WO2020108193A1 - 一种前列腺素e1甲酯注射用冻干制剂及制备和应用 - Google Patents

一种前列腺素e1甲酯注射用冻干制剂及制备和应用 Download PDF

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WO2020108193A1
WO2020108193A1 PCT/CN2019/113557 CN2019113557W WO2020108193A1 WO 2020108193 A1 WO2020108193 A1 WO 2020108193A1 CN 2019113557 W CN2019113557 W CN 2019113557W WO 2020108193 A1 WO2020108193 A1 WO 2020108193A1
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parts
oil
prostaglandin
methyl ester
injection
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PCT/CN2019/113557
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English (en)
French (fr)
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王汝涛
安龙
赵熠
庞菁华
陈涛
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西安力邦肇新生物科技有限公司
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Priority to EP19890056.5A priority Critical patent/EP3888637A4/en
Priority to US17/297,148 priority patent/US20220023219A1/en
Priority to JP2021529847A priority patent/JP7169024B2/ja
Priority to KR1020217019456A priority patent/KR20210095177A/ko
Publication of WO2020108193A1 publication Critical patent/WO2020108193A1/zh

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • 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
    • A61K47/24—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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • 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
    • 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
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • A61P9/08—Vasodilators for multiple indications
    • 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
    • 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
    • A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers

Definitions

  • the present invention relates to the field of medicine. Specifically, the present invention relates to a lyophilized preparation of prostaglandin E1 methyl ester for injection, preparation and application.
  • Prostaglandin E1 is a natural endogenous vasodilator, which can be synthesized by human cells and is an important substance for regulating cell function. It does not accumulate in the body, does not produce tolerance, and is non-toxic and non-damaging side effects. The effect is definite, better than exogenous drugs. PGE1 is extremely physiologically active and has a wide range of pharmacological activities. It can be clinically applied to cardiovascular and cerebrovascular diseases, diabetic complications, respiratory diseases, pulmonary arterial hypertension, hepatorenal syndrome (HRS), liver failure, kidney disease, etc. E1 not only has the effect of dilating blood vessels and reducing heart load, but also has the effects of sodium excretion, diuresis, cardiotonia, improving coronary circulation, protecting myocardium and improving microcirculation.
  • prostaglandin E1 fat emulsion injection currently on the market also has several obvious shortcomings: poor chemical stability, high temperature sterilization causes the content of prostaglandin E1 to be reduced, and the content of degradation product PGA1 is significantly increased, and the product is stored The conditions are harsh (0 ⁇ 5°C), and the duration of effect is only 1 year. Because prostaglandin E1 itself is an inflammatory substance, it has a strong pain in clinical application and human body, causing phlebitis, which limits the promotion of this product.
  • Prostaglandin E1 alkyl esters are currently considered to be prodrugs of prostaglandin E1.
  • U.S. Patent No. US5681850 discloses prostaglandin E1 alkyl ester (C1-4) for the treatment of impotence.
  • prostaglandin E1 alkyl ester can be better absorbed through the skin by enhancing fat solubility, and then is decomposed into the prostate by hydrolase Prostaglandin E1 is effective and belongs to a prodrug;
  • US Patent US6673841 discloses a prostaglandin E1 alkyl ester (C1-5) external preparation, which contains prostaglandin E1 alkyl ester as a prodrug, an oily carrier, a skin penetration enhancer, and an anti-inflammatory agent .
  • U.S. Patent US4849451 discloses a fat milk formulation of alprostadil methyl ester, but we prepared the fat emulsion of alprostadil methyl ester according to its examples during the research process.
  • Method-photoresist method the original commonly used dynamic light scattering method cannot accurately measure the number of particles with a large particle size of more than 5um) and found that there are significantly more large particle size (>5um) milk droplets, which does not meet the requirements of milk droplet size , Large milk droplets (>5um) can block capillaries and cause embolism, which has a greater safety risk.
  • the present invention aims to provide a new type with good stability, low safety risk, and efficacy Better prostaglandin E1 methyl ester products.
  • An object of the present invention is to provide a freeze-dried preparation of prostaglandin E1 methyl ester for injection;
  • Another object of the present invention is to provide a preparation method of prostaglandin E1 methyl ester lyophilized preparation for injection;
  • Another object of the present invention is to provide a freeze-dried preparation of prostaglandin E1 methyl ester prepared by the preparation method
  • Another object of the present invention is to provide the application of the prostaglandin E1 methyl ester lyophilized preparation for injection.
  • the present invention provides a lyophilized preparation of prostaglandin E1 methyl ester for injection, wherein the lyophilized preparation contains the following components by weight: 0.1-10 parts of prostaglandin E1 methyl ester, for injection 500-4000 parts of oil, 500-2000 parts of emulsifier, 0-10 parts of co-emulsifier, 5000-50000 parts of lyoprotectant, and 200-1500 parts of glycerin.
  • the oil for injection is selected from one or more of soybean oil, medium chain oil, olive oil, tea oil, corn oil or castor oil.
  • the emulsifier is selected from egg yolk phospholipids and/or soybean phospholipids.
  • the co-emulsifier is a mixture of one or more selected from oleic acid, palmitic acid, stearic acid, linolenic acid, linoleic acid, and sodium oleate.
  • the lyoprotectant is selected from a mixture of one or more of lactose, sucrose, trehalose, mannitol, glucose, and maltose.
  • the lyophilized formulation contains the following parts by weight of ingredients: prostaglandin E1 methyl ester 0.1-10 parts, injection oil 500-4000 parts, emulsifier 500-1500 parts, co-emulsifier 0 -10 parts, lyophilized protective agent 5000-20000, and glycerin 200-1500 parts.
  • the lyophilized preparation contains the following parts by weight per 100 ml before lyophilization: prostaglandin E1 methyl ester 0.1-10 mg, soybean oil 0.5-4 g, egg yolk phospholipid 0.5-1.5 g, oil Sodium 0-0.01g, lactose 5-20g, and glycerol 0.2-1.5g.
  • the “per 100 ml” in the “the lyophilized preparation contains the following parts by weight per 100 ml before lyophilization” in the present invention refers to the solution per 100 ml prepared before lyophilization; namely An aqueous solution containing each component.
  • the weight ratio of the lyoprotectant to prostaglandin E1 methyl ester is 20-500:0.01.
  • the present invention also provides a method for preparing a lyophilized preparation of prostaglandin E1 methyl ester for injection, wherein the method includes preparing the lyophilized preparation with the following weight percent ingredients as raw materials: prostaglandin E1 methyl ester 0.0001-0.01%, oil for injection 0.5-4%, emulsifier 0.5-2%, co-emulsifier 0-0.01%, lyoprotectant 5-50%, glycerin 0.2-1.5%, appropriate amount of pH regulator, and excess Amount of water for injection.
  • the oil for injection is selected from one or more of soybean oil, medium chain oil, olive oil, tea oil, corn oil or castor oil.
  • the emulsifier is selected from egg yolk phospholipids and/or soybean phospholipids.
  • the co-emulsifier is a mixture of one or more selected from oleic acid, palmitic acid, stearic acid, linolenic acid, linoleic acid, and sodium oleate.
  • the lyoprotectant is selected from a mixture of one or more of lactose, sucrose, trehalose, mannitol, glucose, and maltose.
  • the method includes preparing the lyophilized formulation with the following weight percent ingredients as raw materials: prostaglandin E1 methyl ester 0.0001-0.01%, oil for injection 0.5-4%, emulsifier 0.5- 1.5%, co-emulsifier 0-0.01%, lyoprotectant 5-20%, glycerin 0.2-1.5%, appropriate amount of pH adjuster, and the remaining amount of water for injection.
  • the method includes preparing the lyophilized formulation with the following parts by weight ingredients: prostaglandin E1 methyl ester 0.1-10 parts, soybean oil 500-4000 parts, egg yolk phospholipid 500-1500 , 0-10 parts of sodium oleate, 5000-20000 parts of lactose, 20-1500 parts of glycerin, an appropriate amount of sodium citrate or hydrochloric acid, and the remaining amount of water for injection based on the total weight of raw materials of 100 ⁇ 10 3 parts.
  • the amount of the pH adjusting agent is adjusted to 4.5 to 6.5 after the volume is adjusted to the full amount with water for injection into a mixed solution.
  • the method includes the following steps:
  • Prostaglandin E1 methyl ester and emulsifier are evenly dispersed in an oily solvent as an oil phase;
  • step d is to obtain colostrum by shearing under a constant temperature condition of 20°C-50°C.
  • freeze-drying process of step f includes pre-freezing at -50°C to -35°C for 100-200 minutes, and then evacuating 420 at -25°C to -15°C with 70-90 mTorr -540 minutes, and then evacuate at 50-70mTorr for 240-360 minutes at 5°C to 15°C, and then evacuate at 35-45mTorr for 300-420 minutes at 35°C to 45°C.
  • the lyophilization process in step f includes pre-freezing at -40°C for 150 minutes, then evacuating at 80 mTorr for 480 minutes at -20°C, and then evacuating at 60 mTorr at 10°C 300 minutes, then at 40°C, evacuate at 40mTorr for 360 minutes.
  • the present invention also provides a lyophilized preparation of prostaglandin E1 methyl ester prepared by the preparation method of the present invention.
  • the present invention also provides the use of the prostaglandin E1 methyl ester lyophilized preparation for the preparation of vasodilators, coronary heart disease, angina pectoris, heart failure, pulmonary heart disease, cerebral infarction, amniotic fluid embolism, Or scleroderma).
  • the vasodilator drug is used to treat the following diseases: microcirculation disorder.
  • microcirculation disorder is caused by the following conditions: thromboangiitis obliterans, arteriosclerosis obliterans, diabetes, frostbite, burns, or bedsores.
  • prostaglandin E1 methyl ester itself has strong biological activity, and is superior to prostaglandin E1 in anticoagulation and vasodilator activity, and has better drug development potential.
  • the inventors adopted further preparations In the study, a lyophilizer was selected as the preparation form of prostaglandin E1 methyl ester, and it was unexpectedly found that the prostaglandin E1 methyl ester lyophilizer prepared by the present invention has the following significant characteristics compared with the existing preparations:
  • the prostaglandin E1 methyl ester lyophilizer of the present invention shows better drug activity and therapeutic effect than that of superior diur in specific experimental examples.
  • the prostaglandin E1 methyl ester lyophilizing agent of the present invention significantly reduces the average particle size of milk droplets and the proportion of large-diameter milk droplets, unexpectedly improves the pharmacokinetic behavior, thereby enhancing the efficacy of the drug.
  • Figure 3 is the relationship between the diameter of mesenteric arterioles and the administration time in Experimental Example 3;
  • FIG. 4 is a plasma concentration-time curve of alprostadil after intravenous injection of 40 ⁇ g/kg alprostadil in rats.
  • the preparation process is as follows:
  • Oil phase Weigh 2g of soybean oil, add 0.5g of egg yolk phospholipid, 0.5mg of prostaglandin E1 methyl ester, shear to dissolve at 50°C;
  • Aqueous phase Weigh 90g of water for injection, add 0.75g of glycerin, 12.5g of lactose, 0.01g of sodium oleate, mix by shearing, adjust pH to 6.5 with 0.1M sodium citrate, then add 2g of phospholipid, and continue to shear for 10min;
  • the homogenized emulsion was sterile filtered, prepacked, pre-frozen at -40°C for 150 minutes, then evacuated at -20°C, 80mTorr for 480 minutes, 10°C, 60mTorr for 300 minutes, 40°C, 40mTorr for 360 Minutes, press the lid under vacuum to get lyophilized powder injection.
  • Prostaglandin E1 methyl ester lyophilizer with different co-emulsifiers and contents Prostaglandin E1 methyl ester lyophilizer with different co-emulsifiers and contents
  • Inhibition rate% (solvent aggregation rate-compound aggregation rate) / solvent aggregation rate ⁇ 100%
  • Rabbits were used to prepare isolated aortic ring specimens in the experiment: New Zealand white rabbits, male, body weight (2.5 ⁇ 0.3) kg. Stun the rabbit with a blunt instrument, fix it on the dissecting table of the rabbit, quickly separate the thoracic aorta, and put in Kerbs solution saturated with 37°C (containing NaCl 6.9g, KCl 0.35g, MgSO 4 ⁇ 7H 2 O 0.29 per 1000mL g, KH 2 PO 4 0.16g, NaHCO 3 2.1g, CaCl 2 0.28g, glucose 2g) and continuous ventilation of the mixed gas (95% O 2 , 5% CO 2 ) in the petri dish, the residual in the blood vessel The blood was squeezed out, and the peripheral fat and connective tissue were carefully peeled off, and cut into several sections of 0.5 cm long arterial ring for use.
  • Kerbs solution saturated with 37°C (containing NaCl 6.9g, KCl 0.35g, MgSO 4 ⁇ 7H 2 O
  • the vascular ring is penetrated by the two stainless steel L-shaped hooks through the lumen of the blood vessel, suspended horizontally in a 20mL bath tube, fixed at the bottom, and connected with a thin steel wire to the tension transducer above. Give 3.00g tension and adjust the tension level continuously to maintain it at about 3.00g and stabilize for 2h (change Kerbs solution every 15min along the bath wall).
  • the BL-420S biological function experiment system (Chengdu Taimeng Technology) was used to record the changes of vascular ring tension. After the contraction of the vascular ring has stabilized, the prostaglandin E1 and the example compound are added cumulatively, so that the final mass concentration of prostaglandin E1 in the bath tube is sequentially increased to 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, 25.6nM, the diastolic efficacy of the vascular ring was recorded.
  • Rats were anesthetized with 2.5% pentobarbital sodium 30 mg/kg ip, fixed in the dorsal position, a 3 to 4 cm long incision was made at the midline of the abdomen, and a small intestinal mesentery was gently pulled back to the blind part and placed in an organic solution filled with 37°C saline In the glass constant temperature water bath, keep the mesentery moist, and lay it flat on the organic convex observation table in the center of the bath, press the fixed plate, use the biological microscope (40 times magnification) camera to collect the video image under the microscope, and observe with BI-2000 microcirculation The system analyzes the collected video images under the mirror in real time.
  • the preparation group of Example 3 can significantly improve the microcirculation disorder caused by epinephrine, and there is a significant difference between the model group and the model group from 0.5 minutes to 10 minutes (P ⁇ 0.05), Compared with the model control group at 4 minutes, the Udil group had no significant difference (P>0.05).
  • the effect of improving adrenaline-induced circulatory disorder was significantly higher than that of superior diur, and there was a significant difference between the two in 0.5-10 minutes (P ⁇ 0.05).
  • Sample processing method each example of lyophilized milk is added with 10ml of re-milk, and a portion is simultaneously diluted with water 10 times, and an appropriate amount is accurately placed in a 20ml brown stoppered test tube, 2.5ml of tetrahydrofuran is added, mixed, and the phosphoric acid solution is added (1 ⁇ 1000) 15ml, mix well, and pass through a pretreatment column [filler is octadecylsilane-bonded silica gel, particle size is 70 ⁇ m, ⁇ 10mm ⁇ 9mm polypropylene tube (SEP-PAK C18 column, Waters).
  • Examples 3 to 18 and Youdier were reconstituted with physiological saline at a concentration of 1ug/mL, and then sampled and diluted 10 times to 0.1ug/mL. After taking the above reconstituted appropriate amount of each preparation, it was placed in an ultrafiltration centrifuge tube In the experiment, after centrifugal ultrafiltration at 2800r ⁇ min-1 for 30min, 1ml of the filtrate was taken to prepare the sample solution according to the above sample processing method, and 20 ⁇ l was injected. The measured drug content is the free drug content.
  • Free rate% total free drug / total drug amount ⁇ 100%
  • a Appearance Average particle size of final product >5um milk drops (%)
  • Example 3 Loose lumps, good reconstitution 94 0.0021
  • Example 4 Loose lumps, good reconstitution 93 0.0019
  • Example 5 Loose lumps, good reconstitution 96 0.0022
  • Example 6 Loose lumps, good reconstitution 77 0.0018
  • Example 7 Loose lumps, good reconstitution 89 0.0019
  • Example 8 Loose lumps, good reconstitution 148 0.0053
  • Example 9 Loose lumps, good reconstitution 93 0.0037
  • Example 10 Loose lumps, good reconstitution 104 0.0018
  • Example 11 Loose lumps, good reconstitution 86 0.0025
  • Example 12 Loose lumps, good reconstitution 86 0.0022
  • Example 13 Loose lumps, good reconstitution 101 0.0019
  • Example 14 Loose lumps, good reconstitution 113 0.0033
  • Example 15 Loose lumps, good reconstitution 92 0.0024
  • Example 16 Loose lumps, good reconstitution 99 0.0018
  • Example 17 Loose lumps, good reconstitution 91 0.0038
  • Example 18 Loose lumps, good reconstitution 89 0.0023 Comparative Example 1
  • the average particle size was measured using PSS's 380ZLS particle size analyzer (dynamic light scattering method), and the PSS company's Accusizer 780 instrument (photoresist method) was used to measure the large particle size milk droplets, and the percentage of milk droplets larger than 5um was calculated.
  • the results show that the samples prepared in Examples 3 to 18 have a good appearance after freeze-drying, the average particle size after reconstitution is significantly smaller than that of Comparative Example 1, and the percentage of large emulsion droplets (>5um) is far less than 0.05%, and the large particle size of Comparative Example 1 (>5um) exceeds the standard (>0.05%, USP standard), while Comparative Example 2 cannot be freeze-dried.
  • the peak blood drug concentration of Example 3 after administration was significantly higher than that of Comparative Example 1, the half-life was significantly extended, and the bioavailability was also significantly improved (see Table 6) .
  • Example 3 178.1 ⁇ 24.5 606.1 ⁇ 100.4 6.6 ⁇ 1.3
  • Example 6 165.3 ⁇ 31.8 671.1 ⁇ 110.9 6.2 ⁇ 0.9
  • Example 7 168.5 ⁇ 26.9 625.3 ⁇ 82.4 7.1 ⁇ 1.6
  • Example 8 151.6 ⁇ 24.6 591.6 ⁇ 68.2 6.7 ⁇ 1.1 Comparative Example 1 125.3 ⁇ 41.9 233.3 ⁇ 78.8 2.3 ⁇ 0.3
  • the obvious improvement in the pharmacokinetic behavior of the preparations of Examples 3 and 6-8 compared with Comparative Example 1 may be that the metabolic inactivation of the drug in the pulmonary circulation is inhibited, and this phenomenon may be related to the particle size of the drug.

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Abstract

提供一种前列腺素E1甲酯注射用冻干制剂及制备和应用。所述冻干制剂包括如下重量份成分：前列腺素E1甲酯0.1-10份、注射用油500-4000份、乳化剂500-2000份、助乳化剂0-10份、冻干保护剂5000-50000份、以及甘油200-1500份。所述前列腺素E1甲酯冻干剂具有血管刺激小、药物稳定性好。

Description

一种前列腺素E1甲酯注射用冻干制剂及制备和应用 技术领域
本发明涉及医药领域，具体的说，本发明涉及一种前列腺素E1甲酯注射用冻干制剂及制备和应用。
背景技术
前列腺素E1(PGE1)为天然内源性血管舒张药，人体细胞均能合成，是调节细胞功能的重要物质，在体内不积累，不产生耐受性，且无毒、无损害性副作用，治疗效果确切，优于外源性药物。PGE1生理活性极强，具有广泛的药理活性，临床上可应用于心脑血管疾病、糖尿病并发症、呼吸系统疾病、肺动脉高压、肝肾综合征(HRS)、肝衰竭、肾病等，研究发现前列腺素E1不仅有扩张血管、减轻心脏负荷的作用,同时有排钠、利尿、强心、改善冠状循环、保护心肌、改善微循环等功效。
但目前市售常见的前列腺素E1脂肪乳注射液本身也存在几个明显的缺点：化学稳定性差，高温灭菌致使前列腺素E1含量有所降低，而降解产物PGA1含量明显升高，且产品贮存条件苛刻(0～5℃)，效期短仅有1年；由于前列腺素E1本身是致炎物质，临床应用与人体时具有强烈的疼痛感，引起静脉炎，限制了本品的推广。
针对上述缺点，重庆药友开发了前列腺素E1冻干乳剂(ZL201010168597.2)—优帝尔，该上市产品通过过滤除菌避免了高温灭菌带来的含量损失，并克服了前列腺素E1在含水制剂中稳定性差的缺点，延长了效期，但其处方中采用环糊精作为冻干保护剂，由于环糊精作为注射用辅料存在一定的安全风险，尤其是β-环糊精注射给药会引起明显的肾毒性、溶血以及注射部位坏死，制剂安全隐患大，并且注射疼痛的问题并没有得到解决(优帝尔说明书)。
前列腺素E1烷酯目前被认为是前列腺素E1的前药。例如美国专利US5681850公开了前列腺素E1烷酯(C1-4)用于治疗阳痿，专利中认为前列腺素E1烷酯通过增强脂溶性可以更好地透过皮肤被吸收，随后被水解酶分解成前列腺素E1起效，属于前药；美国专利US6673841公开了前列腺素E1烷酯(C1-5)外用制剂，制剂包含作为前药的前列腺素E1烷酯，油性载体，皮肤渗透增强剂以及抗炎剂。
然而发明人在研究中意外发现，前列腺素E1甲酯本身具有很强的生物活性，具有良好的成药前景。美国专利US4849451中公开了前列地尔甲酯的脂肪乳制剂，但我们在研究过程中依据其实施例制备获得的前列地尔甲酯的脂肪乳，在使用美国药典关于乳滴粒径测定 的新方法-光阻法(原常用动态光散射法无法准确测定5um以上大粒径粒子的数量)进行测定后发现其大粒径(>5um)乳滴明显较多，不符合乳滴粒径的要求，大乳滴(>5um)可堵塞毛细血管，引起栓塞，具有较大的安全隐患，美国曾发生注射脂肪乳大粒子导致病人死亡的医疗事故，调查研究证明为乳剂中存在大于5um乳粒引起，因此于2004年美国药典制定了测定乳剂大粒径的检测方法和标准，鉴于现有产品存在的缺点和不足，本发明旨在提供一种新型的，稳定性好，安全风险小，药效更优的前列腺素E1甲酯产品。
发明内容
本发明的一个目的在于提供一种前列腺素E1甲酯注射用冻干制剂；
本发明的另一目的在于提供一种前列腺素E1甲酯注射用冻干制剂的制备方法；
本发明的再一目的在于提供所述的制备方法制备得到的前列腺素E1甲酯注射用冻干制剂；
本发明的再一目的在于提供所述的前列腺素E1甲酯注射用冻干制剂的应用。
为达上述目的，一方面，本发明提供了一种前列腺素E1甲酯注射用冻干制剂，其中，所述冻干制剂包含如下重量份成分：前列腺素E1甲酯0.1-10份、注射用油500-4000份、乳化剂500-2000份、助乳化剂0-10份、冻干保护剂5000-50000份、以及甘油200-1500份。
根据本发明一些具体实施方案，其中，所述注射用油选自大豆油、中链油、橄榄油、茶油、玉米油或蓖麻油的一种或多种的混合。
根据本发明一些具体实施方案，其中，所述乳化剂选自蛋黄磷脂和/或大豆磷脂。
根据本发明一些具体实施方案，其中，所述助乳化剂选自油酸、棕榈酸、硬脂酸、亚麻酸、亚油酸和油酸钠中的一种或多种的混合。
根据本发明一些具体实施方案，其中，所述冻干保护剂选自乳糖、蔗糖、海藻糖、甘露醇、葡萄糖和麦芽糖中的一种或多种的混合。
根据本发明一些具体实施方案，其中，所述冻干制剂包含如下重量份成分：前列腺素E1甲酯0.1-10份、注射用油500-4000份、乳化剂500-1500份、助乳化剂0-10份、冻干保护剂5000-20000、以及甘油200-1500份。
根据本发明一些具体实施方案，其中，所述冻干制剂在冻干前每100ml包含如下重量份成分：前列腺素E1甲酯0.1-10mg、大豆油0.5-4g、蛋黄磷脂0.5-1.5g、油酸钠0-0.01g、乳糖5-20g、以及甘油0.2-1.5g。
其中可以理解的是，本发明所述的“所述冻干制剂在冻干前每100ml包含如下重量份成分”中的“每100ml”是指在冻干前所配制的每100ml的溶液；即含有各组分的水溶液。
根据本发明一些具体实施方案，其中，所述冻干保护剂与前列腺素E1甲酯的重量比为 20～500:0.01。
另一方面，本发明还提供了一种前列腺素E1甲酯注射用冻干制剂的制备方法，其中，所述方法包括以如下重量百分比成分为原料制备所述冻干制剂：前列腺素E1甲酯0.0001-0.01％、注射用油0.5-4％、乳化剂0.5-2％、助乳化剂0-0.01％、冻干保护剂5-50％、甘油0.2-1.5％、pH调节剂适量、以及余量的注射用水。
根据本发明一些具体实施方案，其中，所述注射用油选自大豆油、中链油、橄榄油、茶油、玉米油或蓖麻油的一种或多种的混合。
根据本发明一些具体实施方案，其中，所述乳化剂选自蛋黄磷脂和/或大豆磷脂。
根据本发明一些具体实施方案，其中，所述助乳化剂选自油酸、棕榈酸、硬脂酸、亚麻酸、亚油酸和油酸钠中的一种或多种的混合。
根据本发明一些具体实施方案，其中，所述冻干保护剂选自乳糖、蔗糖、海藻糖、甘露醇、葡萄糖和麦芽糖中的一种或多种的混合。
根据本发明一些具体实施方案，其中，所述方法包括以如下重量百分比成分为原料制备所述冻干制剂：前列腺素E1甲酯0.0001-0.01％、注射用油0.5-4％、乳化剂0.5-1.5％、助乳化剂0-0.01％、冻干保护剂5-20％、甘油0.2-1.5％、pH调节剂适量、以及余量的注射用水。
根据本发明一些具体实施方案，其中，所述方法包括以如下重量份成分为原料制备所述冻干制剂：前列腺素E1甲酯0.1-10份、大豆油500-4000份、蛋黄磷脂500-1500、油酸钠0-10份、乳糖5000-20000份、甘油20-1500份、柠檬酸钠或盐酸适量、以及以原料总重量为100×10 3份计的余量的注射用水。
根据本发明一些具体实施方案，其中，所述pH调节剂用量是在用注射用水定容至全量配制成混合溶液后，将混合溶液pH调至4.5～6.5。
根据本发明一些具体实施方案，其中，所述方法包括如下步骤：
a.将前列腺素E1甲酯和乳化剂均匀分散于油性溶剂中作为油相；
b.将渗透压调节剂和冻干保护剂溶于适量注射用水中，作为水相；
c.将助乳化剂溶于油相或水相；
d.将油相在搅拌的条件下加入水相，或水相在搅拌的条件下加入油相中，剪切获得初乳；
e.将初乳均质得到均匀的乳状液，然后用注射用水定容至全量，用pH调节剂将pH调至4.5～6.5；
f.将获得的乳状液添加冻干保护剂搅拌溶解，过滤除菌、分装、冻干、封口，获得冻干剂。
根据本发明一些具体实施方案，其中，步骤d是在20℃-50℃的恒温条件下剪切获得初乳。
根据本发明一些具体实施方案，其中，步骤f的冻干过程包括-50℃至-35℃下预冻100-200分钟，然后在-25℃至-15℃下，以70-90mTorr抽真空420-540分钟，再在5℃至15℃下，以50-70mTorr抽真空240-360分钟，然后在35℃至45℃下，以35-45mTorr抽真空300-420分钟。
根据本发明一些具体实施方案，其中，步骤f的冻干过程包括-40℃下预冻150分钟，然后在-20℃下，以80mTorr抽真空480分钟，再在10℃下，以60mTorr抽真空300分钟，然后在40℃下，以40mTorr抽真空360分钟。
再一方面，本发明还提供了本发明所述的制备方法制备得到的前列腺素E1甲酯注射用冻干制剂。
再一方面，本发明还提供了本发明所述的前列腺素E1甲酯注射用冻干制剂在制备扩血管药物中的应用、冠心病、心绞痛、心力衰竭、肺心病、脑梗死、羊水栓塞、或硬皮病)。
根据本发明一些具体实施方案，其中，所述扩血管药物用于治疗如下疾病：微循环障碍。
根据本发明一些具体实施方案，其中，所述微循环障碍由如下病症引起：血栓闭塞性脉管炎、闭塞性动脉硬化症、糖尿病、冻伤、烧伤或褥疮。
发明人在在研究中意外发现，前列腺素E1甲酯本身具有很强的生物活性，抗凝以及扩血管活性方面都优于前列腺素E1，具有较好的药物开发潜力，发明人通过进一步的制剂研究，选择冻干剂作为前列腺素E1甲酯的制剂形式，并意外发现本发明制备的前列腺素E1甲酯冻干剂相比较现有制剂具有以下显著特点：
1.本发明前列腺素E1甲酯冻干剂在具体实验例中表现出了比优帝尔更优的药物活性和治疗效果。
2.本发明前列腺素E1甲酯冻干剂相比脂肪乳剂明显降低了乳滴的平均粒径和减少了大粒径乳滴比例，意外改善了药代动力学行为，从而提高药效。
2.显著降低了冻干剂复溶后的游离药物含量，尤其是按照说明书给药方式稀释10倍后游离药物没有明显增加，避免了血管注射刺激。
3.显著提高了制剂的稳定性，降低了对生产、运输和贮存条件的需求，延长了效期。
附图说明
图1为实验例1的化合物对ADP诱导血小板聚集的抑制率与孵育时间关系曲线；
图2为实验例2的血管环的舒张效能与前列腺素E1浓度关系曲线；
图3为实验例3的肠系膜微动脉的管径与给药时间关系曲线；
图4为大鼠静脉注射40μg/kg前列地尔后前列地尔的血浆浓度-时间曲线。
具体实施方式
以下结合附图及实施例详细说明本发明的技术方案，但本发明的保护范围包括但是不限于此。
实施例1
本发明化合物1([(1R,2R,3R)-3-羟基-2-(S,E)-3-羟基-1-烯基)-5-氧代环戊基]庚酸甲酯)的合成(前列腺素E1甲酯)
Figure PCTCN2019113557-appb-000001
原料PGE1(63mg，0.18mmol)加入三口烧瓶中，然后加入配置好的1M的干燥THF/Et2O溶液溶液搅拌溶解，冰浴条件下，向反应液中缓慢中滴加MeI(26mg，1M)溶液，滴加完后加入KOH(10mg，0.18mmol)和Bu 4NBr(6mg，0.018mmol)。反应搅拌1h后，加热至室温，TLC监控至反应结束。加水20ml猝灭反应，用EtOAc(10mL×3)萃取，合并有机相，用无水Na 2SO 4干燥并过滤。滤液减压浓缩，用柱层析纯化(洗脱液正己烷/EA＝1/1)，得到白色固体产物(24.8mg，38％收率)。
LCMS(MS Found:391.3[M+Na] +。
1HNMR(400MHZ,DMSO)(ppm):5.46(s,2H),5.01(s,1H),4.57(s,1H),3.88(s,2H),3.57(s,3H),1.9-2.3(m,5H)1.2-1.48(m,19H),0.85(s,3H)。
实施例2
化合物2([(1R,2R,3R)-3-羟基-2-(S,E)-3-羟基-1-烯基)-5-氧代环戊基]庚酸乙酯)的合成(前列腺素E1乙酯)
Figure PCTCN2019113557-appb-000002
原料PGE1(63mg，0.18mmol)加入三口烧瓶中，然后加入配置好的1M的干燥THF/Et 2O 溶液溶液搅拌溶解，冰浴条件下，向反应液中缓慢中滴加EtBr(20mg，1M)溶液，滴加完后加入KOH(10mg，0.18mmol)和Bu 4NBr(6mg，0.018mmol)。反应搅拌1h后，加热至室温，TLC监控至反应结束。加水20ml猝灭反应，用EtOAc(10mL×3)萃取，合并有机相，用无水Na 2SO 4干燥并过滤。滤液减压浓缩，用柱层析纯化(洗脱液正己烷/EA＝1/1)，得到白色固体产物(20.5mg，29.8％收率)。
LCMS(MS Found:405[M+Na] +。
1HNMR(400MHZ,DMSO)(ppm):5.46(s,2H),5.01(s,1H),4.57(s,1H),3.88(s,2H),3.57(s,3H),1.9-2.3(m,7H)1.2-1.48(m,19H),0.85(s,3H)。
不同主药含量的前列腺素E1甲酯冻干剂
实施例3
Figure PCTCN2019113557-appb-000003
制备工艺如下：
油相：称取大豆油2g，加入蛋黄磷脂0.5g，前列腺素E1甲酯0.5mg，50℃下剪切至溶解；
水相：称取注射用水90g，加入甘油0.75g，乳糖12.5g，油酸钠0.01g，剪切混匀，0.1M柠檬酸钠调节pH到6.5，再加入2g磷脂，继续剪切10min；
将油相缓慢加入到水相，继续在50℃下剪切10min，得到初乳，加水至100mL；
将初乳过均质机，850bar压力下，均质8次；
将均质后的乳剂进行无菌过滤，分装、-40℃下预冻150分钟，然后-20℃，80mTorr抽真空480分钟，10℃，60mTorr抽真空300分钟，40℃，40mTorr抽真空360分钟，抽真空下压盖，即得冻干粉针剂。
实施例4
Figure PCTCN2019113557-appb-000004
制备工艺同实施例3
实施例5
Figure PCTCN2019113557-appb-000005
制备工艺同实施例3
不同注射液用油、磷脂及含量的前列腺素E1甲酯冻干剂
实施例6
Figure PCTCN2019113557-appb-000006
Figure PCTCN2019113557-appb-000007
制备工艺同实施例3
实施例7
Figure PCTCN2019113557-appb-000008
制备工艺同实施例3
实施例8
Figure PCTCN2019113557-appb-000009
制备工艺同实施例3
实施例9
Figure PCTCN2019113557-appb-000010
制备工艺同实施例3
实施例10
Figure PCTCN2019113557-appb-000011
制备工艺同实施例3
不同助乳化剂及含量的的前列腺素E1甲酯冻干剂
实施例11
Figure PCTCN2019113557-appb-000012
Figure PCTCN2019113557-appb-000013
制备工艺同实施例3
实施例12
Figure PCTCN2019113557-appb-000014
制备工艺同实施例3
实施例13
Figure PCTCN2019113557-appb-000015
制备工艺同实施例3
实施例14
Figure PCTCN2019113557-appb-000016
制备工艺同实施例3
不同冻干保护剂及含量的的前列腺素E1甲酯冻干剂
实施例15
Figure PCTCN2019113557-appb-000017
制备工艺同实施例3
实施例16
Figure PCTCN2019113557-appb-000018
Figure PCTCN2019113557-appb-000019
制备工艺同实施例3
实施例17
Figure PCTCN2019113557-appb-000020
制备工艺同实施例3
实施例18
Figure PCTCN2019113557-appb-000021
制备工艺同实施例3
对比例1
依据美国专利US4849451中实施例2制备获得。
对比例2
将对比例1所得制剂添加本发明处方所用冻干保护剂进行冷冻干燥。
实验例1
体外抗血小板凝集试验中的作用
健康成年SD大鼠腹腔注射10％水合氯醛麻醉后，腹主动脉采集大鼠新鲜全血加入使用3.8％枸橼酸钠溶液抗凝的离心管中，900转离心10min取上层富含血小板的血浆(PRP)备用。取出PRP的试管继续4000转离心10分钟，取上层澄清血浆(PPP)备用。实验选用泰利康信LBY-NJ4型4通道血小板聚集仪测定各化合物的抗凝效能。
含有300μL PRP样品杯中先加入，2μL 100μM的PGE1、实施例化合物1、实施例化合物2和甲醇(溶剂)，孵育不同的时间(0、1、2、4、7、10、15min)后加入聚集诱导剂180μM ADP溶液20μL，测定各样品的聚集率，并计算化合物对ADP诱导血小板聚集的抑制率。
抑制率％＝(溶剂聚集率-化合物聚集率)/溶剂聚集率×100％
由结果(图1所示)可知，PRP中加入PGE1与实施例化合物1后立即生效，抑制率相当，随孵育时间延长，实施例化合物抗凝效能缓慢降低，10分钟后抑制率仍为43.66％，PGE1孵育4分钟后抑制率仅为4.93％。实施例化合物2加入后并未立即生效，随时间延长效能逐渐加强，孵育10分钟后达到最大抑制率51.86％。故实施例化合物1为具有活性的非前药化合物，其抗凝作用时长超过PGE12倍，而实施例化合物2为典型的前药型化合物。
实验例2
体外血管条扩张试验
实验选用家兔制备离体主动脉环标本：新西兰大白兔,雄性,体重(2.5±0.3)kg。用钝器击昏兔子,固定于兔解剖台,迅速分离出胸主动脉,放入盛有37℃饱和的Kerbs液(每1000mL中含NaCl 6.9g,KCl 0.35g,MgSO 4·7H 2O 0.29g,KH 2PO 4 0.16g,NaHCO 3 2.1g,CaCl 2 0.28g,葡萄糖2g)并持续通入通混合气体(95％O 2,5％CO 2)的培养皿中,将血管内的残存血液挤出,并小心剥去外围的脂肪及结缔组织,剪成0.5cm长的动脉环数段备用。血管环用两根不锈钢L型挂钩贯穿血管管腔,横向悬挂在20mL浴管内,下方固定,上方以一细钢丝连于张力换能器,先调解静息张力为0.00g,稳定20min后,再给予3.00g张力,不断调整张力水平,使之维持在3.00g左右,稳定2h(每15min沿浴槽壁换Kerbs液一次)。
使用BL-420S生物机能实验系统(成都泰盟科技)记录血管环张力变化。待血管环收缩达到稳定后,累积加入前列腺素E1和实施例化合物,使浴管中的前列腺素E1终质量浓度依次递增为0.05、0.1、0.2、0.4、0.8、1.6、3.2、6.4、12.8、25.6nM,记录血管环的舒张效 能。
结果显示(图2所示)在本实验条件下实施例1化合物对家兔离体血管的舒张效能(EC 50＝1.090nM)明显强于PGE 1(EC 50＝9.767nM)。
实验例3
肠系膜微循环障碍模型
2.5％戊巴比妥钠30mg/kg ip麻醉大鼠，背位固定，于腹正中线作3～4cm长切口，轻拉回盲部一段小肠系膜，置于充满37℃生理盐水液的有机玻璃恒温水浴槽中，保持肠系膜湿润，并平铺在浴槽中央的有机凸型观察台上，压上固定板，利用生物显微镜(放大40倍)摄像头采集显微镜下视频图像利用BI-2000微循环观测系统对所采集的镜下视频图像进行实时分析。
固定一视野，平衡10min后，观察选定区域的微动脉和微静脉管径、流速，然后向选定区域滴加1∶100稀释盐酸肾上腺素生理盐水溶液100μL/只，正常对照组滴加等体积生理盐水，同时立即经尾静脉给与10μg/kg的实施例制剂和优帝尔，测量给药后0.5、1.2、4、6、8、10、12、15min时肠系膜微动脉的管径。
如图3所示，经管径测量，实施例3制剂组可极为明显改善肾上腺素引起的微循环障碍，给药后0.5分钟-10分钟均与模型组有明显的差异(P<0.05)，优帝尔组4分钟时与模型对照组相比差异已不显著(P>0.05)。实施例组改善肾上腺素引起循环障碍的效能明显高优帝尔，两者比较0.5-10min均具有显著性差异(P<0.05)。
实验例4
制剂游离药物测定
样品处理方法：各实施例冻干乳分别加10ml复乳同步取一份以水稀释10倍，精密量取适量置20ml棕色具塞试管中，加四氢呋喃2.5ml，混匀，加磷酸溶液(1→1000)15ml，混匀，通过预处理柱[填充物为十八烷基硅烷键合硅胶，粒径为70μm，φ10mm×9mm聚丙烯管(SEP-PAK C18柱，Waters)。使用前用甲醇10ml、水10ml冲洗]，试管用10ml水冲洗并通过预处理柱。再用甲醇7ml洗脱，洗脱液全部移入10ml棕色蒸馏瓶中，50℃减压蒸馏10分钟，蒸干溶剂，残渣用内标溶液1ml溶解，摇匀，即得样品。
用十八烷基键合硅胶为填充剂，以0.0067mol/L磷酸盐缓冲液(pH＝6.3)(取磷酸二氢钾9.07g，加水使溶解，制成1000ml，另取无水磷酸氢二钠9.46g，加水使溶解，制成1000ml，将后者加入到前者中，直到pH为6.3，取此液100ml加水至1000ml，摇匀，即得)-乙腈(70:30)为流动相；流速为每分钟1ml；柱后反应液为1mol·L-1KOH溶液，柱后反应管为聚四氟乙烯管
Figure PCTCN2019113557-appb-000022
柱温60℃；检测波长278nm。供试品和对照品溶液各 进样20μl，按内标峰面积法计算。
将实施例3～18和优帝尔用生理盐水复溶，浓度均为1ug/mL，再取样均稀释10倍至0.1ug/mL，取上述复溶后各制剂适量，置于超滤离心管中，以2800r·min-1离心超滤30min后，取滤液1ml按上述样品处理方法配制样品溶液，进样20μl，测得的各药物含量即为游离药物含量。
同时测定各实施例样品1mL复乳(1×)和10倍稀释(10×)后样品的游离率。
游离率％＝游离药物总量/总药物量×100％
测得的各实施例游离率如下表1所示：
表1
Figure PCTCN2019113557-appb-000023
实验结果表明，实施例3～18药物复溶后游离药物显著低于优帝尔，且优帝尔按照说明书使用方法稀释10倍后，游离率显著上升，而本发明实施例制备制剂稀释后游离药物无明显增加。
实验例5
血管刺激试验
选取新西兰大白兔20只，分为2组体重约2kg。实施例制剂与优帝尔均采用生理盐水复乳为1μg/ml,并继续用生理盐水稀释至0.1μg/ml。按照前列腺素E1临床用量折算，0.5μg/kg耳缘静脉缓慢滴注，对侧耳缘静脉静注同样体积的生理盐水，每天1次，连续滴注7天。末次给药后2小时目视评价血管刺激程度，所有动物处死后取注射部位做组织病理学检查，结果如下表2和表3所示。
表2、血管刺激性试验大体检查表
Figure PCTCN2019113557-appb-000024
结果显示实施例组基本无刺激性(P>0.05)，优帝尔组出现了明显的血管刺激(P<0.05)。
表3
Figure PCTCN2019113557-appb-000025
*P<0.05与本组对照耳对比；#P<0.05与实施例组给药耳对比
实验例6
制剂稳定性试验
实施例3～18，对比例1、2所制备的样品外观以及粒径检测如下表4所示：
表4
  外观性状 终产品平均粒径(nm) >5um乳滴(％)
实施例3 疏松块状物，复溶良好 94 0.0021
实施例4 疏松块状物，复溶良好 93 0.0019
实施例5 疏松块状物，复溶良好 96 0.0022
实施例6 疏松块状物，复溶良好 77 0.0018
实施例7 疏松块状物，复溶良好 89 0.0019
实施例8 疏松块状物，复溶良好 148 0.0053
实施例9 疏松块状物，复溶良好 93 0.0037
实施例10 疏松块状物，复溶良好 104 0.0018
实施例11 疏松块状物，复溶良好 86 0.0025
实施例12 疏松块状物，复溶良好 86 0.0022
实施例13 疏松块状物，复溶良好 101 0.0019
实施例14 疏松块状物，复溶良好 113 0.0033
实施例15 疏松块状物，复溶良好 92 0.0024
实施例16 疏松块状物，复溶良好 99 0.0018
实施例17 疏松块状物，复溶良好 91 0.0038
实施例18 疏松块状物，复溶良好 89 0.0023
对比例1 白色均一乳状液体 206 0.2502
对比例2 无法成型 / /
采用PSS公司的380ZLS粒度仪(动态光散射法)测定平均粒径，采用PSS公司的Accusizer 780仪器(光阻法)测定大粒径乳滴，计算大于5um乳滴的百分比。结果显示，实施例3～18所制备样品冻干后外观良好，复溶后平均粒径明显小于对比例1，且大乳滴(>5um)占百分比远小于0.05％，对比例1大粒径(>5um)超标(>0.05％，USP标准)，而对比例2无法冻干成型。
取实施例3、5、8、15制备的样品进行24h长期稳定性试验(温度25℃±2℃，湿度60％±10％)，结果如下表5所示：
表5
Figure PCTCN2019113557-appb-000026
Figure PCTCN2019113557-appb-000027
结果显示，本发明实施例制备的前列腺素E1甲酯冻干剂在24个月长期稳定试验中产品质量稳定。
实验例7
实施例3、6、7、8和对比例1在大鼠体内药代动力学研究
药动学实验采用SD雄性大鼠每组12只，给药前禁食12h，自由饮水。分别按40μg/kg的剂量静脉给与两种制剂，给药后0.5min、1.25min、3min、5min、8min、12min、16min、20min、30min、45min眼眶采血，测定各时间点的血药浓度(前列腺素E1甲酯与前列腺素之和)如图4所示。
根据血药浓度曲线计算所得的两种制剂的药动学参数，实施例3给药后峰值血药浓度 明显高于对比例1组，半衰期明显延长，生物利用度也明显提高(见表6)。
表6大鼠静脉注射40μg/kg实施例3、6-8和对比例1制剂的药代动力学参数
  Cmax(ng/mL) AUC(0-t)(ng/mL*min) t1/2(min)
实施例3 178.1±24.5 606.1±100.4 6.6±1.3
实施例6 165.3±31.8 671.1±110.9 6.2±0.9
实施例7 168.5±26.9 625.3±82.4 7.1±1.6
实施例8 151.6±24.6 591.6±68.2 6.7±1.1
对比例1 125.3±41.9 233.3±78.8 2.3±0.3
实施例3、6-8相比对比例1制剂体内药动学行为的明显改善，可能是抑制了药物在肺循环中的代谢灭活，而这种现象可能与药物的粒径有关。

Claims (10)

  1. 一种前列腺素E1甲酯注射用冻干制剂，其中，所述冻干制剂包含如下重量份成分：前列腺素E1甲酯0.1-10份、注射用油(优选所述注射用油选自大豆油、中链油、橄榄油、茶油、玉米油或蓖麻油的一种或多种的混合)500-4000份、乳化剂(优选所述乳化剂选自蛋黄磷脂和/或大豆磷脂)500-2000份、助乳化剂(优选所述助乳化剂选自油酸、棕榈酸、硬脂酸、亚麻酸、亚油酸和油酸钠中的一种或多种的混合)0-10份、冻干保护剂(优选所述冻干保护剂选自乳糖、蔗糖、海藻糖、甘露醇、葡萄糖和麦芽糖中的一种或多种的混合)5000-50000份、以及甘油200-1500份。
  2. 根据权利要求1所述的冻干制剂，其中，所述冻干制剂包含如下重量份成分：前列腺素E1甲酯0.1-10份、注射用油500-4000份、乳化剂500-1500份、助乳化剂0-10份、冻干保护剂5000-20000、以及甘油200-1500份。
  3. 根据权利要求1所述的冻干制剂，其中，所述冻干制剂在冻干前每100ml包含如下重量份成分：前列腺素E1甲酯0.1-10mg、大豆油0.5-4g、蛋黄磷脂0.5-1.5g、油酸钠0-0.01g、乳糖5-20g、以及甘油0.2-1.5g。
  4. 根据权利要求1～3任意一项所述的冻干制剂，其中，所述冻干保护剂与前列腺素E1甲酯的重量比为20～500:0.01。
  5. 一种前列腺素E1甲酯注射用冻干制剂的制备方法，其中，所述方法包括以如下重量百分比成分为原料制备所述冻干制剂：前列腺素E1甲酯0.0001-0.01％、注射用油(优选所述注射用油选自大豆油、中链油、橄榄油、茶油、玉米油或蓖麻油的一种或多种的混合)0.5-4％、乳化剂(优选所述乳化剂选自蛋黄磷脂和/或大豆磷脂)0.5-2％、助乳化剂(优选所述助乳化剂选自油酸、棕榈酸、硬脂酸、亚麻酸、亚油酸和油酸钠中的一种或多种的混合)0-0.01％、冻干保护剂(优选所述冻干保护剂选自乳糖、蔗糖、海藻糖、甘露醇、葡萄糖和麦芽糖中的一种或多种的混合)5-50％、甘油0.2-1.5％、pH调节剂适量、以及余量的注射用水。
  6. 根据权利要求5所述的制备方法，其中，所述方法包括以如下重量百分比成分为原料制备所述冻干制剂：前列腺素E1甲酯0.0001-0.01％、注射用油0.5-4％、乳化剂0.5-1.5％、助乳化剂0-0.01％、冻干保护剂5-20％、甘油0.2-1.5％、pH调节剂适量、以及余量的注射用水。
  7. 根据权利要求5所述的制备方法，其中，所述方法包括以如下重量份成分为原料制 备所述冻干制剂：前列腺素E1甲酯0.1-10份、大豆油500-4000份、蛋黄磷脂500-1500、油酸钠0-10份、乳糖5000-20000份、甘油20-1500份、柠檬酸钠或盐酸适量、以及以原料总重量为100×10 3份计的余量的注射用水。
  8. 根据权利要求5～7任意一项所述的制备方法，其中，所述方法包括如下步骤：
    a.将前列腺素E1甲酯和乳化剂均匀分散于油性溶剂中作为油相；
    b.将甘油和冻干保护剂溶于适量注射用水中，作为水相；
    c.将助乳化剂溶于油相或水相；
    d.将油相在搅拌的条件下加入水相，或水相在搅拌的条件下加入油相中，剪切获得初乳(优选是在20℃-50℃的恒温条件下剪切获得初乳)；
    e.将初乳均质得到均匀的乳状液，然后用注射用水定容至全量，用pH调节剂将pH调至4.5～6.5；
    f.将获得的乳状液添加冻干保护剂搅拌溶解，过滤除菌、分装、冻干(优选冻干过程包括-50℃至-35℃下预冻100-200分钟，然后在-25℃至-15℃下，以70-90mTorr抽真空420-540分钟，在5℃至15℃下，以50-70mTorr抽真空240-360分钟，然后在35℃至45℃下，以35-45mTorr抽真空300-420分钟)、封口，获得冻干剂。
  9. 权利要求5～8任意一项所述的制备方法制备得到的前列腺素E1甲酯注射用冻干制剂。
  10. 权利要求1～4、或者权利要求9任意一项所述的前列腺素E1甲酯注射用冻干制剂在制备扩血管药物中的应用(优选所述扩血管药物用于治疗如下疾病：微循环障碍(优选所述微循环障碍由如下病症引起：血栓闭塞性脉管炎、闭塞性动脉硬化症、糖尿病、冻伤、烧伤或褥疮)、冠心病、心绞痛、心力衰竭、肺心病、脑梗死、羊水栓塞、或硬皮病)。
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CN109288848A (zh) * 2018-11-27 2019-02-01 西安力邦肇新生物科技有限公司 前列腺素e1甲酯在制备扩张血管药物中的应用

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US20220023219A1 (en) 2022-01-27
JP7169024B2 (ja) 2022-11-10
CN109394704B (zh) 2021-09-17
EP3888637A1 (en) 2021-10-06

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