CN102579352A - Clarithromycin freeze-dried liposome and preparation method thereof - Google Patents
Clarithromycin freeze-dried liposome and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a clarithromycin freeze-dried liposome which is prepared by carrying out freeze-drying after mixing liposome solution and a cryoprotectant. 5 g to 20 g of cryoprotectant is added into each 100 mL of the liposome solution; 100 ml of the liposome solution is prepared from the following raw materials: 0.05 g to 2.0 g of clarithromycin, 0.01 g to 2.0 g of sterol substance, 0.2 g to 10g of phospholipid, 0.01 g to 1.0 g of fatty acid and the balance of phosphate buffered solution; and the cryoprotectant is formed by one or more of cane sugar, glucose, trehalose, maltose and mannitol. The invention further discloses a preparation method of the liposome. The clarithromycin freeze-dried liposome disclosed by the invention can be used for obviously reducing the pain of the administration sites and the vascular irritation which are caused by clarithromycin, various indexes of the clarithromycin freeze-dried liposome all meet the requirement on research and development of new drug by the State Food and Drug Administration, and the foundation is laid for the industrial production and the clinical application of aclarithromycin liposome preparation.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of clarithromycin freeze-dried lipidosome and preparation method thereof.
Background technology
Clarithromycin (clarithromycin, CLM) chemistry 6-O-erythromycin by name, be 1981 be raw material with erythromycin by the big positive drugmaker of Japan, 6 hydroxyls replaced with methoxyl group form.Chemical structural formula is as follows:
Clarithromycin belongs to macrolide antibiotics, and its mechanism is the connection through block cell nucleoprotein 50S subunit, CKIs matter synthetic and produce bacteriostasis.Be mainly used in the upper and lower respiratory infections due to the sensitive bacterial, Genito-urinary system infects HIV sufferers's atypical mycobacterial infection etc.
Mostly the clinical dosage form of clarithromycin is oral formulations, comprises tablet, capsule, syrup, suspensoid.Its injection is that U.S. Abbott makes production; Commodity are bright to be
, and the clarithromycin vein uses zest bigger, is prone to cause chemical phlebitis; And because medicine itself has than strong and stimulating, also can cause vasospasm, blood reduces; Local drug concentration increases relatively; Increase the weight of phlebitis, and if transfusion speed greater than velocity of blood flow, then phlebitic incidence rate obviously increases; Be used for periphery superficial vein when transfusion clinical, the ratio that superficial phlebitis takes place is quite high.Therefore,, reduce the zest that intravenously administrable causes, demand developing the preparation of market prospect urgently in order to enlarge the route of administration of clarithromycin.
Summary of the invention
Technical problem to be solved by this invention is the deficiency to above-mentioned prior art, and a kind of clarithromycin freeze-dried lipidosome is provided.Particle diameter after this freeze-dried lipidosome lyophilizing is redissolved is below 200nm, and the pH value after the lyophilizing, envelop rate, content all keep stable.Compare with solution, clarithromycin freeze-dried lipidosome of the present invention can reduce medicine-feeding part pain and the blood vessel irritation that clarithromycin causes significantly, and its each item index has all reached the requirement of state food drug administration about the new drug research exploitation.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of clarithromycin freeze-dried lipidosome, it is characterized in that, and mix the back lyophilizing with freeze drying protectant by liposome solutions and process, add 5g～20g freeze drying protectant in every 100mL liposome solutions; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 0.05g～2.0g, and steroid material 0.01g～2.0g, phosphatidase 10 .2g～10g, fatty acid 0.01g～1.0g, surplus is a PBS; Said freeze drying protectant is one or more in sucrose, glucose, trehalose, maltose and the mannitol; Said steroid material is cholesterol and/or cholesterol sodium sulfate salt; Said fatty acid is one or more in caproic acid, adipic acid and the benzoic acid; Said phospholipid is natural phospholipid and/or synthetic phospholipid.
Above-mentioned a kind of clarithromycin freeze-dried lipidosome, the said liposome solutions of 100mL is processed by following raw material: clarithromycin 1.5g, steroid material 1.5g, phosphatidase 15 .4g, fatty acid 0.75g, surplus is a PBS.
Above-mentioned a kind of clarithromycin freeze-dried lipidosome; Said natural phospholipid is one or more among the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, injection stage Ovum Gallus domesticus Flavus lecithin PL-100M and the lecithin E-80, and said synthetic phospholipid is one or more among synthetic phospholipid POPC (1-palmityl-2-oleoyl lecithin), synthetic phospholipid SOPC (1-stearyl-2-oleoyl lecithin) and the synthetic phospholipid MOPC (1-myristoyl-2-oleoyl lecithin).
Above-mentioned a kind of clarithromycin freeze-dried lipidosome, said steroid material is the cholesterol sodium sulfate salt, and said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, and said fatty acid is a caproic acid.
The present invention also provides the method for preparing above-mentioned clarithromycin freeze-dried lipidosome, it is characterized in that, this method may further comprise the steps:
Step 1, take by weighing clarithromycin, steroid material, phospholipid and fatty acid respectively; Being scattered in the organic solvent phospholipid that takes by weighing and steroid material fully, dissolving obtains solution A; Being scattered in the organic solvent clarithromycin that takes by weighing and fatty acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase; Said organic solvent is one or more in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether;
Step 2, the rotary evaporation of oil phase described in the step 1 is removed the organic solvent in the oil phase, dry up with nitrogen then, obtain thin film;
Step 3, secure ph are 6.5～7.5 PBS, then PBS are preheated to 50 ℃～80 ℃;
Step 4, with the PBS after preheating in the step 3 with the complete aquation of thin film described in the step 2, obtain colostrum;
Step 5, colostrum described in the step 4 is used pH value is 6.5～7.5 PBS standardize solution, places high pressure homogenizer to carry out homogenizing then or to place Ultrasound Instrument to carry out ultrasonic, obtains liposome solutions;
Step 6, take by weighing freeze drying protectant and the freeze drying protectant that takes by weighing is dissolved in described in the step 5 in the liposome solutions, the ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Above-mentioned method, controlling homogenizing temperature in the homogenizing process described in the step 5 is 5 ℃～40 ℃, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times; Ultransonic temperature described in the step 5 is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
The invention provides the method for the above-mentioned clarithromycin freeze-dried lipidosome of another kind of preparation, it is characterized in that this method may further comprise the steps:
Step 1, take by weighing clarithromycin, steroid material, phospholipid, fatty acid and freeze drying protectant respectively; Being scattered in the organic solvent phospholipid that takes by weighing and steroid material fully, dissolving obtains solution A; Being scattered in the organic solvent clarithromycin that takes by weighing and fatty acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase; Said organic solvent is one or more in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether;
Step 2, secure ph are 6.5～7.5 PBS, the freeze drying protectant that takes by weighing in the step 1 is dissolved in the PBS and with PBS is preheated to 50 ℃～80 ℃ then;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back and under heating condition, continue stirring organic solvent in solution and volatilizees fully, obtain liposome turbid liquor; The temperature of said heating is 50 ℃～80 ℃;
Step 4, place high pressure homogenizer to carry out homogenizing or to place Ultrasound Instrument to carry out ultrasonic liposome turbid liquor described in the step 3; To sterilize through the liposome turbid liquor ultrafiltration behind the homogenizing or after ultrasonic then; Packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Above-mentioned method, controlling homogenizing temperature in the homogenizing process described in the step 4 is 5 ℃～40 ℃, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times; Ultransonic temperature described in the step 4 is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
Provided by the invention the third prepares the method for above-mentioned clarithromycin freeze-dried lipidosome, it is characterized in that, this method may further comprise the steps:
Step 1, take by weighing clarithromycin, steroid material, phospholipid and fatty acid respectively; Being scattered in the organic solvent phospholipid that takes by weighing and steroid material fully, dissolving obtains solution A; Being scattered in the organic solvent clarithromycin that takes by weighing and fatty acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase; Said organic solvent is one or more in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether;
Step 2, secure ph are 6.5～7.5 PBS, then PBS are preheated to 50 ℃～80 ℃;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back reduction vaporization and remove organic solvent, make liposome turbid liquor;
Step 4, place high pressure homogenizer to carry out homogenizing or to place Ultrasound Instrument to carry out ultrasonic, obtain liposome solutions liposome turbid liquor described in the step 3;
Step 5, take by weighing freeze drying protectant and the freeze drying protectant that takes by weighing is dissolved in described in the step 4 in the liposome solutions, the ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Above-mentioned method, controlling homogenizing temperature in the homogenizing process described in the step 4 is 5 ℃～40 ℃, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times; Ultransonic temperature described in the step 4 is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
The present invention compared with prior art has the following advantages:
1, clarithromycin freeze-dried lipidosome of the present invention, its drug loading reaches as high as 20mg/mL, and the particle diameter after lyophilizing is redissolved is below 200nm, and the pH value after the lyophilizing, envelop rate, content all keep stable; Compare with solution, clarithromycin freeze-dried lipidosome of the present invention can reduce medicine-feeding part pain and the blood vessel irritation that clarithromycin causes significantly, and its each item index has all reached the requirement of state food drug administration about the new drug research exploitation.
2, the present invention has improved the drug loading of clarithromycin in liposome, has reduced the blood vessel irritation of clarithromycin intravenously administrable simultaneously to a great extent, for the suitability for industrialized production and the clinical practice of clarithromycin Liposomal formulation are laid a good foundation.
3, the deep research of process among the present invention; Find that fatty acid, especially caproic acid can interact with weakly alkaline medicine clarithromycin, in addition the assosting effect of steroid; Increased the distribution of clarithromycin between phospholipid bilayer; Play pivotal role for improving drug loading, increased the drug loading in liposome of clarithromycin greatly, and through adjusting the proportioning of phospholipid and steroid material; The envelop rate that can make liposome has guaranteed the protective effect to the vein blood vessel of injection site more than 95%.
4, each item physicochemical properties such as the content of the clarithromycin freeze-dried lipidosome of employing method preparation of the present invention, particle diameter, envelop rate, pH value all meet the used for intravenous injection requirement; Not only the long term storage physical and chemical stability is good; And blood vessel irritation significantly reduces; Improve patient's compliance, be suitable for clinical practice, can satisfy industrial production requirement simultaneously.
5, the preferred cholesterol sodium sulfate salt of steroid material of the present invention (SCS); SCS is derived behind Sulfation by cholesterol; It can be used as the composition that a kind of new membrane stability material is participated in liposome, when playing adjusting membrane fluidity function, has emulsification again.
6, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the preferred natural phospholipid injection stage of phospholipid of the present invention, its phosphatidylcholine content (PC) reaches more than 98%, and this high-purity characteristic makes the stability of clarithromycin freeze-dried lipidosome be significantly improved.
7, the result that investigates of clarithromycin freeze-dried lipidosome long-time stability of the present invention showed: 25 ± 2 ℃ condition held 3 months; Its outward appearance, pH value, medicament contg, envelop rate and mean diameter all do not have to take place to change significantly, still meet the requirement of intravenous administration.
Below in conjunction with accompanying drawing and embodiment, technical scheme of the present invention is done further to describe in detail.
Description of drawings
Fig. 1 is the preceding projection electromicroscopic photograph of clarithromycin freeze-dried lipidosome lyophilizing of the embodiment of the invention 1 preparation.
Fig. 2 is the projection electromicroscopic photograph before the clarithromycin freeze-dried lipidosome of the embodiment of the invention 1 preparation redissolves.
Fig. 3 be before the clarithromycin freeze-dried lipidosome lyophilizing of the embodiment of the invention 1 preparation with redissolve after the release in vitro curve.
The negative matched group of Fig. 4 a is apart from the pathological section figure at medicine-feeding part 1cm place.
The negative matched group of Fig. 4 b is apart from the pathological section figure at medicine-feeding part 2cm place.
Fig. 4 c is to be checked group of pathological section figure apart from medicine-feeding part 1cm place of the present invention.
Fig. 4 d is to be checked group of pathological section figure apart from medicine-feeding part 2cm place of the present invention.
The specific embodiment
Embodiment 1
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 1.5g, and steroid material 1.5g, phosphatidase 15 .4g, fatty acid 0.75g, surplus is a PBS; Said freeze drying protectant is a sucrose; The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 12.5g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage; Said fatty acid is a caproic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 1.5g clarithromycin, 1.5g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 5.4g injection stage and 0.75g caproic acid respectively; Being scattered in the dehydrated alcohol the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing and cholesterol sodium sulfate salt fully, dissolving obtains solution A; Being scattered in the dehydrated alcohol clarithromycin that takes by weighing and caproic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, the rotary evaporation of oil phase described in the step 1 is removed the ethanol in the oil phase, dry up with nitrogen then, obtain thin film;
Step 3, secure ph are 7.0 PBS, then PBS are preheated to 65 ℃;
Step 4, with the PBS after preheating in the step 3 with the complete aquation of thin film described in the step 2, obtain colostrum;
Step 5, colostrum described in the step 4 is used the pH value of preparing in the step 3 is that 7.0 PBS is settled to 100mL, is transferred to then and carries out homogenizing in the high pressure homogenizer, obtains liposome solutions; The control homogenizing temperature is 20 ℃ in the said homogenizing process, and homogenization pressure is 50MPa, and the homogenizing cycle-index is 6 times;
Step 6, take by weighing 12.5g sucrose as freeze drying protectant, with the sucrose dissolved that takes by weighing in liposome solutions described in the step 5, then with 0.22 μ m filter membrane ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Embodiment 2
Present embodiment is identical with embodiment 1, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said fatty acid is adipic acid or benzoic acid, perhaps is two or three in caproic acid, adipic acid and the benzoic acid; Said freeze drying protectant is glucose, trehalose, maltose or mannitol, perhaps is at least two kinds in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is absolute methanol, chloroform, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Embodiment 3
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clamycin 2 .0g, and steroid material 2.0g, phosphatidase 11 0g, fatty acid 1.0g, surplus is a PBS; Said freeze drying protectant is a mannitol; The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 20g freeze drying protectant; Said steroid material is a cholesterol; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M; Said fatty acid is an adipic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 2.0g clarithromycin, 2.0g cholesterol, 10g injection stage Ovum Gallus domesticus Flavus lecithin PL-100M and 1.0g adipic acid respectively; Being scattered in the absolute methanol the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing and cholesterol fully, dissolving obtains solution A; Being scattered in the absolute methanol clarithromycin that takes by weighing and adipic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, the rotary evaporation of oil phase described in the step 1 is removed the methanol in the oil phase, dry up with nitrogen then, obtain thin film;
Step 3, secure ph are 7.5 PBS, then PBS are preheated to 80 ℃;
Step 4, with the PBS after preheating in the step 3 with the complete aquation of thin film described in the step 2, obtain colostrum;
Step 5, colostrum described in the step 4 is used pH value is that 7.5 PBS is settled to 100mL, is transferred to then and carries out homogenizing in the high pressure homogenizer, obtains liposome solutions; The control homogenizing temperature is 5 ℃～40 ℃ in the said homogenizing process, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times;
Step 6, take by weighing 20g mannitol and the mannitol that takes by weighing is dissolved in described in the step 5 in the liposome solutions, with 0.22 μ m filter membrane ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Embodiment 4
Present embodiment is identical with embodiment 1, and wherein difference is: said steroid material is the cholesterol sodium sulfate salt or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said fatty acid is caproic acid or benzoic acid, perhaps is two or three in caproic acid, adipic acid and the benzoic acid; Said freeze drying protectant is sucrose, glucose, trehalose or maltose, perhaps is at least two kinds in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is dehydrated alcohol, chloroform, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Embodiment 5
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 0.05g, and steroid material 0.01g, phosphatidase 10 .2g, fatty acid 0.01g, surplus is a PBS; Said freeze drying protectant is sucrose and glucose (mass ratio 1: 1); The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 5g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is synthetic phospholipid POPC; Said fatty acid is a caproic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 0.05g clarithromycin, 0.2g synthetic phospholipid POPC, 0.01g cholesterol sodium sulfate salt and 0.01g caproic acid respectively; The synthetic phospholipid POPC that takes by weighing and cholesterol sodium sulfate salt being scattered in the mixed solvent (volume ratio 1: 1) of chloroform and ether fully, dissolving obtains solution A; The clarithromycin that takes by weighing and caproic acid being scattered in the mixed solvent (volume ratio 1: 1) of chloroform and ether all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, the rotary evaporation of oil phase described in the step 1 is removed chloroform and ether in the oil phase, dry up with nitrogen then, obtain thin film;
Step 3, secure ph are 6.5 PBS, then PBS are preheated to 50 ℃;
Step 4, with the PBS after preheating in the step 3 with the complete aquation of thin film described in the step 2, obtain colostrum;
Step 5, colostrum described in the step 4 is used pH value is that 6.5 PBS is settled to 100mL, is transferred to then to carry out ultrasonicly in the Ultrasound Instrument, obtains liposome solutions; Said ultransonic temperature is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min;
Step 6, take by weighing sucrose and each 2.5g of glucose, and the sucrose that takes by weighing and glucose are dissolved in described in the step 5 in the liposome solutions, with 0.22 μ m filter membrane ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Embodiment 6
Present embodiment is identical with embodiment 5, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said fatty acid is adipic acid or benzoic acid, perhaps is two or three in caproic acid, adipic acid and the benzoic acid; Said freeze drying protectant is a kind of in sucrose, glucose, trehalose, maltose and the mannitol or more than three kinds; Perhaps being two kinds in glucose, trehalose, maltose and the mannitol, perhaps is the mixture of a kind of and sucrose in trehalose, maltose and the mannitol; Said organic solvent is dehydrated alcohol, absolute methanol, chloroform, dichloromethane or ether; Perhaps being two kinds in absolute methanol, dehydrated alcohol, dichloromethane and the chloroform, perhaps is the mixture of a kind of and ether in absolute methanol, dehydrated alcohol and the dichloromethane.
Embodiment 7
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 1.5g, and steroid material 1.5g, phosphatidase 15 .4g, fatty acid 0.75g, surplus is a PBS; Said freeze drying protectant is a sucrose; The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 20g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage; Said fatty acid is a caproic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 1.5g clarithromycin, 1.5g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 5.4g injection stage, 0.75g caproic acid and 20g sucrose respectively; Being scattered in the dehydrated alcohol the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing and cholesterol sodium sulfate salt fully, dissolving obtains solution A; Being scattered in the dehydrated alcohol clarithromycin that takes by weighing and caproic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, secure ph are 6.5 PBS, then the sucrose dissolved that takes by weighing in the step 1 are preheated to 70 ℃ in PBS and with PBS;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back and under heating condition, continue stirring ethanol in solution and volatilizees fully, obtain liposome turbid liquor; The temperature of said heating is 70 ℃;
Step 4, place high pressure homogenizer to carry out homogenizing liposome turbid liquor described in the step 3, then the liposome turbid liquor behind homogenizing is sterilized with 0.22 μ m filter membrane ultrafiltration, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome; The control homogenizing temperature is 5 ℃～40 ℃ in the said homogenizing process, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times.
Embodiment 8
Present embodiment is identical with embodiment 7, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said fatty acid is adipic acid or benzoic acid, perhaps is two or three in caproic acid, adipic acid and the benzoic acid; Said freeze drying protectant is glucose, trehalose, maltose or mannitol, perhaps is at least two kinds in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is absolute methanol, chloroform, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Embodiment 9
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clamycin 2 .0g, and steroid material 0.01g, phosphatidase 11 0g, fatty acid 1.0g, surplus is a PBS; Said freeze drying protectant is trehalose and maltose (mass ratio 2: 3); The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 5g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80 and synthetic phospholipid SOPC (mass ratio 8: 1: 1); Said fatty acid is a benzoic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 2.0g clarithromycin, 0.01g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 8.0g injection stage, 1.0g lecithin E-80,1.0g synthetic phospholipid SOPC, 1.0g benzoic acid, 2.0g trehalose and 3.0g maltose respectively; Being scattered in the chloroform the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing, lecithin E-80 and synthetic phospholipid SOPC fully, dissolving obtains solution A; Being scattered in the chloroform clarithromycin that takes by weighing and benzoic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, secure ph are 7.5 PBS, the freeze drying protectant that takes by weighing in the step 1 is dissolved in the PBS and with PBS is preheated to 50 ℃ then;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back and under heating condition, continue stirring chloroform in solution and volatilizees fully, obtain liposome turbid liquor; The temperature of said heating is 50 ℃;
Step 4, to place Ultrasound Instrument to carry out liposome turbid liquor described in the step 3 ultrasonic, then the liposome turbid liquor behind homogenizing sterilized with 0.22 μ m filter membrane ultrafiltration, and packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome; Said ultransonic temperature is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
Present embodiment is identical with embodiment 9, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid be among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC a kind of, more than two kinds or four kinds; Perhaps be three kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Perhaps being among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC the two kinds mixture with the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, perhaps is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of a kind of and injection stage and the mixture of lecithin E-80 among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, synthetic phospholipid POPC and the synthetic phospholipid MOPC; Said fatty acid is adipic acid or caproic acid, perhaps is two or three in caproic acid, adipic acid and the benzoic acid; Said freeze drying protectant is a kind of in sucrose, glucose, trehalose, maltose and the mannitol or more than three kinds; Perhaps being two kinds in sucrose, glucose, maltose and the mannitol, perhaps is the mixture of a kind of and trehalose in sucrose, glucose and the mannitol; Said organic solvent is dehydrated alcohol, absolute methanol, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Embodiment 11
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 0.05g, and steroid material 2.0g, phosphatidase 10 .2g, fatty acid 0.01g, surplus is a PBS; Said freeze drying protectant is sucrose, glucose, trehalose, maltose and mannitol (waiting mass ratio); The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 10g freeze drying protectant; Said steroid material is cholesterol and cholesterol sodium sulfate salt (waiting mass ratio); Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage; Said fatty acid is an adipic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 0.05g clarithromycin, 1.0g cholesterol, 1.0g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 0.2g injection stage, 0.01g adipic acid, 2.0g sucrose, 2.0g glucose, 2.0g trehalose, 2.0g maltose and 2.0g mannitol respectively; Being scattered in the absolute methanol the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing, cholesterol and cholesterol sodium sulfate salt fully, dissolving obtains solution A; Being scattered in the absolute methanol clarithromycin that takes by weighing and adipic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, secure ph are 7.0 PBS, the sucrose that takes by weighing in the step 1, glucose, trehalose, maltose and mannitol are dissolved in the PBS and with PBS are preheated to 80 ℃ then;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back and under heating condition, continue stirring methanol in solution and volatilizees fully, obtain liposome turbid liquor; The temperature of said heating is 80 ℃;
Step 4, place high pressure homogenizer to carry out homogenizing liposome turbid liquor described in the step 3, then with the sterilization of the liposome turbid liquor ultrafiltration behind homogenizing, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome; The control homogenizing temperature is 5 ℃～40 ℃ in the said homogenizing process, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times.
Embodiment 12
Present embodiment is identical with embodiment 11, and wherein difference is: said steroid material is cholesterol or cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said freeze drying protectant is four kinds at the most in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is dehydrated alcohol, chloroform, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Embodiment 13
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 1.5g, and steroid material 1.5g, phosphatidase 15 .4g, fatty acid 0.75g, surplus is a PBS; Said freeze drying protectant is a sucrose; The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 5g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage; Said fatty acid is a caproic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 1.5g clarithromycin, 1.5g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 5.4g injection stage and 0.75g caproic acid respectively; Being scattered in the dehydrated alcohol the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing and cholesterol sodium sulfate salt fully, dissolving obtains solution A; Being scattered in the dehydrated alcohol clarithromycin that takes by weighing and caproic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, secure ph are 7.5 PBS, then PBS are preheated to 50 ℃;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back reduction vaporization and remove ethanol, make liposome turbid liquor;
Step 4, to place Ultrasound Instrument to carry out liposome turbid liquor described in the step 3 ultrasonic, obtains the 100mL liposome solutions; Said ultransonic temperature is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min;
Step 5, take by weighing 5g sucrose and with the sucrose dissolved that takes by weighing in liposome solutions described in the step 4, with 0.22 μ m filter membrane ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Embodiment 14
Present embodiment is identical with embodiment 13, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said fatty acid is adipic acid or benzoic acid, perhaps is two or three in caproic acid, adipic acid and the benzoic acid; Said freeze drying protectant is glucose, trehalose, maltose or mannitol, perhaps is at least two kinds in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is absolute methanol, chloroform, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Embodiment 15
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clamycin 2 .0g, and steroid material 2.0g, phosphatidase 11 0g, fatty acid 0.01g, surplus is a PBS; Said freeze drying protectant is a maltose; The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 20g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage; Said fatty acid is a benzoic acid.
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 2.0g clarithromycin, 2.0g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 10g injection stage and 0.01g benzoic acid respectively; Being scattered in absolute methanol and the dehydrated alcohol (volume ratio 1: 2) the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing and cholesterol sodium sulfate salt fully, dissolving obtains solution A; Being scattered in absolute methanol and the dehydrated alcohol (volume ratio 1: 2) clarithromycin that takes by weighing and benzoic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, secure ph are 6.5 PBS, then PBS are preheated to 80 ℃;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back reduction vaporization and remove methanol and ethanol, make liposome turbid liquor;
Step 4, to place Ultrasound Instrument to carry out liposome turbid liquor described in the step 3 ultrasonic, obtains liposome solutions; Said ultransonic temperature is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min;
Step 5, take by weighing 20g maltose and the maltose that takes by weighing is dissolved in described in the step 4 in the liposome solutions, with 0.22 μ m filter membrane ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Embodiment 16
Present embodiment is identical with embodiment 15, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said freeze drying protectant is sucrose, glucose, trehalose or mannitol, perhaps is at least two kinds in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is a kind of in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether or more than three kinds; Perhaps being two kinds in absolute methanol, chloroform, dichloromethane and the ether, perhaps is the mixture of a kind of and dehydrated alcohol in chloroform, dichloromethane and the ether.
Embodiment 17
The clarithromycin freeze-dried lipidosome of present embodiment mixes the back lyophilizing by liposome solutions and processes with freeze drying protectant; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 0.05g, and steroid material 0.01g, phosphatidase 10 .2g, fatty acid 1.0g, surplus is a PBS; Said freeze drying protectant is a glucose; The consumption of said freeze drying protectant is that every 100mL liposome solutions is used the 10g freeze drying protectant; Said steroid material is the cholesterol sodium sulfate salt; Said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage; Said fatty acid is caproic acid, adipic acid and benzoic acid (mass ratio 1: 2: 2).
The method for preparing of the clarithromycin freeze-dried lipidosome of present embodiment may further comprise the steps:
Step 1, take by weighing 0.05g clarithromycin, 0.01g cholesterol sodium sulfate salt, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of 0.2g injection stage, 0.2g caproic acid, 0.4g adipic acid and 0.4g benzoic acid respectively; Being scattered in the chloroform the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of the injection stage that takes by weighing fully, dissolving obtains solution A; Being scattered in the chloroform clarithromycin that takes by weighing, caproic acid, adipic acid and benzoic acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase;
Step 2, secure ph are 7.0 PBS, then PBS are preheated to 65 ℃;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back reduction vaporization and remove chloroform, make liposome turbid liquor;
Step 4, place high pressure homogenizer to carry out homogenizing liposome turbid liquor described in the step 3, obtain liposome solutions; The control homogenizing temperature is 5 ℃～40 ℃ in the said homogenizing process, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times;
Step 5, take by weighing the 10g glucose and the glucose that takes by weighing is dissolved in described in the step 4 in the liposome solutions, with 0.22 μ m filter membrane ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
Embodiment 18
Present embodiment is identical with embodiment 17, and wherein difference is: said steroid material is cholesterol or is the mixture of cholesterol and cholesterol sodium sulfate salt; Said phospholipid is injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC or synthetic phospholipid MOPC, perhaps is at least two kinds among injection stage Ovum Gallus domesticus Flavus lecithin PL-100M, the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, lecithin E-80, synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC; Said fatty acid is caproic acid, adipic acid or benzoic acid; Said freeze drying protectant is sucrose, trehalose, maltose or mannitol, perhaps is at least two kinds in sucrose, glucose, trehalose, maltose and the mannitol; Said organic solvent is absolute methanol, dehydrated alcohol, dichloromethane or ether, perhaps is at least two kinds in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether.
Clarithromycin freeze-dried lipidosome of the present invention is carried out following detection test:
One, the outward appearance of clarithromycin freeze-dried lipidosome detects and the release in vitro detection
Clarithromycin freeze-dried lipidosome of the present invention is carried out perusal, and the freeze-dried lipidosome form is for for spongy agglomerate, and its outward appearance is not subsided, not shrinkage, color even, fine and close, any surface finish of hole, and volume keeps the volume before the lyophilizing; Clarithromycin freeze-dried lipidosome of the present invention is redissolved, and the liposome after the redissolution is translucent milky.
Fig. 1 is the preceding transmission electron microscope photo of clarithromycin freeze-dried lipidosome lyophilizing of the embodiment of the invention 1 preparation, and Fig. 2 is the projection electromicroscopic photograph before the clarithromycin freeze-dried lipidosome of the embodiment of the invention 1 preparation redissolves.Can know that by figure the lipidosome freeze-dried front and back of clarithromycin are multiple structure, the profile rounding.
Fig. 3 be before the clarithromycin freeze-dried lipidosome lyophilizing of the embodiment of the invention 1 preparation with redissolve after the release in vitro curve, two curves are quite similar, show that clarithromycin liposome its state before and after lyophilizing does not take place than about-face.
Two, the particle size distribution of clarithromycin freeze-dried lipidosome is investigated
After sample is diluted to suitable concentration respectively after sample and the redissolution before the water for injection of 0.22 μ m microporous filter membrane ultrafiltration is with the clarithromycin freeze-dried lipidosome lyophilizing of the embodiment of the invention 1, embodiment 7 and embodiment 13 preparations, put Nicomp immediately
TMIn 380 particle size analyzers, measure particle size distribution.Compare with the particle size distribution of liposome solutions before the lyophilizing, the result sees table 1.
Table 1 particle size distribution measuring result
| Embodiment | PSD (nm) before the lyophilizing | PSD after the redissolution (nm) |
| 1 | 136.9±57 | 190.9±76 |
| 7 | 140±59 | 196±77 |
| 13 | 142±58 | 192±78 |
Can find out that from table 1 particle size distribution of clarithromycin freeze-dried lipidosome before and after lyophilizing of embodiment 1, embodiment 7 and embodiment 13 preparations changes very little, all below 200nm, satisfies the used for intravenous injection requirement.
Three, the zeta potential measurement of clarithromycin freeze-dried lipidosome
Adopt Nicomp
TM380 carry out the mensuration of zeta current potential.After sample is diluted to suitable concentration respectively before the clarithromycin freeze-dried lipidosome lyophilizing that the embodiment of the invention 1, embodiment 7 and embodiment 13 is prepared through the water for injection of 0.22 μ m microporous filter membrane ultrafiltration, put into Nicomp immediately
TMIn 380 sample cells, measure the zeta current potential, zeta potential measurement result sees table 2 before the three lot sample article lyophilizing.
Table 2zeta potential measurement result
| Embodiment | 1 | 7 | 13 |
| Zeta potential (mv) | -21.32 | -22.52 | -23.16 |
Can find out from table 2, the zeta current potential of the clarithromycin freeze-dried lipidosome of the present invention preparation is-23.16mv～-21.32mv, satisfy stable particle liquid system to the zeta current potential generally be controlled at-20mv～-requirement in the 45mv scope.
Four, the clarithromycin assay of clarithromycin freeze-dried lipidosome and the investigation of envelop rate
Adopt the methanol breakdown of emulsion, with the content of clarithromycin in the clarithromycin freeze-dried lipidosome of the HPLC mensuration embodiment of the invention 1, embodiment 7 and embodiment 13 preparations.Method with external microdialysis is measured envelop rate, and the result is as shown in table 3.
Table 3 clarithromycin content and entrapment efficiency determination result
| Embodiment | 1 | 7 | 13 |
| Content (%) | 100.0 | 98.85 | 99.16 |
| Envelop rate (%) | 95.56 | 96.64 | 94.53 |
Can find out that from table 3 content of the clarithromycin freeze-dried lipidosome of the present invention preparation is all more than 98%, envelop rate all about 95%, steady quality.
Five, the investigation of the long-time stability of clarithromycin freeze-dried lipidosome
With the clarithromycin freeze-dried lipidosome of embodiment 1, embodiment 7 and embodiment 13 preparations be positioned over 25 ℃ ± 2 ℃ with 4 ± 2 ℃ of conditions under store 3 months, respectively at the 1st, 2, take a sample and carry out the inspection and the mensuration of each item physicochemical property March, the result sees table 4 and table 5.
Table 4 clarithromycin freeze-dried lipidosome long-term stable experiment result (25 ℃ ± 2 ℃)
Table 5 clarithromycin freeze-dried lipidosome long-term stable experiment result (4 ± 2 ℃)
Can find out from table 4 and table 5; 25 ℃ ± 2 ℃ and 4 ± 2 ℃ of condition held 3 months; Indexs such as the outward appearance of clarithromycin freeze-dried lipidosome, pH value, medicament contg, envelop rate and redissolution back particle diameter all do not have to take place significant the variation, still meet the intravenous administration requirement.
Six, the zoopery of clarithromycin freeze-dried lipidosome
6.1 mice scratching test
Divide 3 groups at random with experiment mice: negative control group, positive controls and to be checked group; Every group of 6 mices; With the hypodermic mode administration of mouse back, negative control group injecting normal saline, positive controls injection clarithromycin solution; To be checked group of injection is through the water-reducible clarithromycin freeze-dried lipidosome of injection; Injection volume is 0.15mL, the time first and the total degree of every mice scratching medicine-feeding part in the record injection back 15min, and wherein the clarithromycin concentration of the clarithromycin freeze-dried lipidosome after clarithromycin solution and the dilution is 4mg/mL.The result adds up with the T check, sees table 6.
Table 6 mice scratching result of the test (in the 15min)
| Group | Incidence rate | Time (s) first | Number of times | Suppression ratio | n | P |
| Negative control group | 83％(5/6) | 315 | 3.5±3.5 | 94％ | 6 | ＜0.01 |
| |
100％(6/6) | 52 | 56.7±21.7 | 0％ | 6 | |
| To be checked group | 83％(5/6) | 128 | 9.7±10.8 | 83％ | 6 | ＜0.01 |
The result shows that in 95% confidence interval, the clarithromycin freeze-dried lipidosome is compared with solution and had significant difference, and the suppression ratio of pain is reached more than 80%, has obviously reduced pain and zest.To the suppression ratio of pain is that total degree with every mice scratching medicine-feeding part in the 15min is an index, is to get with reference to calculating with the positive controls.
6.2 rat licks sufficient test
With experimental rat (body weight 80g～120g) be divided into 3 groups at random: negative control group, positive controls and to be checked group; Every group 6; With the mode administration of the right back foot injection of rat, negative control group injecting normal saline, positive controls injection clarithromycin solution; To be checked group of injection is through the water-reducible clarithromycin freeze-dried lipidosome of injection; Injection volume is 0.1mL, and every rat licks the sufficient time first and always licks sufficient number of times in the record injection back 15min, and wherein the clarithromycin concentration of the clarithromycin freeze-dried lipidosome after clarithromycin solution and the dilution is 4mg/mL.The result adds up with the T check, sees table 7.
Table 7 rat licks sufficient result of the test (in the 15min)
| Group | Incidence rate | Time (s) first | Number of times | Suppression ratio | n | P |
| Negative control group | 17％(1/6) | --- | 0.5±1.2 | 98.5％ | 6 | ＜0.01 |
| |
100％(6/6) | 243 | 33.8±11.3 | 0％ | 6 | |
| To be checked group | 83％(5/6) | 302 | 6.7±4.8 | 80.3％ | 6 | ＜0.01 |
The result shows that in 95% confidence interval, the clarithromycin freeze-dried lipidosome is compared with solution and had significant difference, and the suppression ratio of pain is reached more than 80%, has obviously reduced pain and zest.To the suppression ratio of pain is that to lick sufficient total degree with every rat in the 15min be index, is to get with reference to calculating with the positive controls.
6.3 intravenous rabbit irritation test
Experimental rabbit (about body weight 3kg) is divided into 2 groups at random, i.e. negative control group and to be checked group, 3 every group; Each group all adopts the administration of ear vein injection system, and wherein negative control group is injected clarithromycin solution, the to be checked group of water-reducible clarithromycin freeze-dried lipidosome of injection injection; Clarithromycin concentration in two groups is 2mg/mL; Every rabbit administration 5mL, injection speed is 2mL/min, successive administration 3 days.Observe whether variable color of medicine-feeding part in the administration process, erythema and swelling occur.Put to death rabbit after the last administration; Rabbit ear vein tissue is processed pathological section (seeing Fig. 4 a～Fig. 4 d); From Fig. 4 a～Fig. 4 d, can find out; There is tangible inflammatory cell infiltration in negative control group, compares with negative control group for to be checked group to have significant difference, and to be checked group can obviously be reduced blood vessel irritation.
In sum, clarithromycin freeze-dried lipidosome of the present invention, its drug loading reaches as high as 20mg/mL, and the particle diameter after lyophilizing is redissolved is below 200nm, and the pH value after the lyophilizing, envelop rate, content all keep stable; Compare with solution; Can reduce medicine-feeding part pain and blood vessel irritation that clarithromycin causes significantly; Its each item index has all reached the requirement of state food drug administration about the new drug research exploitation, for the suitability for industrialized production and the clinical practice of clarithromycin Liposomal formulation are laid a good foundation.
The above; It only is preferred embodiment of the present invention; Be not that the present invention is done any restriction, every technical spirit changes any simple modification, change and the equivalent structure that above embodiment did according to the present invention, all still belongs in the protection domain of technical scheme of the present invention.
Claims (10)
1. a clarithromycin freeze-dried lipidosome is characterized in that, mixes the back lyophilizing with freeze drying protectant by liposome solutions and processes, and adds 5g～20g freeze drying protectant in every 100mL liposome solutions; The said liposome solutions of 100mL is processed by following raw material: clarithromycin 0.05g～2.0g, and steroid material 0.01g～2.0g, phosphatidase 10 .2g～10g, fatty acid 0.01g～1.0g, surplus is a PBS; Said freeze drying protectant is one or more in sucrose, glucose, trehalose, maltose and the mannitol; Said steroid material is cholesterol and/or cholesterol sodium sulfate salt; Said fatty acid is one or more in caproic acid, adipic acid and the benzoic acid; Said phospholipid is natural phospholipid and/or synthetic phospholipid.
2. a kind of clarithromycin freeze-dried lipidosome according to claim 1 is characterized in that the said liposome solutions of 100mL is processed by following raw material: clarithromycin 1.5g; Steroid material 1.5g; Phosphatidase 15 .4g, fatty acid 0.75g, surplus is a PBS.
3. a kind of clarithromycin freeze-dried lipidosome according to claim 1; It is characterized in that; Said natural phospholipid is one or more among the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, injection stage Ovum Gallus domesticus Flavus lecithin PL-100M and the lecithin E-80, and said synthetic phospholipid is one or more among synthetic phospholipid POPC, synthetic phospholipid SOPC and the synthetic phospholipid MOPC.
4. according to the described a kind of clarithromycin freeze-dried lipidosome of claim 1,2 or 3, it is characterized in that said steroid material is the cholesterol sodium sulfate salt, said phospholipid is the high-purity Ovum Gallus domesticus Flavus lecithin PC-98T of injection stage, and said fatty acid is a caproic acid.
5. method for preparing clarithromycin freeze-dried lipidosome according to claim 1 or claim 2 is characterized in that this method may further comprise the steps:
Step 1, take by weighing clarithromycin, steroid material, phospholipid and fatty acid respectively; Being scattered in the organic solvent phospholipid that takes by weighing and steroid material fully, dissolving obtains solution A; Being scattered in the organic solvent clarithromycin that takes by weighing and fatty acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase; Said organic solvent is one or more in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether;
Step 2, the rotary evaporation of oil phase described in the step 1 is removed the organic solvent in the oil phase, dry up with nitrogen then, obtain thin film;
Step 3, secure ph are 6.5～7.5 PBS, then PBS are preheated to 50 ℃～80 ℃;
Step 4, with the PBS after preheating in the step 3 with the complete aquation of thin film described in the step 2, obtain colostrum;
Step 5, colostrum described in the step 4 is used pH value is 6.5～7.5 PBS standardize solution, places high pressure homogenizer to carry out homogenizing then or to place Ultrasound Instrument to carry out ultrasonic, obtains liposome solutions;
Step 6, take by weighing freeze drying protectant and the freeze drying protectant that takes by weighing is dissolved in described in the step 5 in the liposome solutions, the ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
6. method according to claim 5 is characterized in that, controlling homogenizing temperature in the homogenizing process described in the step 5 is 5 ℃～40 ℃, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times; Ultransonic temperature described in the step 5 is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
7. method for preparing clarithromycin freeze-dried lipidosome according to claim 1 or claim 2 is characterized in that this method may further comprise the steps:
Step 1, take by weighing clarithromycin, steroid material, phospholipid, fatty acid and freeze drying protectant respectively; Being scattered in the organic solvent phospholipid that takes by weighing and steroid material fully, dissolving obtains solution A; Being scattered in the organic solvent clarithromycin that takes by weighing and fatty acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase; Said organic solvent is one or more in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether;
Step 2, secure ph are 6.5～7.5 PBS, the freeze drying protectant that takes by weighing in the step 1 is dissolved in the PBS and with PBS is preheated to 50 ℃～80 ℃ then;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back and under heating condition, continue stirring organic solvent in solution and volatilizees fully, obtain liposome turbid liquor; The temperature of said heating is 50 ℃～80 ℃;
Step 4, place high pressure homogenizer to carry out homogenizing or to place Ultrasound Instrument to carry out ultrasonic liposome turbid liquor described in the step 3; To sterilize through the liposome turbid liquor ultrafiltration behind the homogenizing or after ultrasonic then; Packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
8. method according to claim 7 is characterized in that, controlling homogenizing temperature in the homogenizing process described in the step 4 is 5 ℃～40 ℃, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times; Ultransonic temperature described in the step 4 is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
9. method for preparing clarithromycin freeze-dried lipidosome according to claim 1 or claim 2 is characterized in that this method may further comprise the steps:
Step 1, take by weighing clarithromycin, steroid material, phospholipid and fatty acid respectively; Being scattered in the organic solvent phospholipid that takes by weighing and steroid material fully, dissolving obtains solution A; Being scattered in the organic solvent clarithromycin that takes by weighing and fatty acid all, dissolving obtains solution B; With stirring after solution A and the solution B mixing, obtain oil phase; Said organic solvent is one or more in absolute methanol, dehydrated alcohol, chloroform, dichloromethane and the ether;
Step 2, secure ph are 6.5～7.5 PBS, then PBS are preheated to 50 ℃～80 ℃;
Step 3, under stirring condition, oil phase described in the step 1 is dropped in the step 2 in the PBS after preheating, drip and finish the back reduction vaporization and remove organic solvent, make liposome turbid liquor;
Step 4, place high pressure homogenizer to carry out homogenizing or to place Ultrasound Instrument to carry out ultrasonic, obtain liposome solutions liposome turbid liquor described in the step 3;
Step 5, take by weighing freeze drying protectant and the freeze drying protectant that takes by weighing is dissolved in described in the step 4 in the liposome solutions, the ultrafiltration sterilization, packing, lyophilizing obtains the clarithromycin freeze-dried lipidosome.
10. method according to claim 9 is characterized in that, controlling homogenizing temperature in the homogenizing process described in the step 4 is 5 ℃～40 ℃, and homogenization pressure is 30MPa～70MPa, and the homogenizing cycle-index is 2～10 times; Ultransonic temperature described in the step 4 is 5 ℃～40 ℃, and ultrasonic power is 200W～600W, and ultrasonic time is 2min～20min.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104173288A (en) * | 2014-09-03 | 2014-12-03 | 沈阳药科大学 | Clarithromycin ion pair lipidosome injection and preparation method thereof |
| CN111759811A (en) * | 2020-08-06 | 2020-10-13 | 淄博职业学院 | Non-irritant clarithromycin freeze-dried powder and preparation method thereof |
| CN113876696A (en) * | 2021-11-16 | 2022-01-04 | 中国人民解放军海军军医大学第一附属医院 | Clarithromycin hydrogel capable of being locally injected, preparation method and application thereof |
-
2012
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Non-Patent Citations (1)
| Title |
|---|
| 李海刚等: "注射用克拉霉素脂质体的制备及其包封率的测定", 《中国抗生素杂志》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104173288A (en) * | 2014-09-03 | 2014-12-03 | 沈阳药科大学 | Clarithromycin ion pair lipidosome injection and preparation method thereof |
| CN104173288B (en) * | 2014-09-03 | 2017-01-18 | 沈阳药科大学 | Clarithromycin ion pair lipidosome injection and preparation method thereof |
| CN111759811A (en) * | 2020-08-06 | 2020-10-13 | 淄博职业学院 | Non-irritant clarithromycin freeze-dried powder and preparation method thereof |
| CN113876696A (en) * | 2021-11-16 | 2022-01-04 | 中国人民解放军海军军医大学第一附属医院 | Clarithromycin hydrogel capable of being locally injected, preparation method and application thereof |
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