定剂0-0.2％，渗透压调节剂2-3％；更优选的比例为：尼莫地平0.08-0.18％，注射用油8-12％，乳化剂1-2％，络合剂0-0.01％，稳定剂0-0.1％，渗透压调节剂2-2.5％。制成的尼莫地平注射 液组合物pH值为6.0-8.5，优选的pH值为6.5-8.0，更优选的pH值为7.0-7.5。制成的尼莫地平注射液组合物平均粒径为100-600nm，优选的平均粒径为120-400nm，更优选的平均粒径为140-300nm。
所述尼莫地平选自α晶型或β晶型，优选熔点为114-116℃的α晶型。
所述的尼莫地平注射液组合物中，所述乳化剂选自大豆磷脂、蛋黄磷脂、氢化大豆磷脂、氢化蛋黄磷脂中的一种或一种以上组合物。
所述的尼莫地平注射液组合物中，所述络合剂选自依地酸钙钠、依地酸二钠、依地酸钠、依地酸中的一种或一种以上组合物。
所述的尼莫地平注射液组合物中，优选的稳定剂为油酸或油酸钠。
所述的尼莫地平注射液组合物中，所述渗透压调节剂为甘油。
所述的尼莫地平注射液组合物pH值为6.0-8.5，优选的pH值为6.5-8.0，更优选的pH值为7.0-7.5。
所述的尼莫地平注射液组合物平均粒径为100-600nm，优选的平均粒径为120-400nm，更优选的平均粒径为140-300nm。
本发明还提供了一种尼莫地平注射液组合物的制备方法，包括如下步骤：
(1)取注射用水，调节pH，加入络合剂、稳定剂搅拌溶解，再加入渗透压调节剂，搅拌混合均匀，制成水相，水温控制在50～70℃：
所述的络合剂选自依地酸钙钠、依地酸二钠、依地酸钠、依地酸中的一种或一种以上组合物，优选的络合剂为依地酸二钠。渗透压调节剂优选甘油。
(2)取注射用油，加入40％处方量的乳化剂、稳定剂剪切混匀，水浴加热到50～70℃，加入尼莫地平搅拌使其溶解，制成油相。所述尼莫地平选自α晶型或β晶型，优选熔点为114-116℃的α晶型。
所述注射用油选自注射用大豆油或注射用中链甘油三酸酯或二者混合物，优选的注射用大豆油、注射用中链甘油三酸酯混合物，优选的质量比为2:8-5:5，更优选的质量比为4:6-5:5，最优选的质量比为4:6。
(3)稳定剂选用油酸钠时，油酸钠加入水相(1)中，选用油酸时，油酸加入油相(2)中。
(4)取60％处方量的乳化剂，加入水相中，在氮气保护下，剪切乳化剂至分散均匀。
所述的乳化剂选自大豆磷脂或蛋黄磷脂，大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为60-90％，磷脂酰乙醇胺(PE)含量为2-20％。优选的范围为所述大豆磷脂或蛋黄磷脂中磷 脂酰胆碱(PC)含量为70-85％，磷脂酰乙醇胺(PE)含量为5-18％。
(5)将油相缓缓加入至水相中，剪切制成初乳。
(6)将初乳进行均质，循环至乳液粒度符合规定。
(7)取样检测pH值、乳粒粒径，充氮气，灌封，灭菌。
附图说明
图1为本发明组合物高剂量组9号大鼠脑血流变化图，圆点曲线为全颅脑区，菱形曲线为矢状缝区。
图2为尼莫地平水针高剂量组16号大鼠脑血流变化图，圆点曲线为全颅脑区，菱形曲线为矢状缝区。
图3为尼莫地平水针和本发明组合物的药时曲线图。
具体实施方式
下面结合实施例对本发明进一步详细说明，但本发明不限于这些实施例：
实施例1
尼莫地平1g、大豆油40g、中链甘油三酸酯60g、卵磷脂12g、依地酸二钠0.05g、油酸0.3g、甘油22.5g、pH调节剂适量、注射用水至1000ml。制法如下：
(1)取处方量的注射用水，用2％氢氧化钠溶液调节pH，加入依地酸二钠搅拌溶解，再加入甘油，搅拌混合均匀。水温控制在50～70℃：
(2)取处方量大豆油与中链甘油三酸酯混合，加入40％处方量的卵磷脂、油酸剪切混匀，水浴加热到50～70℃，加入处方量尼莫地平搅拌使其溶解。
(3)称取60％处方量的卵磷脂，加入水相中，在氮气保护下，剪切乳化剂至分散均匀。
(4)将油相缓缓加入至水相中，剪切制成初乳。
(5)将初乳进行均质，循环至乳液粒度符合规定。
(6)取样检测pH值、乳粒粒径符合规定后，经滤膜过滤，充氮气，灌封。
(7)进行121℃、12分钟灭菌。
(8)pH值为7.3，平均粒径为155nm。
实施例2
尼莫地平1.5g、大豆油40g、中链甘油三酸酯60g、卵磷脂12g、依地酸二钠0.05g、油酸0.3g、甘油22.5g、pH调节剂适量、注射用水至1000ml。制法如下：
(1)取处方量的注射用水，用2％氢氧化钠溶液调节pH，加入依地酸二钠搅拌溶解，再加入甘油，搅拌混合均匀。水温控制在50～70℃：
(2)取处方量大豆油与中链甘油三酸酯混合，加入40％处方量的卵磷脂、油酸剪切混匀，水浴加热到50～70℃，加入处方量尼莫地平搅拌使其溶解。
(3)称取60％处方量的卵磷脂，加入水相中，在氮气保护下，剪切乳化剂至分散均匀。
(4)将油相缓缓加入至水相中，剪切制成初乳。
(5)将初乳进行均质，循环至乳液粒度符合规定。
(6)取样检测pH值、乳粒粒径符合规定后，经滤膜过滤，充氮气，灌封。
(7)进行121℃、12分钟灭菌。
(8)pH值为6.9，平均粒径为162nm。
实施例3
尼莫地平0.2g、大豆油50g、卵磷脂8g、依地酸钙钠0g、油酸钠0.2g、甘油25g、pH调节剂适量、注射用水至1000ml。制法如下：
(1)取处方量的注射用水，调节pH，加入油酸钠搅拌溶解，再加入甘油，搅拌混合均匀。水温控制在50～70℃。
(2)取处方量大豆油与中链甘油三酸酯混合，水浴加热到50～70℃，加入40％处方量的卵磷脂剪切混合均匀，加入处方量尼莫地平搅拌使其溶解。
(3)称取60％处方量的卵磷脂，加入水相中，在氮气保护下，剪切乳化剂至分散均匀。
(4)将油相缓缓加入至水相中，剪切制成初乳。
(5)将初乳进行均质，循环至乳液粒度符合规定。
(6)取样检测pH值、乳粒平均粒径符合规定后，经滤膜过滤，充氮气，灌封。
(7)进行121℃、12分钟灭菌。
(8)pH值为7.4，平均粒径为150nm。
实施例4
尼莫地平2.3g、大豆油80g、中链甘油三酸酯120g、卵磷脂15g、依地酸二钠0.1g、油酸1g、甘油20g、pH调节剂适量、注射用水至1000ml。
制法：同实施例1。
pH值为7.2，平均粒径为296nm。
实施例5
尼莫地平0.4g、大豆油40g、中链甘油三酸酯60g、卵磷脂8g、依地酸二钠0.03g、油酸 0.3g、甘油22.5g、pH调节剂适量、注射用水至1000ml。
制法：同实施例1。
pH值为7.9，平均粒径为600nm。
实施例6
尼莫地平0.8g、大豆油30g、中链甘油三酸酯70g、卵磷脂30g、依地酸二钠0.0g、油酸0.0g、甘油22.5g、pH调节剂适量、注射用水至1000ml。
制法：同实施例1。
pH值为6.8，平均粒径为146nm。
实施例7
尼莫地平1.2g、大豆油120g、中链甘油三酸酯180g、卵磷脂30g、依地酸二钠0.0g、油酸0.3g、甘油20g、pH调节剂适量、注射用水至1000ml。
制法：同实施例1。
pH值为6.5，平均粒径为352nm。
实施例8
尼莫地平1.2g、大豆油50g、中链甘油三酸酯50g、卵磷脂30g、依地酸二钠0.0g、油酸0.0g、甘油22.5g、pH调节剂适量、注射用水至1000ml。
制法：同实施例1。
pH值为8.5，平均粒径为246nm。
为确定本发明不加助溶剂的最大载药量和不加辅助乳化剂的最优配方和最佳工艺，发明人进行了大量试验研究，研究结果如下：
1、大豆油与中链甘油三酸酯不同比例的最佳载药研究。
由于大豆油与中链甘油三酸酯对尼莫地平的溶解度不同，因此不同比例配比，会造成不同的载药量。另外，中链甘油三酸酯用量过多会造成乳剂出油。在实施例1的处方基础上，进行了不同比例油相的最佳载药研究。
表1不同比例油相对本发明组合物注射液稳定性研究
Figure PCTCN2019104760-appb-000001
结论：大豆油：中链甘油三酸酯4：6时为最佳处方。
2、在实施例1的处方基础上考察磷脂中磷脂酰胆碱(PC)和磷脂酰乙醇胺不同含量对乳剂稳定性的影响。
表2不同PC与PE含量组成的磷脂对本发明组合物注射液的影响
Figure PCTCN2019104760-appb-000002
结论：大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为70％-85％，磷脂酰乙醇胺(PE)含量为5％-18％组成的磷脂乳化能力最佳。
3、在实施例1的处方基础上考察磷脂加水相或油相对本发明组合物注射液的影响。
表3磷脂加水相或油相对本发明组合物注射液的影响
Figure PCTCN2019104760-appb-000003
结论：将磷脂磷脂加水相或油相，本发明组合物δ-电位更高，稳定性更佳。
4、针对实施例6考察络合剂对本发明组合物注射液甲氧基苯胺值的影响。
表4络合剂对本发明组合物注射液甲氧基苯胺值的影响
Figure PCTCN2019104760-appb-000004
结论：使用金属离子络合剂就可以实现抗氧化，还能够大幅降低制剂中甲氧基苯胺值，提高了制剂的安全性，添加其他抗氧剂并不能降低本发明组合物注射液的甲氧基苯胺值。
为确定本发明制备工艺的稳定性，按照实施例1进行了中试试验，并进行了稀释稳定性研究及长期稳定性研究。
各取本发明组合物注射液30ml置250ml量瓶中，分别用0.9％氯化钠注射液及5％葡萄糖注射液稀释至刻度，摇匀，即得配伍实验溶液，在0h，2h，4h，6h，8h及12h分别测定上述溶液的pH值、乳粒粒径、有关物质、含量。检测结果见下表。
表5本发明组合物注射液与0.9％氯化钠注射液稀释配伍试验
Figure PCTCN2019104760-appb-000005
表6本发明组合物注射液与5％葡萄糖注射液稀释配伍试验
Figure PCTCN2019104760-appb-000006
从稀释后样品的主要项目测定的结果显示：12小时内所有稀释样品的pH值、乳粒粒径、有关物质、含量均无明显变化。本发明组合物注射液在临床使用时用葡萄糖注射液或氯化钠注射液稀释，在12小时内使用是安全可靠的。
本发明组合物注射液在温度25±2℃的条件下遮光放置，分别于0、3、6、9、12、18个月取样一次，按稳定性重点考察项目检测，与0月相比较。
表7本发明组合物注射液长期稳定性考察
Figure PCTCN2019104760-appb-000007
由试验结果可知，本发明组合物注射液经18个月25±2℃的长期稳定性考察，各项指标均符合规定，表明本发明组合物注射液在2～25℃保存条件下质量稳定。
为验证本发明组合物注射液的非临床安全效果，针对本发明实施例1制备得到的组合物注射液进行了过敏性、溶血性和局部(血管、皮肤、粘膜、肌肉等)刺激性等特殊安全性试验，斑马鱼试验及对大鼠蛛网膜下腔出血模型的药效学试验，实验结果如下：
一、对本发明组合物注射液进行了过敏性、溶血性和局部(血管、皮肤、粘膜、肌肉等)刺激性等特殊安全性试验。
1、过敏性试验
1.1大鼠被动过敏试验
健康清洁级SD大鼠皮内注射相应的抗血清0.1ml被动致敏48h后，各组再分别静脉注射本发明组合物注射液8、4mg/kg剂量，采用现有技术专利工艺制备的尼莫地平乳剂8、4mg/kg剂量(申请号：200910021091.6处方中含吐温80)牛血清白蛋白100mg/kg剂量和0.9％NaCl注射液10ml/kg剂量，以及1％依文思蓝溶液1ml/只进行激发。30分钟后解剖结果显示：本发明组合物注射液8、4mg/kg剂量组和0.9％NaCl注射液阴性对照组大鼠背部皮肤内层均未出现蓝斑。而采用专利工艺制备的尼莫地平乳剂8、4mg/kg剂量、牛血清白蛋白阳性对照组大鼠背部皮肤内层均出现明显蓝斑(直径均大于5mm)，蓝斑反应为阳性。在本试验剂量条件下，现有技术尼莫地平乳剂对致敏大鼠有被动皮肤过敏反应，本发明组合物注射液对致敏大鼠无被动皮肤过敏反应。
表8本发明组合物注射液大鼠被动皮肤过敏试验蓝斑反应结果
剂量组别 蓝斑反应(mm，±s)
本发明组合物注射液8mg/kg -
本发明组合物注射液4mg/kg -
现有技术尼莫地平乳剂8mg/kg +(18.0±3.6)
现有技术尼莫地平乳剂4mg/kg +(11.6±3.3)
0.9％NaCl注射液10ml/kg -
牛血清白蛋白100mg/kg +(22.0±4.3)
注：-表示阴性，+表示阳性。
1.2豚鼠全身主动过敏试验
健康白色豚鼠分别腹腔注射本发明组合物注射液4、2mg/kg剂量，隔日一次，连续3次进行致敏。末次致敏后第13天由静脉单次注射致敏剂量的本发明组合物注射液进行激发， 现有技术尼莫地平乳剂的致敏和激发试验方法同上，本发明组合物注射液高剂量组豚鼠即刻(约1分钟)出现俯卧不动、继而死亡等类似过敏反应，死亡率100％，低剂量组豚鼠未见明显异常反应。而现有技术尼莫地平乳剂高、低剂量组均出现俯卧不动、继而死亡等类似过敏反应，高剂量组死亡率100％，低剂量组死亡40％。本发明组合物注射液高、低剂量组、现有技术尼莫地平乳剂、牛血清白蛋白阳性对照组豚鼠在致敏期间未见明显异常反应，其首次致敏、末次致敏和激发时的平均体重与相应时间的0.9％NaCl注射液阴性对照组相似(P>0.05)。牛血清白蛋白阳性对照组豚鼠在激发后均出现明显的过敏反应症状，主要表现为不安宁、竖毛、发抖、搔鼻、喷嚏、咳嗽、呼吸急促、排尿、流泪、呼吸困难、步态不稳、喘息、跳跃、痉挛、甚至死亡，过敏反应强度为++～++++，死亡时间在给药后5分钟左右，有过敏反应的存活豚鼠在给药后30分钟内逐渐恢复正常，其反应出现率为100％，死亡率为50％。在本试验条件下，现有技术尼莫地平乳剂2mg/kg剂量对豚鼠有过敏反应，本发明组合物注射液2mg/kg剂量对豚鼠无过敏反应。
2、溶血性研究
按药物常规溶血试验方法，本发明组合物注射液0.1～0.5ml各管在3小时内均未出现溶血和凝集，与0.9％NaCl注射液阴性对照管相同。现有技术尼莫地平乳剂和蒸馏水阳性对照管各时间点观察均出现完全溶血。在本试验条件下，现有技术尼莫地平乳剂有溶血和凝集作用，本发明组合物注射液无溶血和凝集作用。
3、兔血管刺激性试验
兔耳缘静脉(血管)每天1次，连续7天分别缓慢注射本发明组合物注射液和现有技术尼莫地平乳剂1mg(10ml)/(kg.d)剂量，停药96小时剖检，大体观未见明现有技术尼莫地平乳剂显异常，病理组织学检查仅见2只兔紧靠注射局部耳缘静脉血管内皮细胞浊肿，周围间质轻度水肿，均未见血栓、栓塞和大范围出血、水肿、坏死等严重刺激性病变，与0.9％NaCl注射液对照组比较无明显差异。根据血管刺激试验评分标准综合判定为“<1”。在本试验剂量条件下，按“血管刺激强度判断”，本发明组合物注射液和现有技术尼莫地平乳剂对兔耳缘静脉(血管)均无明显刺激作用。
4、兔肌肉刺激性试验
兔左右腿股四头肌每天1次，连续7天分别注射本发明组合物注射液和现有技术尼莫地平乳剂0.1mg(1ml)/(点.d)剂量，停药48小时大体观检查8只兔单侧股四头肌注射局部可见0.5×1.0cm以下充血灶病灶，其余兔注射局部均未见明显充血、肿胀和坏死等病变，其所有股四头肌反应级之和为4，小于6；组织病理学检查注射局部股四头肌出现以小灶性或微 灶性间质增生、单核细胞浸润为主病变，部分病灶内可见肌纤维浊肿或嗜碱性变但程度轻微，与同期检查0.9％NaCl注射液对照组比较病变略严重，但总体病灶范围局限，且均未出现明显大范围肌纤维变性、坏死和间质炎性浸润、充血、出血、水肿等严重刺激性病变。在本试验剂量条件下，根据肌肉刺激反应分级标准判断，本发明组合物注射液和现有技术尼莫地平乳剂对兔股四头肌均无明显刺激反应。
5、小结：
(1)大鼠被动过敏试验本发明组合物注射液对致敏大鼠无被动皮肤过敏反应，现有技术尼莫地平乳剂对致敏大鼠有被动皮肤有过敏反应；豚鼠全身主动过敏试验本发明组合物注射液高剂量组豚鼠即刻(约1分钟)出现俯卧不动、继而死亡等类似过敏反应，死亡率100％，低剂量组豚鼠未见明显异常反应。而现有技术尼莫地平乳剂高、低剂量组均出现俯卧不动、继而死亡等类似过敏反应，高剂量组死亡率100％，低剂量组死亡40％。
(2)现有技术尼莫地平乳剂有溶血和凝集作用，本发明组合物注射液无溶血和凝集作用。
(3)兔血管刺激性试验：本发明组合物注射液和现有技术尼莫地平乳剂对兔耳缘静脉(血管)均无明显刺激作用。
(4)兔肌肉刺激性试验：本发明组合物注射液和现有技术尼莫地平乳剂对兔股四头肌均无明显刺激反应。
二、本发明组合物注射液斑马鱼模型心血管毒性研究，试验情况如下：
1、溶于鱼水给药
1.1药物
本项目的待测药物有尼莫地平原料药、尼莫地平水针剂(尼膜同，拜耳生产)、本发明组合物注射液(按实施例1制备)及空白乳剂，采用现有技术专利工艺制备的尼莫地平乳剂(申请号:200910021091.6处方中含吐温80)。均由浙江九旭药业有限公司提供。
1.2实验仪器与试剂
解剖显微镜(SMZ645，Nikon公司，日本)；精密电子天平(CP214，奥豪斯)；六孔板(Nest Biotech)。
斑马鱼与养鱼用水
实验观察的斑马鱼幼鱼为生育力实验所产胚胎自然孵育而成。养鱼用水水质：每1L反渗透水中加入200mg速溶海盐，电导率为480～510μS/cm；pH为6.9～7.2；硬度为53.7～71.6mg/LCaCO 3。实验完成后，用0.25mg/ml的三卡因甲磺酸对各个发育阶段的斑马鱼进行麻醉处死。麻醉处死的操作步骤符合美国兽医协会(AVMA)对动物麻醉处死的规范要 求。
1.3实验方法
用待测药物处理一定阶段的野生型AB系斑马鱼幼鱼24小时，五个初始检测浓度分别为：0.1μg/ml、1μg/ml、10μg/ml、100μg/ml和500μg/ml，同时设置空白对照组，每个浓度均处理30尾斑马鱼。药物处理结束后，统计各实验组的斑马鱼死亡数量，为下一步实验浓度设计提供依据。待测药物的检测浓度范围，上限为1000μg/ml(若最大溶解度小于1000μg/ml则以最高溶解度为准)，下限为0.001μg/ml。
1.4斑马鱼致死率比较
尼莫地平原料药和尼莫地平水针剂最大非致死浓度为2.5μg/ml，现有技术尼莫地平乳剂最大非致死浓度为5.0μg/ml，而本发明组合物注射液的最大非致死浓度为10μg/ml，为尼莫地平原料药和水剂的4倍，为现有技术尼莫地平乳剂的2倍，由此可见，本发明组合物注射液对斑马鱼的毒性远小于尼莫地平原料药、尼莫地平水针剂和现有技术尼莫地平乳剂。
表9三种尼莫地平药物对斑马鱼的致死率比较
Figure PCTCN2019104760-appb-000008
1.5斑马鱼心率减慢比较
随着药物浓度的增加，斑马鱼心率减慢。其中尼莫地平原料药、尼莫地平水针剂和现有技术尼莫地平乳剂诱导的斑马鱼心率随药物浓度的增加减慢幅度较大，而本发明组合物注射液诱导的斑马鱼心率随药物浓度的增加减慢幅度较小。
表10三种药物诱导的斑马鱼心率减慢比较
Figure PCTCN2019104760-appb-000009
1.6斑马鱼心房停止跳动发生率比较
尼莫地平原料药、尼莫地平水针剂和现有技术尼莫地平乳剂在2.5μg/ml即可诱导部分斑马鱼发生心房跳动停止，在5.0μg/ml时，尼莫地平原料药、尼莫地平水针剂和现有技术尼莫地平乳剂诱导的斑马鱼心房跳动停止发生率分别达到了90％、100％和70％，而本发明组合物注射液诱导部分斑马鱼发生心房跳动停止的浓度为20μg/ml，100％发生心房跳动停止的浓度为40μg/ml，发生浓度均明显高于尼莫地平原料药、尼莫地平水针剂和现有技术尼莫地平乳剂。
表11三种药物诱导的斑马鱼心房停止跳动发生率比较
Figure PCTCN2019104760-appb-000010
1.7斑马鱼心包水肿发生率比较
四种药物诱导斑马鱼心包水肿发生率随着药物浓度的增加而升高。其中尼莫地平水针剂诱导的斑马鱼心包水肿发生率随浓度增加上升最快，在2.5μg/ml时即可诱导全部斑马鱼发生心包水肿；尼莫地平原料药次之，在5.0μg/ml时诱导全部斑马鱼发生心包水肿；现有技术尼莫地平乳剂再次之，在10.0μg/ml时诱导全部斑马鱼发生心包水肿；而本发明组合物注射液诱导的斑马鱼心包水肿发生率随药物浓度增加上升最慢，浓度直至20μg/ml时才诱导全部斑 马鱼发生心包水肿。
表12三种药物诱导的斑马鱼心包水肿发生率比较
Figure PCTCN2019104760-appb-000011
1.8小结
实验结果表明尼莫地平原料药、尼莫地平水针剂、现有技术尼莫地平乳剂和本发明组合物注射液均可诱导斑马鱼发生心率减慢、心房跳动停止、心包水肿以及循环缺陷，四种药物均有心血管毒性。尼莫地平原料药和尼莫地平水针剂的最大非致死浓度为2.5μg/ml，现有技术尼莫地平乳剂最大非致死浓度为5.0μg/ml，而本发明组合物注射液的最大非致死浓度为10μg/ml，为尼莫地平原料药和尼莫地平水针剂的4倍，为现有技术尼莫地平乳剂的2倍，表明本发明组合物注射液对斑马鱼的毒性远小于尼莫地平原料药、尼莫地平水针剂以及现有技术尼莫地平乳剂。
2、卵黄囊注射给药
2.1实验设计
利用卵黄囊注射给药法给药，最大给药体积40nl，待测药物处理野生型AB系斑马鱼幼鱼24小时，五个初始检测浓度分别为：0.1mg/ml、1mg/ml、10mg/ml、100mg/ml和500mg/ml，同时设置空白对照组，每个浓度均处理30尾斑马鱼。药物处理结束后，统计各实验组的斑马鱼死亡数量，为下一步实验浓度设计提供依据。待测药物的检测浓度范围，上限为500mg/ml(若最大溶解度小于500mg/ml则以最高溶解度为准)，下限为0.001mg/ml。
2.2尼莫地平原料药对斑马鱼心血管的影响。
2.2.1通过卵黄囊注射给药的方式注射给药10nl。处理一定时间后随机挑选10尾斑马鱼对其进行计数心率，并计算其相对心率进行比较，结果表明当浓度达到32mg/ml时心率发生减慢，其余药物处理组均正常。结果见表13。
表13尼莫地平原料药对斑马鱼心率的影响
Figure PCTCN2019104760-appb-000012
注：“*”表示经统计分析，处理组与对照组差异显著(p＜0.05)
阳性对照：特非那定片
2.2.2实验结束时进行死亡率统计，4.0mg/ml及以上浓度组随浓度增加致死率升高。结果见表14。
表14尼莫地平原料药对斑马鱼的致死率
分组 死亡率(％)
空白对照 0(0/30)
DMSO 0(0/30)
阳性对照 0(0/30)
0.5mg/ml 0(0/30)
1.0mg/ml 0(0/30)
2.0mg/ml 0(0/30)
4.0mg/ml 20(6/30)*
8.0mg/ml 33.3(10/30)*
16mg/ml 50(15/30)*
32mg/ml 80(24/30)*
注：本研究中斑马鱼心房心室均不跳动则视其死亡；
“*”表示经fisher精确检验法检验，处理组与对照组差异显著(p＜0.05)
阳性对照：特非那定片
2.2.3每组随机挑选10尾斑马鱼置于解剖显微镜下观察，结果表明随着处理组浓度的增加心包水肿发生率增加，最高浓度32mg/ml组心包水肿发生率达到了100％，同时还伴随斑马鱼的血液循环缺失。结果见表15。
表15尼莫地平原料药诱导的斑马鱼水肿及循环缺陷
Figure PCTCN2019104760-appb-000013
注：“*”表示经fisher精确检验法检验，处理组与对照组差异显著(p＜0.05)
阳性对照：特非那定片
2.3尼莫地平水针剂(尼膜同)对斑马鱼心血管的影响
2.3.1由于尼膜同最高浓度仅为0.2mg/ml，且无法配得其溶剂，故采用最高浓度注射不同体积的方法给药。而溶剂中含量最高的成分为23.7％乙醇，为证明该浓度的乙醇不会影响实验，故配制24％的乙醇溶液作为溶剂对照组。处理一定时间后随机挑选10尾斑马鱼对其进行计数心率，并计算其相对心率进行比较，结果除阳性对照组(特非那定片)外，其余各组未观察到异常。结果见表16。
表16尼莫地平水针剂对斑马鱼心率的影响
Figure PCTCN2019104760-appb-000014
注：“*”表示经fisher精确检验法检验，处理组与对照组差异显著(p＜0.05)
2.3.2实验结束时进行死亡率统计，未发现死亡个体。结果见表17。
表17尼莫地平水针剂对斑马鱼的致死率
分组 死亡率(％)
空白对照 0(0/30)
24％乙醇 0(0/30)
阳性对照 0(0/30)
10nl 0(0/30)
20nl 0(0/30)
40nl 0(0/30)
注：本研究中斑马鱼心房心室均不跳动则视其死亡；阳性对照：特非那定片
2.3.3每组随机挑选10尾斑马鱼置于解剖显微镜下观察，结果表明除阳性对照组(特非那定片)外仅40nl组有40％个体发生心包水肿并伴随循环确实和血流速度减慢。结果见表18。
表18尼莫地平水针剂诱导的斑马鱼水肿及循环缺陷
Figure PCTCN2019104760-appb-000015
注：“*”表示经fisher精确检验法检验，处理组与对照组差异显著(p＜0.05)
2.4本发明组合物注射液对斑马鱼心血管的影响
设置了三个正式实验浓度。处理一定时间后随机挑选10尾斑马鱼对其进行计数心率，并计算其相对心率进行比较，结果除阳性对照组(特非那定片)外，未观察到异常。实验结束时，未见死亡个体，除阳性对照组外其余各组未见异常。结果见表19。
表19本发明组合物注射液对斑马鱼心率的影响
Figure PCTCN2019104760-appb-000016
2.5小结
卵黄囊注射给药实验中尼莫地平原料药4.0mg/ml及以上浓度组对斑马鱼的致死率随浓度增加而升高，同时可诱发心包水肿和循环缺陷。尼莫地平原料药给药浓度2.0mg/ml(67mg/kg)诱发30％个体心包水肿，尼莫地平水针剂最大给药剂量40nl(26.7mg/kg)可诱发40％个体心包水肿，而本发明组合物注射液最大给药剂量下(106.7mg/kg)未观察到心血管毒性，说明本发明组合物注射液对斑马鱼的心血管毒性小于尼莫地平水针剂和尼莫地平原料药。
三、本发明组合物注射液对大鼠蛛网膜下腔出血模型的药效学研究。
3.1药效试验方法
3.1.1造模方法
雄性SD大鼠，体重160-200g，麻醉后暴露颅骨，用约1mm骨钻在颅骨(人字缝后的顶骨)处打孔，但勿打穿硬脑膜。同时，自眼底血管丛采血，用1mL注射器吸取自体血500μL，随后沿骨孔插入颅骨，深度3-4mm，生物胶封口防止泄露，缓慢注血，注意观察。随后采用Pericam PSI型激光微循环血流成像仪测定大鼠脑血流。
3.1.2分组和给药
实验动物随机分为8组，分别为只暴露颅骨但不注血的对照组；尼莫地平水针剂高中低剂量组；本发明组合物注射液高中低剂量组；生理盐水的模型组。每组成膜后存活10只以上。各组给药方法和剂量如下。
尼莫地平水针剂组：高剂量2mg/kg体重，中剂量1mg/kg体重，低剂量0.5mg/kg体重；药物浓度为1mg/5mL。
本发明组合物注射液组：本发明组合物注射液，用前用生理盐水稀释至浓度为1mg/5mL。高剂量2mg/kg体重，中剂量1mg/kg体重，低剂量0.5mg/kg体重。
模型组：尾静脉注射生理盐水1mL/100g体重。
给药体积和方法：高剂量1mL/100g体重，中剂量0.5mL/100g体重，低剂量0.25mL/100g体重。分别于造模后约30min、48h、96h后，按上述剂量，由尾静脉注射给药。
3.2试验结果
3.2.1脑血流的测定结果
各组造模后给药前脑血流测定结果见表20。
表20.各组造模后给药前的脑血流量表(mean±SD)
分组 矢状缝区(基底动脉区) 全颅脑区
正常组 294.44±92.40★ 275.43±48.84★★★
模型组 219.63±45.48▲ 193.30±32.01▲▲▲
尼莫地平水针低-给药前 190.93±36.31▲▲▲ 202.01±31.24▲▲▲
尼莫地平水针中-给药前 177.55±22.52★▲▲▲ 165.93±24.62▲▲▲
尼莫地平水针高-给药前 198.12±32.19▲▲▲ 186.57±26.78▲▲▲
本发明组合物低-给药前 211.81±66.28▲▲ 179.08±45.33▲▲▲
本发明组合物中-给药前 189.98±41.52▲▲▲ 177.53±37.70▲▲▲
本发明组合物高-给药前 193.04±32.48▲▲▲ 181.40±27.53▲▲▲
造模后各组与模型组比较P＜0.05★，P＜0.01★★，P＜0.001★★★。
造模后各组与正常组间比较P＜0.05▲，P＜0.01▲▲，P＜0.001▲▲▲。
造模后，各组与正常组比较，全颅脑区和矢状缝区(基底动脉)的血流量均有极显著性差异。说明模型成功，各组脑血流灌注量均大幅度下降。
与模型组比较，本发明组合物各组全颅脑区和矢状缝区(基底动脉)的血流量均无显著性差异。尼莫地平水针中剂量组矢状缝区(基底动脉)的血流量与模型组比较有显著性差异，其余各组均无显著性差异，显示尼莫地平水针中剂量组造模后基底动脉血流量与模型组比较有明显差异。
测定脑血流后给药，计时，给药后30min再次测定全颅脑区和矢状缝区血流量。采用EXCEL软件进行单因素方差分析，结果见下表21和表22。
表21各组给药前后的脑血流量改善值(mean±SD)
分组 矢状缝区 全颅脑区
尼莫地平水针低-给药前后差 33.75±28.83 7.41±42.29
尼莫地平水针中-给药前后差 15.17±27.47 4.04±33.32
尼莫地平水针高-给药前后差 19.75±62.89 20.54±48.32
本发明组合物低-给药前后差 13.99±39.85 30.94±25.90
本发明组合物中-给药前后差 14.11±63.07 25.40±52.50
本发明组合物高-给药前后差 26.42±28.53 23.42±32.45
各组间给药前后比较P＜0.05★，P＜0.01★★，P＜0.001★★★。
由表21可见，与给药前相比，给药后30min各组脑血流量均有一定程度的提高。尼莫地平水针各剂量组和本发明组合物各剂量组给药前后的脑血流改善情况无显著性差异。
表22.给药后不同组间的脑血流量表(mean±SD)
分组 矢状缝区 全颅脑区
正常组 294.44±92.40★ 275.43±48.84★★★
模型组 219.63±45.48 193.30±32.01
尼莫地平水针低 223.93±55.42 209.42±56.52
尼莫地平水针中 192.71±27.18 169.96±28.03
尼莫地平水针高 219.42±65.60 209.08±57.46
本发明组合物低 225.80±49.14 210.02±41.22
本发明组合物中 204.09±44.66 202.93±33.18
本发明组合物高 217.53±43.15 205.22±39.65
各组与模型组比较P＜0.05★，P＜0.01★★，P＜0.001★★★。
由表22可见，给药后各组间脑血流量无显著性差异。尼莫地平水针与本发明组合物同剂量组间比较也均无显著性差异。
造模10d后，尼莫地平水针高剂量组(3只)和本发明组合物高剂量组(4只)激光微循环血流成像仪测定全脑和脑中部血流量。采用EXCEL软件进行单因素方差分析，结果见下表23.。
表23.造模10d后本发明组合物和尼莫地平水针高剂量组的脑血流量表(mean±SD)
分组 矢状缝区 全颅脑区
本发明组合物高-10d(n＝4) 198.20±100.07 181.81±51.06
尼莫地平水针高-10d(n＝3) 222.13±4.03 192.04±24.83
由表23.可见，尼莫地平水针和本发明组合物高剂量组(给药3次)10d后全颅脑区和矢状缝区(基底动脉)的血流量基本一致，无显著性差异。
尼莫地平水针高剂量组和本发明组合物高剂量组造模前后和给药前后的脑血流值见表24.，脑血流变化见附图1和2.，
表24.尼莫地平水针和本发明组合物高剂量组的脑血流量表(mean±SD)
分组 矢状缝区 全颅脑区
本发明组合物高造模前 315.94±93.82 314.77±73.00
本发明组合物高造模后 193.04±32.48 181.40±27.53
本发明组合物高造模后降低值 122.90 133.37
本发明组合物高给药后 217.53±43.15 205.22±39.65
本发明组合物高给药前后差 26.42±28.53 23.42±32.45
尼莫地平水针高造模前 306.10±102.35 301.90±86.88
尼莫地平水针高造模后 198.12±32.19 186.57±26.78
尼莫地平水针高造模后降低值 107.98 115.33
尼莫地平水针高给药后 219.42±65.60 209.08±57.46
尼莫地平水针高给药前后差 19.75±62.89 20.54±48.32
结果表明，尼莫地平水针和本发明组合物高剂量组动物造模后全颅脑区和矢状缝区(基底动脉)的脑血流值均明显下降，给药后又均有一定程度升高。本发明组合物的改善效果略优于同剂量的尼莫地平水针，但两组基本一致，无显著性差异。
3.2.2脑血管痉挛程度的测定结果
脑组织经脱水，石蜡包埋，截取包含基底动脉的脑干组织切片，HE染色和封片后显微镜下观察，并用图像分析系统测定基底动脉血管周长和面积，判定脑部血管痉挛的程度。结果见下表25.。
表25.基底动脉周长和面积(mean±SD)
分组 周长(μm) 面积(μm 2)
正常组 551.73±126.69 18669.27±7277.06
模型组 447.24±127.94 13934.68±6852.99
尼莫地平水针低 594.26±135.17★ 22730.60±11828.17
尼莫地平水针中 592.57±136.58★ 23304.12±10688.16
尼莫地平水针高 647.09±140.65★★ 27385.99±10778.41★★
本发明组合物低 567.43±104.38★ 18825.13±6587.00
本发明组合物中 595.82±164.43★ 22503.74±11587.18
本发明组合物高 603.41±123.66★ 24031.65±9632.43★
各组与模型组比较P＜0.05★，P＜0.01★★，P＜0.001★★★。
由上表可见，与正常组比较，模型组的基底动脉周长和面积均有较大下降，说明颅内注血造成了大鼠基底动脉收缩。对于基底动脉截面周长，与模型组相比，尼莫同高中低剂量均有显著性改善作用，其中尼莫同高剂量有非常明显的改善作用；脂肪乳低中高剂量也有显著性改善作用。
因血管在后期制片时易变形，造成部分动物的血管截面积变化很大，加大了组内的SD。与模型组比较，基底动脉血管截面积仅尼莫同高剂量组和脂肪乳高剂量组有显著性改善。
同剂量各给药组间比较均没有显著性差异，但有一定量效关系。随着剂量的增加，基底动脉的周长和面积均增大。
根据各组对脑血管痉挛程度(基底动脉周长和面积)的改善情况，疗效强弱排序为：尼莫地平水针高剂量≈本发明组合物高剂量＞本发明组合物中≈尼莫地平水针中≈尼莫地平水针低＞本发明组合物低。
3.2.3行为学指标的测定结果
实验动物分别于给药后24h、72h、5d后进行转棒耐力试验。结果见表26.。
表26.转棒耐力时间(秒，mean±SD)
分组 24h后 72h后 5d后
正常组 51.0±3.9★★★ 46.8±9.1★★★ 46.4±5.3★
模型组 10.2±2.2 12.7±3.4 19.7±7.2
尼莫地平水针低 16.2±4.6 19.2±6.9 26.2±8.6
尼莫地平水针中 20.4±3.8★★ 18.7±6.2 24.5±4.5
尼莫地平水针高 23.4±7.2★★★ 22.8±4.8★★ 27.5±4.4
本发明组合物低 15.0±5.1 13.3±7.5 20.7±1.2
本发明组合物中 19.0±6.9★ 20.8±2.8★ 22.7±5.3
本发明组合物高 21.4±6.3★★ 26.3±6.6★★★ 56.5±45.0★★
各组与模型组比较P＜0.05★，P＜0.01★★，P＜0.001★★★
由转棒耐力试验结果可见，与正常组比较，造模后模型组的转棒耐力显著下降，随着时间的推移有慢慢恢复的趋势。
第一次给药24h后，与模型组比较，给药各组均有一定恢复。其中尼莫地平水针中和高剂量组，本发明组合物中和高剂量组有显著性改善。不同剂量间有明显量效关系。
给药2次(72h后)，与模型组比较，给药各组也均有一定恢复。其中尼莫地平水针高剂 量组，本发明组合物中和高剂量组有显著性改善。不同剂量间有一定量效关系。
给药3次(5d后)，与模型组比较，给药各组也均有一定恢复。其中本发明组合物高剂量组有显著性改善。不同剂量间有一定量效关系。
实验动物分别于给药后24h、72h、5d后进行开场的自主活动实验，开启视频监测系统，记录5min内大鼠的活动距离和活动时间。结果见表27和28.。
表27.活动距离表(cm/5min，mean±SD)
分组 24h后 72h后 5d后
正常组 1557.2±306.8★ 1557.2±306.8★ 1557.2±306.8★
模型组 1131.9±514.2 1301.5±410.3 920.3±462.6
尼莫地平水针低 1490.9±553.2 1268.6±627.1 841.6±583.5
尼莫地平水针中 1241.9±580.0 1326.3±821.2 1288.0±353.1
尼莫地平水针高 1592.5±299.3 1421.0±38.9 869.3±453.7
本发明组合物低 1408.4±862.1 1724.3±644.5 1166.8±648.7
本发明组合物中 1441.9±354.4 1478.0±1022.6 955.2±466.7
本发明组合物高 954.2±768.2 1111.2±508.5 1526.3±179.0★
各组与模型组比较P＜0.05★，P＜0.01★★，P＜0.001★★★
表28.活动时长表(秒/5min，mean±SD)
分组 24h后 72h后 5d后
正常组 195.40±15.72★ 195.40±15.72★ 195.40±15.72★
模型组 157.19±37.95 157.19±37.95 120.50±34.74
尼莫地平水针低 199.82±31.11 160.43±63.80 123.61±70.94
尼莫地平水针中 156.98±57.56 181.09±36.51 160.88±30.87
尼莫地平水针高 196.84±36.54★ 206.21±14.84★ 124.66±41.69
本发明组合物低 106.44±44.73 129.91±43.42 129.03±64.83
本发明组合物中 200.73±20.92★ 179.42±52.92 146.13±33.15
本发明组合物高 146.49±53.32 171.67±26.82 183.36±18.88
各组与模型组比较P＜0.05★，P＜0.01★★，P＜0.001★★★
由表27和28的结果可见，与正常组比较，造模后模型组的5min内活动距离和活动时长均显著下降，随着时间的推移活动能力并无明显趋势性改变。
给药后活动距离和活动时长有所改善，总体活动能力增强，随着给药次数的增加也略有改善。与模型组比较，其中给药后5d的本发明组合物高剂量组活动距离明显改善；尼莫地平水针高剂量和本发明组合物中剂量组的活动时长也有显著性改善，但无显著性差异。
3.3药效试验中的不良反应
尼莫地平水针剂注射液含高浓度乙醇，静脉注射有很强的“麻醉”作用，注射后大鼠约需10-30min后方能缓慢恢复。其中，尼莫地平水针剂高剂量组出现明显毒副作用，在注射给药时有多只死亡。尼莫地平水针剂多次给药后多只动物出现明显静脉炎和尾巴溃烂。本发明组合物注射液各剂量组均未在给药时出现死亡和其它可见不良反应。
综上所述，同剂量的本发明组合物注射液与尼莫地平水针剂疗效基本相当，无明显差异。但试验过程中本发明组合物注射液未出现毒副作用和其他可见不良反应。因此，本发明组合物注射液与市售尼莫地平注射液及现有技术尼莫地平乳注射液药效相当，但安全性更高。
四、本发明组合物注射液的血浆药物浓度测定。
选取雄性SD大鼠，适应性饲养2天后，以5％的水合氯醛腹腔注射麻醉(以生理盐水配制)，麻醉剂量同上，麻醉后分别将动物编号分组，分为本发明组合物组和尼莫酮组，大鼠分别尾静脉注射给药，给药剂量均为2mg/kg体重。分别于给药后10、30、90、120min于大鼠目内眦眼底静脉从取血0.3ml，置于预先肝素化的采血管中，以4000rpm离心10min，取上层血浆于-80℃保存。
采用UPLC-PDA分析方法测定尼莫地平血浆样品。将样品血浆逐一处理并进样分析，计算血浆中尼莫地平的浓度，结果见表29.。以血药浓度(ng/ml)对时间(min)作图，绘制血药浓度时间曲线图，结果见附图3。
表29.尼莫地平血浆浓度表(ng/ml)
Figure PCTCN2019104760-appb-000017
由表29.及附图3可见，本发明组合物组与尼莫地平水针组的血浆药物浓度基本相当，药时曲线也基本一致。

Claims (13)

  1. 一种尼莫地平注射液组合物，其特征在于，以质量浓度计，包括如下组分：尼莫地平0.02-0.23％，注射用油2-30％，乳化剂0.8-3％，络合剂0-0.1％，稳定剂0-0.3％，渗透压调节剂1-3％。
  2. 根据权利要求1所述的注射液组合物，其特征在于，优选的比例为：尼莫地平0.04-0.20％，注射用油3-20％，乳化剂1-2.5％，络合剂0-0.05％，稳定剂0-0.2％，渗透压调节剂2-3％；更优选的比例为：尼莫地平0.08-0.18％，注射用油8-12％，乳化剂1-2％，络合剂0-0.01％，稳定剂0-0.1％，渗透压调节剂2-2.5％。
  3. 根据权利要求1-2任一项所述的注射液组合物，其特征在于，注射液组合物pH值为6.0-8.5，优选的pH值为6.5-8.0，更优选的pH值为7.0-7.5。
  4. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，所述尼莫地平选自α晶型或β晶型，优选熔点为114-116℃的α晶型。
  5. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，所述乳化剂选自大豆磷脂或蛋黄磷脂。
  6. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，所述大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为60-90％，磷脂酰乙醇胺(PE)含量为2-20％；优选的范围为所述大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为70-85％，磷脂酰乙醇胺(PE)含量为5-18％。
  7. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，所述络合剂选自依地酸钙钠、依地酸二钠、依地酸钠、依地酸中的一种或一种以上组合物；优选的络合剂为依地酸二钠。
  8. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，所述注射用油为注射用大豆油或注射用中链甘油三酸酯或二者混合物，优选注射用大豆油、注射用中链甘油三酸酯混合物，优选的质量比为2:8-5:5，更优选的质量比为4:6-5:5，最优选的质量比为4:6。
  9. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，优选的稳定剂为油酸或油酸钠。
  10. 根据权利要求1-4任一项所述的注射液组合物，其特征在于，渗透压调节剂为甘油。
  11. 根据权利要求1-10任一项所述的注射液组合物，其特征在于，注射液组合物平均粒径为100-600nm，优选的平均粒径为120-400nm，更优选的平均平均粒径为140-300nm。
  12. 根据权利要求1-10所述本发明组合物，其特征在于，本发明组合物在蛛网膜下腔出血后的脑血管痉挛和进行脑血管病恢复期的血液循环改善方面的应用。
  13. 一种尼莫地平注射液组合物的制备方法，其特征在于，包括如下步骤：
    (1)取注射用水，调节pH，加入络合剂、稳定剂搅拌溶解，再加入渗透压调节剂，搅拌混 合均匀，制成水相，水温控制在50～70℃；
    所述的络合剂选自依地酸钙钠、依地酸二钠、依地酸钠、依地酸中的一种或一种以上组合物，优选的络合剂为依地酸二钠；渗透压调节剂优选甘油；
    (2)取注射用油，加入40％处方量的乳化剂、稳定剂剪切混匀，水浴加热到50～70℃，加入尼莫地平搅拌使其溶解，制成油相；所述尼莫地平选自α晶型或β晶型，优选熔点为114-116℃的α晶型；比例为尼莫地平0.02-0.23％，尼莫地平0.04-0.20％，尼莫地平0.08-0.18％
    所述注射用油选自注射用大豆油或注射用中链甘油三酸酯或二者混合物，优选注射用大豆油、注射用中链甘油三酸酯混合物，优选的质量比为2:8-5:5，更优选的质量比为4:6-5:5，最优选的质量比为4:6；
    (3)稳定剂选用油酸钠时，油酸钠加入水相(1)中，选用油酸时，油酸加入油相(2)中；
    (4)取60％处方量的乳化剂，加入水相中，在氮气保护下，剪切乳化剂至分散均匀；
    所述的乳化剂选自大豆磷脂或蛋黄磷脂，大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为60-90％，磷脂酰乙醇胺(PE)含量为2-20％；优选的范围为所述大豆磷脂或蛋黄磷脂中磷脂酰胆碱(PC)含量为70-85％，磷脂酰乙醇胺(PE)含量为5-18％；
    (5)将油相缓缓加入至水相中，剪切制成初乳；
    (6)将初乳进行均质，循环至乳液粒度符合规定；
    (7)取样检测pH值、乳粒粒径，充氮气，灌封，灭菌。
PCT/CN2019/104760 2018-09-08 2019-09-06 一种尼莫地平注射液组合物及其制备方法 WO2020048533A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP19857267.9A EP3848021A4 (en) 2018-09-08 2019-09-06 NIMODIPINE INJECTION COMPOSITION AND PROCESS FOR PRODUCTION THEREOF
CN201980063913.9A CN112912066B (zh) 2018-09-08 2019-09-06 一种尼莫地平注射液组合物及其制备方法
JP2021537460A JP2021535932A (ja) 2018-09-08 2019-09-06 ニモジピン注射用組成物及びその調製方法
US17/274,299 US20210338649A1 (en) 2018-09-08 2019-09-06 Nimodipine injection composition and preparation method therefor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201811046629.4 2018-09-08
CN201811046629 2018-09-08

Publications (1)

Publication Number Publication Date
WO2020048533A1 true WO2020048533A1 (zh) 2020-03-12

Family

ID=69721490

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/104760 WO2020048533A1 (zh) 2018-09-08 2019-09-06 一种尼莫地平注射液组合物及其制备方法

Country Status (5)

Country Link
US (1) US20210338649A1 (zh)
EP (1) EP3848021A4 (zh)
JP (1) JP2021535932A (zh)
CN (1) CN112912066B (zh)
WO (1) WO2020048533A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114557960A (zh) * 2022-03-16 2022-05-31 陕西省人民医院 一种治疗蛛网膜下腔出血的药物及其应用
CN115990262A (zh) * 2022-06-27 2023-04-21 北京德立福瑞医药科技有限公司 不含乙醇和磷脂的湿热灭菌的尼莫地平组合物及其制备方法
CN116754711B (zh) * 2023-08-17 2023-11-07 蓝星安迪苏南京有限公司 饲料用乳化剂的乳化能力评估方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101797226A (zh) * 2009-02-10 2010-08-11 上海医药工业研究院 一种高抗氧化性中/长链脂肪乳注射液及其制备方法
CN103893119A (zh) * 2014-03-07 2014-07-02 广东药学院 一种尼莫地平的脂肪乳注射液及其制备方法
CN107661294A (zh) * 2016-07-27 2018-02-06 武汉科福新药有限责任公司 抗高血压药物脂肪乳注射剂及其制备方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100386078C (zh) * 2004-07-08 2008-05-07 上海医药工业研究院 尼莫地平乳注射液及制备方法
CN101199522A (zh) * 2006-12-15 2008-06-18 重庆药友制药有限责任公司 注射用尼莫地平冻干乳剂及其制备方法
CN101416942A (zh) * 2008-12-02 2009-04-29 沈阳万爱普利德医药科技有限公司 尼莫地平亚微乳注射液及其制备方法
CN101904814A (zh) * 2009-06-04 2010-12-08 上海恒瑞医药有限公司 制备载药乳剂的方法
US9399019B2 (en) * 2012-05-09 2016-07-26 Evonik Corporation Polymorph compositions, methods of making, and uses thereof
US10092557B2 (en) * 2016-04-13 2018-10-09 Nortic Holdings Inc. Stable nimodipine parenteral formulation
WO2018039039A1 (en) * 2016-08-23 2018-03-01 Edge Therapeutics, Inc. Scalable microparticulate formulations containing polymorphic nimodipine form 2 prepared by a solvent evaporation process

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101797226A (zh) * 2009-02-10 2010-08-11 上海医药工业研究院 一种高抗氧化性中/长链脂肪乳注射液及其制备方法
CN103893119A (zh) * 2014-03-07 2014-07-02 广东药学院 一种尼莫地平的脂肪乳注射液及其制备方法
CN107661294A (zh) * 2016-07-27 2018-02-06 武汉科福新药有限责任公司 抗高血压药物脂肪乳注射剂及其制备方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP3848021A4 *

Also Published As

Publication number Publication date
EP3848021A1 (en) 2021-07-14
EP3848021A4 (en) 2023-11-29
CN112912066A (zh) 2021-06-04
US20210338649A1 (en) 2021-11-04
JP2021535932A (ja) 2021-12-23
CN112912066B (zh) 2022-03-29

Similar Documents

Publication Publication Date Title
DE69534693T2 (de) Sublinguale Zusammensetzung enthaltend Apomorphine zur Diagnose der funktionellen Impotenz
WO2020048533A1 (zh) 一种尼莫地平注射液组合物及其制备方法
CA3113462A1 (en) Formulations of reproxalap for the treatment of dry eye disease
EA014852B1 (ru) Система контролируемой доставки
US9737524B2 (en) Prophylactic and/or therapeutic agent for mild cognitive impairment
CN104248628B (zh) 一种利西拉来缓释微球及其制备方法
CN101653413B (zh) 他克莫司眼用乳剂及其制备方法
JP3603129B2 (ja) 糖尿病性角膜症の治療剤
EP2400838B1 (en) Narcotic emulsion formulations for treatment of cancer pain
BR112019017724A2 (pt) Sistemas depot de glatiramer para tratar formas progressivas de esclerose múltipla
BR112015032534B1 (pt) Uso de compostos ou sal ou solvato farmaceuticamente aceitável dos mesmos
EP2813215A1 (en) 2,2&#39;,6,6&#39;-tetraisopropyl-4,4&#39;-2-biphenol lipid microsphere preparation and preparation method therefor
CN103664936A (zh) 一类治疗创伤性脑损伤疾病的化合物及其用途
RU2504347C1 (ru) Инъекционная лекарственная форма для лечения и профилактики заболеваний печени у животных
CN101152147B (zh) 一种川芎嗪类化合物乳剂及其制备方法
CN113694017A (zh) 一种氟维司群注射制剂及其制备方法
KR20210019402A (ko) 주사용 조성물
KR102619489B1 (ko) 폴마콕시브 및 트라마돌을 포함하는 급, 만성 통증 치료용 약제학적 조성물
CN100508987C (zh) 玻璃酸在制备用于预防或改善眼玻璃体变性疾病的口服产品中的应用
US20220409560A1 (en) Pharmaceutical Compositions, Formulations And Methods For The Treatment Of Retinoblastoma
BR112019023511A2 (pt) uso de compostos peptídeos no tratamento de pancreatite aguda
CN114557960A (zh) 一种治疗蛛网膜下腔出血的药物及其应用
Yoo et al. Osmotically Balanced, Large Unilamellar Liposomes for Sustained Bupivacaine Release: From the Development of Liposomes to Their in Vivo Application
CN117085112A (zh) 一种无针注射用齐考诺肽及其制备方法
KR20230021613A (ko) 안구건조증 치료를 위한 레코플라본 함유 점안 조성물 및 이의 제조방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 19857267

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2021537460

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2019857267

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2019857267

Country of ref document: EP

Effective date: 20210408