CN101637450B - Levocarnitine liposomes injection and its preparation method - Google Patents
Levocarnitine liposomes injection and its preparation method Download PDFInfo
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- CN101637450B CN101637450B CN2009100177689A CN200910017768A CN101637450B CN 101637450 B CN101637450 B CN 101637450B CN 2009100177689 A CN2009100177689 A CN 2009100177689A CN 200910017768 A CN200910017768 A CN 200910017768A CN 101637450 B CN101637450 B CN 101637450B
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Abstract
The invention discloses levocarnitine liposomes injection, which is characterized by comprising the following active ingredients in parts by weight: 1 part of levocarnitine, 3-15 parts of soybean lecithin, 0.4-7.5 parts of cholesterol and 0.02-1 part of antioxidant, and a pharmaceutically acceptable carrier, wherein, the antioxidant is one or more of L-cysteine, thiourea, vitamin E and butylated hydroxyanisole and is most preferably the vitamin E. The invention also discloses a preferable preparation method of the levocarnitine liposomes injection, namely ammonium sulphate pH gradient method. The invention provides levocarnitine liposomes injection which has excellent stability, high entrapment rate and low leakage rate in the process of long-term storage. The acute toxicity tests, unusual toxicity tests and heat source tests adopting the levocarnitine liposomes injection all conform to the specifications and are applicable to industrialized production.
Description
Technical field
The present invention relates to a kind of Liposomal formulation, be specifically related to levocarnitine liposomes injection and method for making thereof, belong to medical technical field.
Background technology
Levocarnitine claims L-carnitine again, and chemical name is: (R)-3-carboxyl-2-hydroxy-n, N, N-trimethyl-1-third ammonium hydroxide, inner salt, molecular formula: C
7H
15NO
3, molecular weight: 161.20, structural formula is:
Being a kind of special acid that extensively is present in the when injected organism tissue, is the essential nutrient substance of the mankind and animal, is the biostearin nutrient, is equivalent to vitamin B complex.Discover that levocarnitine is a kind of and the relevant chemical compound of animal body fat acid metabolic, its basic function is that auxiliary long-chain fatty acid penetrates the carrier that mitochondrial inner membrane carries out beta oxidation.Levocarnitine is a kind of essential nutrients, and the metabolism of its function and organs of living beings and tissue is closely related.People also find, treat some disease with exogenous levocarnitine, can improve clinical symptoms.Engel in 1973 reports the first levocarnitine defective disease and begins to treat with levocarnitine that just clear and definite levocarnitine was a kind of very important nutrient up to 1984.
As far back as the sixties, D, the L-carnitine has been used for clinical as the stomach medicine, be mainly used in aid digestion, appetite stimulator.Found afterwards to have only that the L-carnitine works, the D-carnitine has antagonism, therefore substituted by L-carnitine gradually.The clinical research of L-carnitine has been reported the first carntine deficiency since Engel in 1973.L-carnitine can be widely used in the treatment of various ischemic heart desease.For the treatment of general ischemic heart desease, the consumption of L-carnitine is 1-3g every day.For acute myocardial infarction, acute heart failure, need 3-6g every day.The PRELIMINARY RESULTS of clinical proof patients with coronary heart disease levocarnitine on probation also is positive.For patient with angina pectoris, levocarnitine can improve kinergety, reduces ST section decline degree, reduces the use of angina pectoris attacks frequency and nitroglycerin.In addition, levocarnitine all has better effect for shock, acute and chronic cardiac insufficiency, myocarditis, arrhythmia.The renal failure patient that nephropathy and diabetes cause is because secular hemodialysis causes carnitine to lack, and it is essential that these patients replenish carnitine.Carnitine can be used for hepatopath's auxiliary treatment, and its mechanism is the ammonia concentration that carnitine can reduce the hepatopath, reduces portal hypertension, reduces esters and accumulates in liver, all right blood fat reducing of levocarnitine, fat-reducing, treatment angiopathy.Its another one important function is to be used to perform the operation or the gastrointestinal fistula malignant tumor is accepted patient and the neonate that total parenteral nutrition is supported.
Along with the continuous development of science, levocarnitine has vast market prospect aspect nourishing healthy, defatting beauty, therapeutic treatment and the feedstuff interpolation new achievement in research being arranged.Principal agent is used for former and secondary levocarnitine shortage at present.Shock, acute and chronic cardiac insufficiency, ischemic cardiomyopathy, myocarditis, arrhythmia, angina pectoris, myocardial infarction; Acute, chronic hepatitis, liver cirrhosis, the auxiliary treatment of chronic hepatic insufficiency; Total parenteral nutrition and wound.Also be used to reduce the toxicity of antitumor drug in addition to cardiac toxicity and minimizing valproic acid; The neonate malnutrition, the bad auxiliary treatment of uterine contraction in puerperal.
At present existing levocarnitine injection and lyophilized injectable powder listing, but product quality is very unstable, can not reach the prescription of effect duration.
A kind of levocarnitine preparation of drug combination method is disclosed at Chinese patent CN101011373A, by dissolving, suspendible, emulsive means the active substance in the composite formula is mixed with into fluid composition with corresponding adjuvant, further is prepared into soft capsule and Emulsion.Disclose a kind of preparation method of levocarnitine lyophilized injectable powder among the Chinese patent CN1864673A, adopted hydrochloric acid to regulate pH value, mannitol is as excipient, and lyophilization makes levo-carnitine for injection powder pin.In the above-mentioned described patent, just the dissolving of levocarnitine is studied, the preparation stability of preparation is relatively poor, levocarnitine goes bad medium-term and long-term placement of Emulsion, has influenced drug quality, and freeze-dried powder then solubility is relatively poor, can not obtain clear and bright solution, off quality.
Therefore, existing further needs, so that better levocarnitine preparation of stability to be provided.The inventor has unexpectedly found a kind of novel form liposome of targeting drug delivery system is applied to solve the stability problem of levocarnitine in the levocarnitine ejection preparation, thereby has finished the present invention through long-term conscientious research.
Liposome (liposomes) is dispersed in phospholipid by Britain scholar Bangham and Standish at first and finds when carrying out electron microscopic observation in the water.Phospholipid is dispersed in and forms multilamelar vesicles, every layer of bilayer that is lipid in the water naturally; Separated by water between vesicle central authorities and each layer, the about 4nm of bilayer thickness, the bimolecular folliculus with this similar biofilm structure became liposome afterwards.
Liposome research is that liposome is meant that the earliest the natural grease compounds is suspended in the vesicle with double seal structure that forms in the water, now also can be prepared by the phosphatide cpd of synthetic when previous very active field.People such as late 1960s Rahman at first use liposome as pharmaceutical carrier, in recent years, continuous progress along with biotechnology, liposome preparation technology is progressively perfect, the liposome mechanism of action is further illustrated, liposome is fit to vivo degradation, avirulence and non-immunogenicity in addition, and particularly great number tested data proof liposome can improve the Drug therapy index, reduces drug toxicity and reduce drug side effect as pharmaceutical carrier, and reduces advantage such as drug dose.Yet the liposome ubiquity is assembled easily, is merged, and causes entrapped drug to leak, and the instability of liposome has become the subject matter of its pharmaceuticals industry, therefore needs to optimize the liposome prescription, to obtain the liposome of excellent in stability.
In fact, the technical staff that pharmaceutical field has universal experience knows clearly all difficulties that face aspect preparation liposome-medicinal composition, all these existing absolutely not theories can be expected will obtain solution meeting stable medicinal liposome needs and overcome many difficult problems.
Summary of the invention
Perishable at the long-term placement of present levocarnitine solution, the problem of injectable powder solubility difference, the object of the present invention is to provide a kind of levocarnitine liposomes injection, it wraps up with liposome by the pH value gradient method, thereby has solved the problem of above-mentioned existence.
Liposomal formulation can be used the methods known in the art preparation, preferably adopts ammonium sulfate pH gradient method.
Phospholipid can be selected natural phospholipid and synthetic phospholipid for use usually, and described natural phospholipid is one or more in Ovum Gallus domesticus Flavus lecithin, hydrogenation egg yolk lecithin, EPG, egg yolk lecithin acyl serine, egg yolk lecithin acyl inositol, soybean lecithin, hydrogenated soya phosphatide, soybean phospholipid acyl glycerol, soy phosphatidylserine, the soybean phospholipid acyl inositol; Described synthetic phospholipid is one or more in dioleoyl phospholipid phatidylcholine, distearyl acid phosphatidylcholine, dipalmitoyl phosphatidyl choline, dimyristoyl phosphatidyl choline, two Laurel phosphatidyl cholines, DOPG, distearyl acid phosphatidyl glycerol, two palmityl phosphatidyl glycerols, GLYCEROL,DIMYRISTOYL PHOSPHATIDYL, the two lauroyl phosphatidyl glycerols.The present invention confirms that through trial test phospholipid is preferably soybean lecithin.
The inventor is that membrane material prepares levocarnitine liposomes adopting general lipidosome injection phospholipid and cholesterol commonly used, the levocarnitine liposomes of finding to obtain like this is under 40 ℃ of high temperature, relative humidity 75% ± 5% accelerated test, the stability of levocarnitine liposomes is not good, and percolation ratio is up to more than 30%.
Bound by theory not, the inventor finds to add part antioxidant unexpectedly and has beyond thought effect, thereby has obtained the levocarnitine liposomes of excellent in stability.Described antioxidant is selected from one or more in L-cysteine, thiourea, vitamin E, the butylated hydroxyarisol, preferred vitamin E.
The technical scheme that the present invention solves is as follows:
A kind of levocarnitine liposomes injection, form by active component levocarnitine, soybean lecithin, cholesterol, antioxidant and pharmaceutically acceptable carrier, wherein the ratio of weight and number of each component is: 1 part of levocarnitine, soybean lecithin 3-15 part, cholesterol 0.4-7.5 part, antioxidant 0.02-1 part.
As the present invention's one preferred embodiment, the ratio of weight and number of described each component of levocarnitine liposomes injection is: 1 part of levocarnitine, soybean lecithin 5-9 part, cholesterol 0.8-6 part, antioxidant 0.1-0.6 part.
As the present invention's preferred embodiment further, the ratio of weight and number of described each component of levocarnitine liposomes injection is: 1 part of levocarnitine, 7.2 parts of soybean lecithins, 2.8 parts in cholesterol, 0.36 part in antioxidant.
Wherein, antioxidant in the said components is selected from sodium sulfite, sodium pyrosulfite, L-cysteine, thiourea, formaldehyde and closes in sodium sulfite, vitamin E, ascorbyl palmitate, the butylated hydroxyarisol one or more, in preferred L-cysteine, thiourea, vitamin E, the butylated hydroxyarisol one or more, more preferably vitamin E.
Above-mentioned described levocarnitine liposomes injection, pharmaceutically acceptable carrier can comprise osmotic pressure regulator, buffer solution, proppant.
Wherein, osmotic pressure regulator is the solution that adjusting medicinal liquid and blood plasma have identical osmotic pressure, can not cause any ANOMALOUS VARIATIONS of hemogram.Osmotic pressure regulator commonly used has sodium chloride, glucose, glycerol, mannitol, sorbitol, potassium chloride, sodium lactate, amino acids, dextran, gelatin, polyvidone class, starch derivatives etc., osmotic pressure regulator among the present invention is selected from one or more in sodium chloride, glucose, mannitol, sorbitol, glycerol, the xylitol etc., preferred sodium chloride, glucose and glycerol, more preferably sodium chloride; The consumption of Osmolyte regulator is the 1.08-10 weight portion, and concentration is 0.9%-10% (g/ml).
Wherein, buffer solution is used for regulating the pH value of liposome solutions to the scope of standard-required, can be selected from the phosphate buffer, citrate buffer, carbonate buffer solution, borate buffer solution, acetate buffer of pH value 6.0-6.5 one or more, preferably phosphoric acid sodium dihydrogen-sodium hydrogen phosphate buffer, acetic acid-sodium-acetate buffer, citric acid-sodium citrate buffer, more preferably sodium dihydrogen phosphate-sodium hydrogen phosphate buffer.
Wherein, the parts by weight of proppant are 4-10 part, are selected from one or more of mannitol, glucose, trehalose, lactose, sucrose, sorbitol, sodium chloride, glycine, preferred mannitol and trehalose, more preferably trehalose.
The technical scheme that the present invention solves also comprises:
A kind of preparation method of levocarnitine liposomes injection, step comprises:
(1) soybean lecithin, cholesterol and the antioxidant with recipe quantity is dissolved in the organic solvent, slowly injects ammonium sulfate, and heated and stirred is steamed and removed organic solvent, and is ultrasonic, gets blank liposome;
(2) blank liposome is placed bag filter, seal, bag filter is placed osmotic pressure solution, the ammonium sulfate among the weeding of grease plastid foreign minister;
(3) the good 60 ℃ of insulations of blank liposome of will dialysing under agitation slowly add the aqueous solution of levocarnitine, continue insulation 20-30min, add buffer solution and osmotic pressure regulator solution, and mixing promptly gets the levocarnitine liposomes suspension;
(4) proppant of recipe quantity is water-soluble, add in the above-mentioned suspension, stirring and evenly mixing filters, and lyophilization gets the levocarnitine liposomes lyophilized preparation; Perhaps, above-mentioned suspension is filtered, packing, sterilization gets the levocarnitine liposomes injection.
In the above-mentioned described preparation method, organic solvent is selected from one or more in chloroform, ethanol, methanol, the tert-butyl alcohol, n-butyl alcohol, isopropyl alcohol, acetone, ether, benzyl alcohol, the normal hexane, preferred tertiary butanols, isopropyl alcohol, ethanol, more preferably isopropyl alcohol.The amount of organic solvent is selected according to the amount of the soybean lecithin, cholesterol and the antioxidant that add, to dissolve the requirement that mentioned component is a minimum flow fully, preferably based on 1 of soybean lecithin, cholesterol, antioxidant three gross weight meter: the organic solvent of 5-10 (g/ml) volume.
In the above-mentioned described preparation method, the concentration of described ammonium sulfate is 0.01-0.1mol/L, and the amount of its consumption and organic solvent is identical.
In above-mentioned described preparation method, the amount of dissolving levocarnitine water gets final product for it is dissolved fully, is preferably the water based on 1 of levocarnitine weight meter: 20-50 (g/ml) volume; The amount of dissolving proppant water gets final product for it is dissolved fully, is preferably the water based on 1 of proppant weight meter: 5-12 (g/ml) volume.
As another preferred embodiment of the present invention, the preparation method of levocarnitine liposomes injection of the present invention is:
(1) soybean lecithin, cholesterol and the antioxidant of recipe quantity is dissolved in the organic solvent based on 1 of three's gross weight meter: 5-10 (g/ml) volume, slowly inject the ammonium sulfate of 0.01-0.1mol/L, heated and stirred is steamed and is removed organic solvent, ultrasonic 10-20 minute, gets blank liposome; The volume of ammonium sulfate is identical with volume of organic solvent;
(2) blank liposome is placed bag filter, seal, it is 0.9%-10% (g/ml) osmotic pressure solution that bag filter is placed the concentration of 0.72-2.5 weight portion, the ammonium sulfate among the weeding of grease plastid foreign minister;
(3) levocarnitine is dissolved in the water based on 1 of levocarnitine weight meter: 20-50 (g/ml) volume, 60 ℃ of insulations of blank liposome that dialysis is good, the aqueous solution that under agitation slowly adds levocarnitine, continue insulation 20-30min, add buffer solution and osmotic pressure regulator solution, mixing promptly gets the levocarnitine liposomes suspension;
(4) proppant of 4-10 weight portion is dissolved in the water based on 1 of proppant weight meter: 5-12 (g/ml) volume, adds in the above-mentioned suspension, stirring and evenly mixing filters, lyophilization, the levocarnitine liposomes lyophilized preparation; Perhaps, above-mentioned suspension is filtered, packing, sterilization gets the levocarnitine liposomes injection.
Levocarnitine liposomes injection provided by the invention carries out stability test and investigates, and accelerated test is 6 months under 40 ℃ of high temperature, relative humidity 75% ± 5% condition, and every detection index does not have significant change; Long term test is 18 months under 25 ℃ of high temperature, relative humidity 60% ± 10% condition, and every detection index does not have significant change.
Levocarnitine liposomes injection provided by the invention carries out acute toxicity test, abnormal toxicity test and heat source check, and is all up to specification, and safety obtains proof.
Levocarnitine liposomes injection provided by the invention compared with prior art, has beyond thought effect, and major advantage is as follows:
(1) levocarnitine is wrapped in the liposome, has improved the stability in the aqueous solution greatly, guaranteed product quality;
(2) pharmaceutical carrier liposome vivo degradation, avirulence and non-immunogenicity, and can improve the Drug therapy index, reduce drug toxicity and reduce drug side effect; Obtained the liposome of excellent in stability;
(3) adopt conventional process equipment, but commercial scale, high efficiency production, constant product quality, be a kind of uniqueness with blanket, the low-cost industrial preparation method.
The specific embodiment
Further specify the present invention by the following examples, but should not be construed as limitation of the present invention.
Embodiment 1
The preparation of levocarnitine liposomes injection
Prescription (100 bottles): levocarnitine 25g
Soybean lecithin 180g
Cholesterol 45g
Vitamin E 6g
Preparation technology
(1) 180g soybean lecithin, 45g cholesterol and 6g vitamin E are dissolved in the 1500ml isopropyl alcohol, slowly inject 0.1mol/L ammonium sulfate 1500ml under stirring, heated and stirred is steamed and is removed isopropyl alcohol, puts ultrasonic 10min in the ice bath, gets blank liposome;
(2) blank liposome is placed bag filter, seal, bag filter is placed 0.9% sodium chloride solution 3000ml dialysis 20 hours, the ammonium sulfate among the weeding of grease plastid foreign minister;
(3) the 25g levocarnitine is dissolved in the 800ml water, 60 ℃ of insulations of blank liposome that dialysis is good, the aqueous solution that under agitation slowly adds levocarnitine, continue insulation 20min, add 0.9% sodium chloride solution 1000ml and pH value 6.5 sodium dihydrogen phosphate-sodium hydrogen phosphate buffer 500ml, mixing promptly gets the levocarnitine liposomes suspension;
(4) above-mentioned suspension is filtered, be distributed into 100 bottles, sterilization gets the levocarnitine liposomes injection.
Embodiment 2
The preparation of levocarnitine liposomes injection
Prescription (100 bottles): levocarnitine 50g
Soybean lecithin 250g
Cholesterol 40g
Butylated hydroxyarisol 5g
Preparation technology
(1) 250g soybean lecithin, 40g cholesterol and 5g butylated hydroxyarisol are dissolved in the 2000ml tert-butyl alcohol, stir and slowly inject 0.05mol/L ammonium sulfate 2000ml down, heated and stirred is steamed and is removed the tert-butyl alcohol, puts ultrasonic 20min in the ice bath, gets blank liposome;
(2) blank liposome is placed bag filter, seal, bag filter is placed 10% glycerite 4000ml dialysed the ammonium sulfate among the weeding of grease plastid foreign minister 24 hours;
(3) the 50g levocarnitine is dissolved in the 2000ml water, 60 ℃ of insulations of blank liposome that dialysis is good, the aqueous solution that under agitation slowly adds levocarnitine, continue insulation 30min, the glycerite 1000ml and the pH value 6.0 acetic acid-sodium-acetate buffer 500ml of adding 10%, mixing promptly gets the levocarnitine liposomes suspension;
(4) above-mentioned suspension is filtered, be distributed into 100 bottles, sterilization gets the levocarnitine liposomes injection.
Embodiment 3
The preparation of levocarnitine liposomes injection
Prescription (100 bottles): levocarnitine 100g
Soybean lecithin 900g
Cholesterol 600g
Vitamin E 36g
Preparation technology
(1) 900g soybean lecithin, 600g cholesterol and 36g vitamin E are dissolved in the 10000ml ethanol, slowly inject 0.03mol/L ammonium sulfate 10000ml under stirring, heated and stirred is steamed and is removed ethanol, puts ultrasonic 20min in the ice bath, gets blank liposome;
(2) blank liposome is placed bag filter, seal, bag filter is placed 5% glucose solution 15000ml dialysis 26 hours, the ammonium sulfate among the weeding of grease plastid foreign minister;
(3) the 100g levocarnitine is dissolved in the 5000ml water, 60 ℃ of insulations of blank liposome that dialysis is good, the aqueous solution that under agitation slowly adds levocarnitine, continue insulation 20min, add 5% glucose solution 1000ml and pH value 6.2 citric acid-sodium citrate buffer 500ml, mixing promptly gets the levocarnitine liposomes suspension;
(4) above-mentioned suspension is filtered, be distributed into 100 bottles, sterilization gets the levocarnitine liposomes injection.
Embodiment 4
The preparation of levocarnitine liposomes lyophilized preparation
Prescription (100 bottles): levocarnitine 50g
Soybean lecithin 360g
Cholesterol 140g
Thiourea 18g
Trehalose 300g
Preparation technology
(1) 360g soybean lecithin, 140g cholesterol and 18g thiourea are dissolved in the 3000ml isopropyl alcohol, slowly inject 0.05mol/L ammonium sulfate 3000ml under stirring, heated and stirred is steamed and is removed isopropyl alcohol, puts ultrasonic 20min in the ice bath, gets blank liposome;
(2) blank liposome is placed bag filter, seal, bag filter is placed 0.9% sodium chloride solution 5000ml dialysis 20 hours, the ammonium sulfate among the weeding of grease plastid foreign minister;
(3) the 50g levocarnitine is dissolved in the 2000ml water, 60 ℃ of insulations of blank liposome that dialysis is good, the aqueous solution that under agitation slowly adds levocarnitine, continue insulation 30min, add 0.9% sodium chloride solution 1000ml and pH value 6.0 sodium dihydrogen phosphate-sodium hydrogen phosphate buffer 800ml, mixing promptly gets the levocarnitine liposomes suspension;
(4) the 300g trehalose is dissolved in the 2000ml water, adds in the above-mentioned suspension, stirring and evenly mixing filters, and lyophilization gets the levocarnitine liposomes lyophilized preparation.
Comparative Examples 1
The preparation of levocarnitine liposomes injection
Prescription (100 bottles): levocarnitine 25g
Soybean lecithin 180g
Cholesterol 45g
Preparation method makes the levocarnitine liposomes injection that does not contain antioxidant with embodiment 1.
Comparative Examples 2
The preparation of levocarnitine liposomes injection
Prescription (100 bottles): levocarnitine 50g
Soybean lecithin 250g
Ovum Gallus domesticus Flavus lecithin 40g
Cholesterol 40g
Preparation method is with embodiment 2, makes to contain the levocarnitine liposomes injection that two kinds of phospholipid do not contain antioxidant.
Comparative Examples 3
The preparation of levocarnitine liposomes injection
Prescription (100 bottles): levocarnitine 100g
Soybean lecithin 900g
Cholesterol 600g
Butylated hydroxytoluene 36g
Preparation method is with embodiment 3, and application butylated hydroxytoluene replacement vitamin E makes the levocarnitine liposomes injection outside the preferred antioxidant of the present invention.
Comparative Examples 4
The preparation of levocarnitine liposomes lyophilized preparation
Prescription (100 bottles): levocarnitine 50g
Soybean lecithin 360g
Cholesterol 140g
Thiourea 18g
Trehalose 300g
Select the preparation technology who is different from embodiment 4 for use, make the levocarnitine liposomes lyophilized preparation, as follows: (1) is dissolved in 360g soybean lecithin, 140g cholesterol and 18g thiourea in the 3000ml isopropyl alcohol, mix homogeneously, isopropyl alcohol is removed in decompression on rotary film evaporator, makes immobilized artificial membrane;
(2) add pH value 6.0 sodium dihydrogen phosphate-sodium hydrogen phosphate buffer 800ml, 40min is stirred in jolting, make the complete aquation of immobilized artificial membrane, adopt the even at a high speed matter emulsifying 20min of tissue mashing machine, rotating speed 15000r/min, reuse 0.45 μ m filtering with microporous membrane makes the blank liposome suspension;
(3) the 50g levocarnitine is dissolved in the 2000ml water, with 0.45 μ m filtering with microporous membrane, filtrate adds in the blank liposome suspension, is incubated 60 ℃ and stirs 40 minutes, gets the levocarnitine liposomes suspension;
(4) the 300g trehalose is dissolved in the 2000ml water, adds in the above-mentioned suspension, stirring and evenly mixing filters, and lyophilization gets the levocarnitine liposomes lyophilized preparation.
Liposomal formulation can be used the methods known in the art preparation, confirm that by the routine described method of following test Application Example 4 described methods are better than the method for Comparative Examples 4, so the present invention preferably adopts ammonium sulfate pH gradient method.
Test example 1
The mensuration of particle diameter
Particle diameter to the Liposomal formulation of embodiment 1-4 and Comparative Examples 1-4 preparation detects, concrete grammar is: the Liposomal formulation of getting the present invention's preparation, be dissolved in water or be diluted to the solution that every 1ml contains levocarnitine 30 μ g, detect with H3LA920 laser light scattering particle size analyzer.As a result, the Liposomal formulation of embodiment 1-4 preparation is a spherical shape, and particle diameter is even, and 80% particle diameter is less than 150nm, and 90% particle diameter meets the related request of used for intravenous injection Liposomal formulation less than 180nm; The Liposomal formulation particle diameter of Comparative Examples 1-3 preparation is inhomogeneous, not of uniform size, and shape is indefinite, and is disorderly and unsystematic, and 85% particle diameter does not meet the intravenous injection requirement greater than 850nm., the Liposomal formulation of Comparative Examples 4 is not so good as embodiment 4 described liposomees though meeting the requirements.
Test example 2
The mensuration of envelop rate
The Liposomal formulation of embodiment 1-4 and Comparative Examples 1-4 preparation is dissolved in water or is diluted to the solution that every 1ml contains levocarnitine 30 μ g, high speed centrifugation, 5000r/min, centrifugal 30min, get clear liquid 1ml, use dissolve with methanol,, determine entrapped content M with Levocarnitine determined by HPLC content
1, the levocarnitine total amount is M in the Liposomal formulation
0, envelop rate N is:
N＝M
1/M
0×100％
As a result, the Liposomal formulation envelop rate of present embodiment 1-4 preparation is all greater than 88%, and the Liposomal formulation envelop rate of Comparative Examples 1-3 preparation is all less than 60%, and also not as embodiment 4, envelop rate is less than 75% for the Liposomal formulation of Comparative Examples 4.
Test example 3
Study on the stability
With (the Italian Sigma-Tau company production of the sample of above each embodiment preparation and the levocarnitine injection of listing, lot number 070611057) and the levocarnitine injectable powder (Jiutai Pharmaceutical Co Ltd, Jinzhou City produces, lot number 20071019) under 40 ℃ of high temperature, relative humidity 75% ± 5% condition 6 month, carry out accelerated test and investigate, the results are shown in Table 1; Under 25 ℃ of high temperature, relative humidity 60% ± 10% condition 18 months, carry out long term test and investigate, detect the variation of every quality index, the results are shown in Table 2.
Table 1 accelerated test result
Table 2 long-term test results
Quickened March, June by above found that, long-term December, the levocarnitine clarity of injection of listing is against regulation 18 months the time, and pH value descends bigger, and content reduces obviously, and related substance raises; The levocarnitine injectable powder clarity of listing is against regulation, and other indexs also change more obvious; And the sample appearance character of the present invention's preparation does not have significant change, and clarity, pH value, content and related substance do not have obvious variation yet.The sample stable quality after long time storage that the present invention's preparation is described is better.
Test example 4
The percolation ratio test
Get the sample of embodiment 1-4 and Comparative Examples 1-4 preparation, place 4 ℃ of low temperature, 25 ℃ of room temperature, following 180 days of 40 ℃ of conditions of high temperature respectively, make regular check on, measure the method for envelop rates with test example 2 and measure retention volume, with the dose of sealing in 0 day relatively, by formula Q
Ooze%=(W
Bag-W
Storage)/W
Bag* 100% calculates percolation ratio, result such as following table 3.
Table 3 percolation ratio result of the test (%)
By above experimental result as can be known, the sample of embodiment of the invention preparation percolation ratio in the long term store process changes little, satisfy the requirement of stability under the storage at normal temperature fully, and the sample percolation ratio of the preparation of the Comparative Examples outside the scope of the invention increases gradually, the liposome seepage is serious, and the high more percolation ratio of storage temperature is big more, has proved absolutely the superiority of the present invention's combination, wherein antioxidant has brought beyond thought effect, is in theory can't rational expectation.
Claims (7)
1. levocarnitine liposomes injection, it is characterized in that adopting the preparation of ammonium sulfate pH gradient method, form by active component levocarnitine, soybean lecithin, cholesterol, antioxidant and pharmaceutically acceptable carrier, wherein the ratio of weight and number of each component is: 1 part of levocarnitine, soybean lecithin 5-9 part, cholesterol 0.8-6 part, antioxidant 0.1-0.6 part, described antioxidant is selected from a kind of in thiourea, vitamin E and the butylated hydroxyarisol.
2. levocarnitine liposomes injection according to claim 1 is characterized in that the ratio of weight and number of each component is: 1 part of levocarnitine, 7.2 parts of soybean lecithins, 2.8 parts in cholesterol, 0.36 part in antioxidant.
3. according to each described levocarnitine liposomes injection of claim 1-2, it is characterized in that pharmaceutically acceptable carrier is osmotic pressure regulator, buffer solution, proppant.
4. levocarnitine liposomes injection according to claim 3 is characterized in that osmotic pressure regulator is selected from a kind of in sodium chloride, glucose, the glycerol.
5. levocarnitine liposomes injection according to claim 3 is characterized in that buffer solution is selected from a kind of in sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution of pH value 6.0-6.5, acetic acid-sodium acetate buffer solution, the citric acid-sodium citrate buffer solution.
6. levocarnitine liposomes injection according to claim 3, its proppant are selected from a kind of in mannitol and the trehalose.
7. a method for preparing each described levocarnitine liposomes injection of claim 1-6 comprises the steps:
(1) soybean lecithin, cholesterol and the antioxidant with recipe quantity is dissolved in the organic solvent, slowly injects ammonium sulfate, and heated and stirred steam to be removed organic solvent, and is ultrasonic, blank liposome, described organic solvent is selected from a kind of in the tert-butyl alcohol, isopropyl alcohol, the ethanol;
(2) blank liposome is placed bag filter, seal, bag filter is placed osmotic pressure solution, the ammonium sulfate among the weeding of grease plastid foreign minister;
(3) the good 60 ℃ of insulations of blank liposome of will dialysing under agitation slowly add the aqueous solution of levocarnitine, continue insulation 20-30min, add buffer solution and osmotic pressure regulator solution, and mixing promptly gets the levocarnitine liposomes suspension;
(4) proppant is water-soluble, add in the above-mentioned suspension, stirring and evenly mixing filters, and lyophilization gets the levocarnitine liposomes lyophilized preparation; Perhaps, above-mentioned suspension is filtered, packing, sterilization gets the levocarnitine liposomes injection.
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| CN101637450A CN101637450A (en) | 2010-02-03 |
| CN101637450B true CN101637450B (en) | 2011-11-09 |
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| CN102309475B (en) * | 2011-07-10 | 2013-05-15 | 长春海悦药业有限公司 | Levocarnitine for injection and preparation method thereof |
| CN104189058B (en) * | 2014-08-25 | 2017-10-10 | 西安力邦肇新生物科技有限公司 | A kind of fat emulsion injection containing levocarnitine |
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