CN103932994B - A kind of gemcitabine hydrochloride freeze-dried composition and preparation method thereof - Google Patents
A kind of gemcitabine hydrochloride freeze-dried composition and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to medical technical field, specifically disclose a kind of gemcitabine hydrochloride freeze-dried composition and preparation method thereof. Described composition is made up of gemcitabine hydrochloride, sweet mellow wine and malate, wherein gemcitabine hydrochloride is 1:0.5 ~ 2 by gemcitabine with the weight proportion of sweet mellow wine, gemcitabine hydrochloride is 1:0.05 ~ 0.2 by gemcitabine with the weight proportion of malate, and the pH value of described composition is 2.7 ~ 3.3. The present invention, using malate as pH value conditioning agent and stabilizing agent, can improve the stability of gemcitabine hydrochloride. In addition, the preparation technology of this composition is simple, cost is low, is applicable to suitability for industrialized production, clinical use safety.
Description
Technical field
The invention belongs to medical technical field, more specifically relate to a kind of gemcitabine hydrochloride freeze-dried composition, also relate to simultaneouslyAnd a kind of preparation method of gemcitabine hydrochloride freeze-dried composition. Product of the present invention is applicable to non-small cell lung cancer, cancer of pancreas, breastThe Several Kinds of Malignancy patients' such as gland cancer, prostate cancer, carcinoma of urinary bladder, cervical carcinoma, oophoroma treatment.
Background technology
Gemcitabine hydrochloride (Gemcitabinehydrochloride) is that the one of nineteen eighty-three Li Lai company development is novelDifluoronucleosides class antimetabolic antineoplastic, mainly kills and wounds synthetic (S phase) cell of DNA, also can block cell by G1Enter to the S phaseExhibition. It is converted into the activated diphosphate of tool (dFdCDP) and triphosphate (dFdCTP) by nucleoside kinase in cell, presses downDNA's processed is synthetic. On the one hand, dFdCDP suppresses ribonucleotide reductase, makes the essential deoxidation NTP of synthetic DNA(including dCTP) generates minimizing. On the other hand, dFdCDP and dCTP competition are mixed in DNA molecular, and in cell, dCTP is denseThe reduction of degree promotes dFdCTP mixing to DNA more. DFdCTP mixes after DNA, mixes because archaeal dna polymerase ε can not removeDFdCTP and repair DNA chain in extension, it is synthetic that the dFdCTP mixing in extended chain can further suppress DNA. In addition, it canMake CEM(CEM) T lymphoblast like cell nuclear dna be fragment sample change, thereby cause cellApoptosis.
The nineteen ninety-five state approval hydrochloride for injection gemcitabine listing such as South Africa, Sweden, Holland, Australia, 1996FDA ratifies the clinical first-line treatment that is used for the treatment of non-small cell lung cancer and cancer of pancreas, approval of import Lilly company of China in 1999Hydrochloride for injection gemcitabine. Gemcitabine is one of the most effective first-line drug for the treatment of non-small cell lung cancer, has effectThe advantages such as mechanism uniqueness, wide, the high remission rate of antitumor spectra, prolongation life cycle and toxic reaction are low, with other chemotherapeutics without intersectingResistance and toxic reaction are without features such as stacks, and particularly it can effectively extend patient's life, improve quality of life of patients. OrderBefore, hydrochloride for injection gemcitabine obtains extensive clinical practice in more than 100 countries.
1998, Li Lai company announced the product of its listing product hydrochloride for injection gemcitabine first in FDA websiteDescription, and disclose the formula of product: every bottle containing 200mg gemcitabine hydrochloride (representing with free alkali), 200mg sweet mellow wine and12.5mg sodium acetate, is prepared into aseptic freeze-dried powder.
Chinese patent CN102302462B discloses a kind of gemcitabine hydrochloride lyophilized formulations, by gemcitabine hydrochloride, freezeDry protective agent and antioxidant composition, wherein: gemcitabine hydrochloride accounts for 20～70 weight portions, freeze drying protectant accounts for 15～60 weightPart, antioxidant accounts for 0.005～0.5 weight portion. Add the object of antioxidant to be further to improve the stability of medicine, this is specialIn profit, selected antioxidant is any one in pyrosulfurous acid hydrogen sodium, citric acid, vitamin C, Cys. Burnt sulfurousAcid hydrogen sodium is harmful to human body, has reported clinically at present many bad reactions, modal be sulphite allergy (especiallyAsthma patient), symptom is bronchial spasm, stridulate, expiratory dyspnea, pernicious laryngeal edema, low blood pressure, shock are even dead.In addition, also have some research reports, sulphite may cause damage to chromosome and DNA, and cystine linkage in proteinIrreversible reaction etc.
Chinese patent CN102144981A discloses a kind of gemcitabine hydrochloride lyophilized powder injection and preparation method thereof. This powderInjection comprises gemcitabine hydrochloride 20-30 part, sweet mellow wine 5-9 part, sodium acetate 3-10 part, lactose 5-9 part. Though this freeze drying powder injectionSo have process route simple, freeze-drying time is short, easy to operate advantage, but owing to having increased lactose as additives, breastSugar easily causes " lactose intolerance ", is especially mainly in Asia. In addition, also may introduce residual protein and assorted sugar etc. hasThe anaphylactogen that related substance brings, may there is certain risk in clinical practice.
Chinese patent CN101564381A discloses a kind of gemcitabine hydrochloride lyophilized powder injection, by gemcitabine hydrochloride,Sweet mellow wine and sodium acetate composition, wherein gemcitabine hydrochloride and sweet mellow wine weight proportion are 1: 0.5～5, gemcitabine hydrochloride andSodium acetate weight proportion is 1: 0.01～0.1.
Find by studying us, malate is better to the stabilizing effect of gemcitabine hydrochloride than acetate, its workFor pH value conditioning agent time, also can be used as stabilizing agent, in addition, also may there is the physiological action that is better than sodium acetate: A and anti-Cancer drug is combined while use, can reduce cancer therapy drug to Normocellular infringement, is used for clinically auxiliary after cancer Radiotherapy chemotherapyHelp medicine. B and amino acid preparation are combined while use, can improve amino acid whose bioavilability. C, can promote ammonia metabolism, reduceAmmonia concentration, has protective effect to liver, and treatment dyshepatia, hepatic failure, liver cancer especially hepatosis causeThe good medicine of hyperammonemia. D, can be used as one for the treatment of heart disease basal liquid composition, for K+、Mg2+Supplement, keep myocardium energyAmount metabolism, plays a protective role to the ischemic myocardium of miocardial infarction. E, can improve the energetic supersession of brain tissue, adjust brainInterior neurotransmitter, is conducive to the recovery of learning and memory function, and learning and memory is improved significantly. F, can be used for treating poorThe diseases such as blood, hypoimmunity, uremia, hypertension.
Find through Destructive Test Study, gemcitabine hydrochloride all has not under illumination, acid, alkali, high temperature and oxidizing conditionWith the destruction of degree, the most obvious with Oxidative demage degraded, be secondly that high irenine destroys, it is less that illumination and acid destroy degraded.Therefore, research and develop a kind of good stability, safe and to be beneficial to the gemcitabine hydrochloride lyophilized powder injection of suitability for industrialized production aobviousObtain particularly important.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to be to provide a kind of gemcitabine hydrochloride freeze-dryingComposition, formula is rationally, easy to use, and the product quality of said composition in 2 years terms of validity is obviously better than having gone on the market at presentHydrochloride for injection gemcitabine preparation.
Another object of the present invention is the preparation side that has been to provide a kind of above-mentioned gemcitabine hydrochloride freeze-dried compositionMethod, easy to implement the method, easy and simple to handle, composition profile is full attractive in appearance, and product yield is up to 99%, in addition, with similar manufacturer phaseRatio, freeze-drying time obviously shortens, and has greatly saved production cost.
In order to realize above-mentioned object, the present invention adopts following technical measures:
A kind of gemcitabine hydrochloride freeze-dried composition, it is prepared from by following raw material:
Unit=the g/mL of the unit/described parts by volume of described weight portion;
For example: in the time that the unit of described weight portion is g, the unit of described parts by volume is mL;
In the time that the unit of described weight portion is kg, the unit of described parts by volume is L;
In raw material composition, gemcitabine hydrochloride (by gemcitabine) is 1:0.5～2 with the weight proportion of sweet mellow wine, saltAcid gemcitabine (by gemcitabine) is 1:0.05～0.2 with the weight proportion of malate, and the pH value of described composition is2.7～3.3。
The invention is characterized in and use malate as pH value conditioning agent and stabilizing agent, malate should be solvableProperty salt, metal ion should not be heavy metal or iron, calcium, magnesium etc., preferably apple acid sodium, potassium malate.
A kind of gemcitabine hydrochloride freeze-dried composition, it is prepared from (preferable range) by following raw material:
A kind of gemcitabine hydrochloride freeze-dried composition, it is prepared from (more preferably scope) by following raw material:
A kind of gemcitabine hydrochloride freeze-dried composition, it is prepared from (optimum range) by following raw material:
A kind of gemcitabine hydrochloride freeze-dried composition, it is prepared from (optimum value) by following raw material:
The preparation technology of gemcitabine hydrochloride freeze-dried composition of the present invention, is characterized in that comprising dissolving, active carbonAbsorption or ultrafiltration except bacterial endotoxin, aseptic filtration, filling, partly jump a queue, freeze-drying, tamponade, outlet, roll lid, lamp inspection, labeling and bagDress etc.
A preparation method for above-mentioned gemcitabine hydrochloride freeze-dried composition, its concrete steps are:
1, get the water for injection of formula ratio 70-90%, temperature is 40 ± 5 DEG C, is accompanied by stirring and adds successively the sweet of formula ratioReveal alcohol and malate to dissolving; Take the gemcitabine hydrochloride of formula ratio, limit edged stirs and makes to dissolve completely, and sampling detects medicineLiquid pH value is in 2.7～3.3 scopes; According to the cumulative volume of formula, get 0.1% medicinal carbon, moistening with a small amount of water for injectionAfter add preparation liquid in, add to the full amount of water for injection, stir and evenly mix; To prepare liquid 40 ± 5 DEG C of insulated and stirred 30 minutes, thenBe cooled to room temperature, with 0.45 μ m filtering with microporous membrane decarburization, then use the filtering with microporous membrane degerming of 0.22 μ m, filtrate is collected into closeIn the sterile chamber of envelope.
2, by filled with solution in the tubular injection bottle of suitable size, after partly jumping a queue, send into freeze drying box, freeze-drying program asUnder:
A, pre-freeze: first solution is cooled to-12 DEG C～-14 DEG C, then by conduction oil with 1 DEG C/(8-12) speed of minBe down to-18～-22 DEG C, insulation 2～3h, is down to after-16～-20 DEG C until products temperature, conduction oil is cooled in 30min-40 ± 2 DEG C, insulation 4～5h;
B, lyophilization: in casing, be evacuated to 7～12Pa, with the speed of 1～2 DEG C/h, conduction oil heated up, whenConduction oil temperature is incubated after rising to 0 DEG C, and temperature retention time is no less than 30h, and the temperature difference of sample and conduction oil is less than 15～20 DEG C;
C, parsing-desiccation: the speed with every 1 DEG C/8～12min heats up to conduction oil, be warming up to 40 ± 2 DEG C by goods, protectsTemperature 3～5h.
3,, after freeze-drying process completes, tamponade, outlet, obtain thing of the present invention after rolling lid, lamp inspection, labeling, packaging.
Gemcitabine hydrochloride all has destruction in various degree under illumination, acid, alkali, high temperature and oxidizing condition, to be oxidized brokenlyBad degraded is the most obvious, is secondly that high irenine destroys. Add malate, regulator solution pH value is at 2.7～3.3 models on the one handIn enclosing, on the other hand with gemcitabine in unsettled group have an effect and produce stabilizer function.
Compared with prior art, the advantage of composition and method of making the same of the present invention and beneficial effect are:
1, malate both can be used as pH value conditioning agent, also can have an effect with unsettled group in gemcitabine andProduce stabilizer function. In addition, malate is better than the multiple physiological action of acetate in addition.
2, formula is rationally, easy to use, and the product quality in 2 years terms of validity is obviously better than other injections of having gone on the market at presentUse gemcitabine hydrochloride preparation.
3, preparation method is easy and simple to handle, compares with similar manufacturer, and freeze-drying time obviously shortens, and has greatly saved productionCost, and product yield is up to 99%.
4, composition profile is full attractive in appearance, and solubility is good.
Detailed description of the invention
Applicant is described in further detail the inventive method in connection with specific embodiment below.
Embodiment 1:
A kind of gemcitabine hydrochloride freeze-dried composition, it is made up of following raw material:
The preparation method of above-mentioned gemcitabine hydrochloride freeze-dried composition, the steps include:
1, measure the water for injection 3400mL that is cooled to 40 ± 5 DEG C, put in appropriate vessel, take sweet mellow wine 100g limit edgedStirring is dissolved it, then adds natrium malicum 7g, is stirred to dissolving; Then add 114g gemcitabine hydrochloride to be stirred to dissolving,Sampling detects liquid pH value (should be in 2.7～3.3 scopes, if do not entered with 1mol/L malic acid or natrium malicum in this scopeRow fine setting, can ignore for malic acid or the natrium malicum consumption of finely tuning); Take 4g medicinal carbon and put appropriate vesselIn, add after moistening with a small amount of water for injection in preparation liquid, inject water to 4000mL, stir and evenly mix; To prepare liquid 40± 5 DEG C of insulated and stirred 30 minutes, are then cooled to room temperature, with 0.45 μ m filtering with microporous membrane decarburization, then use the micropore of 0.22 μ mMembrane filtration degerming, filtrate is collected in the sterile chamber of sealing.
2, then in the tubular injection bottle of 20mL (every bottle of 8mL), after partly jumping a queue, send into freeze drying box by filling filtrate, freezeDry program is as follows:
A, pre-freeze: first composition solution is cooled to-12 DEG C or-13 or-14 DEG C, then by conduction oil with 1 DEG C/8minOr the speed of 1 DEG C/9min or 1 DEG C/10min or 1 DEG C/11min or 1 DEG C/12min is down to-18 or-19 or-20 or-21 or-22DEG C, insulation 2 or 3h, be down to after-16 or-17 or-18 or-19 or-20 DEG C until products temperature, and conduction oil is lowered the temperature in 30minTo-38 or-39 or-40 or-41 or-42 DEG C, insulation 4 or 5h;
B, lyophilization: in casing, be evacuated to 7 or 8 or 9 or 10 or 11 or 12Pa, with the speed of 1 or 2 DEG C/h to heat conductionOil heats up, insulation after conduction oil temperature rises to 0 DEG C, temperature retention time is 30 or 31 or 32 or 33 or 34 or 35h, sample andThe temperature difference of conduction oil is less than 15 or 16 or 17 or 18 or 19 or 20 DEG C;
C, parsing-desiccation: with every 1 DEG C/8min or 1 DEG C/9min or 1 DEG C/10min or 1 DEG C/11min or 1 DEG C/12minSpeed heats up to conduction oil, and goods are warming up to 38 or 39 or 40 or 41 or 42 DEG C, insulation 3 or 4 or 5h.
3,, after freeze-drying process completes, tamponade, outlet, obtain the present composition after rolling lid, lamp inspection, labeling, packaging.
Product yield is 99.4%, and resulting composition is white loose block, and profile is full attractive in appearance, and solubility is good.
Embodiment 2:
A kind of gemcitabine hydrochloride freeze-dried composition, it is made up of following raw material:
The preparation method of above-mentioned gemcitabine hydrochloride freeze-dried composition, the steps include:
1, measure the water for injection 1600mL that is cooled to 40 ± 5 DEG C, put in appropriate vessel, take sweet mellow wine 110g limit edgedStirring is dissolved it, then adds natrium malicum 8g, is stirred to dissolving; Then add 114g gemcitabine hydrochloride to be stirred to dissolving,Sampling detects liquid pH value (should be in 2.7～3.3 scopes, if do not entered with 1mol/L malic acid or natrium malicum in this scopeRow fine setting, can ignore for malic acid or the natrium malicum consumption of finely tuning); Take 2g medicinal carbon and put appropriate vesselIn, add after moistening with a small amount of water for injection in preparation liquid, inject water to 2000mL, stir and evenly mix; To prepare liquid 40± 5 DEG C of insulated and stirred 30 minutes, are then cooled to room temperature, with 0.45 μ m filtering with microporous membrane decarburization, then use the micropore of 0.22 μ mMembrane filtration degerming, filtrate is collected in the sterile chamber of sealing.
2, by filling filtrate in the tubular injection bottle of 50mL (every bottle of 20mL), after partly jumping a queue, send into freeze drying box, freeze-dryingProgram is as follows:
A, pre-freeze: first composition solution is cooled to-12 DEG C～-14 DEG C, then the speed with 1 DEG C/10min by conduction oilDegree is down to-20 DEG C, and insulation 2～3h, is down to after-16～-20 DEG C until products temperature, conduction oil is cooled in 30min to-40 ±2 DEG C, insulation 4～5h;
B, lyophilization: in casing, be evacuated to 7～12Pa, with the speed of 1～2 DEG C/h, conduction oil heated up, whenConduction oil temperature is incubated after rising to 0 DEG C, and temperature retention time is 55h or 56h or 57h or 58h or 59h or 60h, sample and conduction oilThe temperature difference is at 15～20 DEG C;
C, parsing-desiccation: the speed with every 1 DEG C/10min heats up to conduction oil, be warming up to 40 ± 2 DEG C by goods, insulation 3～5h。
3,, after freeze-drying process completes, tamponade, outlet, obtain the present composition after rolling lid, lamp inspection, labeling, packaging.
Product yield is 99.2%, and composition is white loose block, and profile is full attractive in appearance, and solubility is good.
Embodiment 3:
A kind of gemcitabine hydrochloride freeze-dried composition, it is made up of following raw material:
Preparation method is with embodiment 1.
Embodiment 4:
A kind of gemcitabine hydrochloride freeze-dried composition, it is made up of following raw material:
Preparation method is with embodiment 1.
Embodiment 5:
A kind of gemcitabine hydrochloride freeze-dried composition, it is made up of following raw material:
Preparation method is with embodiment 2.
Below by following related tests, the invention will be further described:
One, the stability comparative test of sample and commercially available hydrochloride for injection gemcitabine:
(injection) gemcitabine hydrochloride freeze-dried composition of preparing according to embodiment 1 is as test group, commercially available injectionBy gemcitabine hydrochloride freeze-dried powder (use sodium acetate regulator solution pH value to 2.7～3.3 scope in production process as a control groupIn, gemcitabine hydrochloride (by gemcitabine): sweet mellow wine weight ratio=1:1).
In order to investigate the stability of test group and control sample, by product respectively at illumination 4500lx ± 500lx, high temperatureUnder 60 DEG C of conditions, place 10 days, detect respectively at sampling in the 5th, 10 days, measure its proterties, pH value, related substance and content etc.Order. The results are shown in Table 1～2.
Result is investigated in table 1 hydrochloride for injection gemcitabine 4500lx ± 500lx contrast
Result is investigated in 60 DEG C of contrasts of table 2 hydrochloride for injection gemcitabine
Can find out from upper watch test result, the present composition and commercially available hydrochloride for injection gemcitabine exist respectivelyUnder 4500lx ± 500lx and 60 DEG C of conditions, investigate 10 days, proterties, the pH value of product are unchanged, test group and right under illumination conditionRelated substance and content according to group are not obvious, have further verified that illumination is less to the destruction of gemcitabine hydrochloride, stillGap under 60 DEG C of conditions between the present composition and commercially available product displays gradually, especially related substance, of the present invention groupCompound is obviously better than commercially available product. Indices all meets the regulation of hydrochloride for injection gemcitabine quality standard in USP34 version, tableThe steady quality of the bright present composition, and be better than similar commercially available kind.
Two, the safety testing of sample (anaphylaxis, blood vessel irritation and hemolytic):
In order to investigate the security of the present composition, for clinical research provides security reference information, according to the present inventionThe formula of embodiment 1 and preparation method, prepare hydrochloride for injection gemcitabine, as test sample group, carry out excitant, haemolysis andIrritated grade and specific safety test local, that whole body administration is relevant. Commercially available contrast medicine is the injection salt that Li Lai company producesAcid gemcitabine (trade name: gemzar, specification: 0.2g), 0.9% sodium chloride injection is as negative control product, the white egg of 20% human bloodBai Zuowei positive reference substance.
Test method and result:
1, vascular stimulation tests
1.1 test method
According to preliminary experiment result (this product is 25mg/mL in concentration, when dosage is 75mg/kg animal at 24 hours to 72 littleTime in all dead), get 3 of adult healthy white rabbit, adopt consubstantiality left and right sides self-contrast method, test sample group is from left ear edgeSlowly intravenous injection need testing solution, dosage is 5mg/kg, and concentration is 10mg/mL, and control group is injected from auris dextra edge constant speed equivalentSodium chloride injection. Be administered once every day, continuous 5 days. During administration, visually observe administration local vascular have or not thrombus shape every dayBecome, surrounding tissue has or not the inflammatory reactions such as red and swollen hemostasis. After 48 hours, put to death animal in last administration, get rapidly its ear tissue(from entry point centripetal direction 2～3cm), 10% formalin is fixed, FFPE, conventional film-making, HE dyeing, light Microscopic observationBlood vessel and tissue morphology change.
1.2 result of the test
Visually observe, respectively organize and have no the obviously symptom such as red, swollen, hemostasis during the administration of rabbit auricular vein injection site. SupplyTest product group and control group relatively have no obvious difference. The results are shown in Table 3.
Table 3 vascular stimulation tests visually observes result
Note: without thrombus (-) [Dan 1-4mm(+) middle thrombus 5-14mm(++) large thrombus (+++)
NIP changes (-) mild inflammation 3cm scope (+) moderate inflammation scope 1/3 auricular concha (++)
Intensity inflammation scope 1/2 auricular concha-full ear (+++)
Histological observation result: test sample to rabbit blood vessel without obvious irritation.
2, hypersensitive test
2.1 test method
Get 28 of body weight 250g～350g healthy guinea pigs, be divided at random 4 groups of (test sample high and low dose group, negative controlsGroup, positive controls), wherein 6 every group of 8 every group of test sample high and low dose groups, negative control group and positive controls. ConnectContinuous 3 times, the next day lumbar injection need testing solution (concentration is 10mg/mL, 2mg/mL), sodium chloride injection and the white egg of 20% human bloodWhite 0.5mL/ only carries out sensitization. After last injection, vein on the 12nd is injected respectively need testing solution 0.5mL, sodium chloride injectionOnly excite with 20% human serum albumin 1mL/, after intravenous injection, at once to 30min, describe symptom by table 4 and observe in detail everyThe reaction of cavy, the appearance of symptom and extinction time, the longest observation 3 hours. And the evaluation criterion of pressing table 5 is evaluated allergic reactionOccurrence degree. Calculate allergic reaction incidence. Comprehensively judge according to reaction incidence and symptoms of allergic. Negative controlMust not there is allergic reaction in group cavy, allergic reaction should all appear in positive controls cavy.
2.2 result of the test
Interior each group of 30min after hydrochloride for injection gemcitabine high and low dose solution and negative control treated animal intravenously administrableAll there is not allergic reaction in all cavys, after 20% human serum albumin intravenously administrable 6 cavys all occur coughing, be short of breath,The symptom such as instability of gait, spasm, and have 2 cavy death. The results are shown in Table 6.
Table 4 symptoms of allergic
Table 5 whole body sensitization evaluation criterion
Table 6 hydrochloride for injection gemcitabine Hypersensitive tests result
Result: under above-mentioned experimental condition, systemic anaphylaxis does not appear in hydrochloride for injection gemcitabine hypersensitive test.
3, hemolytic test
3.1 test method
Get 52, test tube, add successively 2% red blood cell suspension and physiological saline by table 7 proportional quantity, after mixing, in 37 ±0.5 DEG C of insulating box is placed half an hour, and then 1～No. 5 pipe adds respectively the need testing solution of different volumes (concentration is 40mg/ML), manage negative control tube No. 6 and add physiological saline, manage positive contrast No. 7 and add distilled water, No. 8-12 pipe respectivelyAdd the need testing solution (concentration is 20mg/mL) of different volumes, No. 13-17 pipe adds respectively the need testing solution of different volumes(concentration is 10mg/mL), No. 18-22 pipe adds respectively the need testing solution (concentration is 7mg/mL) of different volumes, No. 23-27 pipeAdd respectively the need testing solution (concentration is 5mg/mL) of different volumes, No. 28-32 pipe adds respectively the commercially available sample of different volumesProduct solution (concentration is 40mg/mL), No. 33-37 pipe adds respectively the commercially available sample solution (concentration is 20mg/mL) of different volumes,No. 38-42 pipe adds respectively the commercially available sample solution (concentration is 10mg/mL) of different volumes, and No. 43-47 pipe adds respectively differenceThe commercially available sample solution (concentration is 7mg/mL) of volume, No. 48-52 pipe adds respectively the commercially available sample solution (concentration of different volumesFor 5mg/mL), after shaking up, put in 37 ± 0.5 DEG C of insulating boxs. Start to observe once every 15min, after 1 hour, every 1 hourObserve once, Continuous Observation 3 hours, as solution is transparent redness, represents haemolysis. As there being brownish red flocculent deposit in solution,Indicate erythroagglutination.
Table 7 hydrochloride for injection gemcitabine hemolytic test proportional quantity (mL) (tested material 40mg/mL)
Table 8 hydrochloride for injection gemcitabine hemolytic test proportional quantity (mL) (tested material 20mg/mL)
Remarks: equal reference table 8 proportionings of the test sample of variable concentrations and commercially available product control group are tested.
3.2 result of the test
No. 7 pipes add after distilled water soon, and haemolysis appears in solution, and No. 6 sodium chloride injection pipe solution are in regulationTime in be not all transparent redness, there is not brownish red flocculent deposit yet, and add the each of need testing solution and commercially available samplePipe is degree of hemolysis difference under variable concentrations, shows that hydrochloride for injection gemcitabine has rabbit erythrocyte in the time of variable concentrationsCertain impact. The results are shown in Table 9-18.
Table 9 hydrochloride for injection gemcitabine hemolytic test result (tested material 40mg/mL)
| Test tube numbering | No. 1 pipe | No. 2 pipes | No. 3 pipes | No. 4 pipes | No. 5 pipes | No. 6 pipes | No. 7 pipes |
| 37 DEG C of insulation 15min | +++ | +++ | +++ | ++ | - | - | +++ |
| 37 DEG C of insulation 30min | +++ | +++ | +++ | +++ | - | - | +++ |
| 37 DEG C of insulation 45min | +++ | +++ | +++ | +++ | - | - | +++ |
| 37 DEG C of insulation 1h | +++ | +++ | +++ | +++ | - | - | +++ |
| 37 DEG C of insulation 2h | +++ | +++ | +++ | +++ | - | - | +++ |
| 37 DEG C of insulation 3h | +++ | +++ | +++ | +++ | ++ | - | +++ |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 1～No. 4 pipe color becomes dark brown.
Table 10 hydrochloride for injection gemcitabine hemolytic test result (tested material 20mg/mL)
| Test tube numbering | No. 8 pipes | No. 9 pipes | No. 10 pipes | No. 11 pipes | No. 12 pipes |
| 37 DEG C of insulation 15min | +++ | ++ | - | - | - |
| 37 DEG C of insulation 30min | +++ | +++ | ++ | ++ | - |
| 37 DEG C of insulation 45min | +++ | +++ | ++ | ++ | - |
| 37 DEG C of insulation 1h | +++ | +++ | ++ | ++ | - |
| 37 DEG C of insulation 2h | +++ | +++ | ++ | ++ | - |
| 37 DEG C of insulation 3h | +++ | +++ | ++ | ++ | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 8～No. 9 pipe colors become dark brown.
Table 11 hydrochloride for injection gemcitabine hemolytic test result (tested material 10mg/mL)
| Test tube numbering | No. 13 pipes | No. 14 pipes | No. 15 pipes | No. 16 pipes | No. 17 pipes |
| 37 DEG C of insulation 15min | - | - | - | - | - |
| 37 DEG C of insulation 30min | - | - | - | - | - |
| 37 DEG C of insulation 45min | - | - | - | - | - 11 --> |
| 37 DEG C of insulation 1h | - | - | - | - | - |
| 37 DEG C of insulation 2h | - | - | - | - | - |
| 37 DEG C of insulation 3h | - | - | - | - | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 13～No. 14 pipe colors become light coffee color.
Table 12 hydrochloride for injection gemcitabine hemolytic test result (tested material 7mg/mL)
| Test tube numbering | No. 18 pipes | No. 19 pipes | No. 20 pipes | No. 21 pipes | No. 22 pipes |
| 37 DEG C of insulation 15min | - | - | - | - | - |
| 37 DEG C of insulation 30min | - | - | - | - | - |
| 37 DEG C of insulation 45min | - | - | - | - | - |
| 37 DEG C of insulation 1h | - | - | - | - | - |
| 37 DEG C of insulation 2h | - | - | - | - | - |
| 37 DEG C of insulation 3h | - | - | - | - | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 18～No. 19 pipe colors become light coffee color.
Table 13 hydrochloride for injection gemcitabine hemolytic test result (tested material 5mg/mL)
| Test tube numbering | No. 23 pipes | No. 24 pipes | No. 25 pipes | No. 26 pipes | No. 27 pipes |
| 37 DEG C of insulation 15min | - | - | - | - | - |
| 37 DEG C of insulation 30min | - | - | - | - | - |
| 37 DEG C of insulation 45min | - | - | - | - | - |
| 37 DEG C of insulation 1h | - | - | - | - | - |
| 37 DEG C of insulation 2h | - | - | - | - | - |
| 37 DEG C of insulation 3h | - | - | - | - | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
Table 14 hydrochloride for injection gemcitabine hemolytic test result (commercially available thing 40mg/mL)
| Test tube numbering | No. 28 pipes | No. 29 pipes | No. 30 pipes | No. 31 pipes | No. 32 pipes |
| 37 DEG C of insulation 15min | +++ | +++ | +++ | ++ | - |
| 37 DEG C of insulation 30min | +++ | +++ | +++ | +++ | - |
| 37 DEG C of insulation 45min | +++ | +++ | +++ | +++ | - |
| 37 DEG C of insulation 1h | +++ | +++ | +++ | +++ | - |
| 37 DEG C of insulation 2h | +++ | +++ | +++ | +++ | - |
| 37 DEG C of insulation 3h | +++ | +++ | +++ | +++ | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 28～No. 31 pipe colors become dark brown.
Table 15 hydrochloride for injection gemcitabine hemolytic test result (commercially available thing 20mg/mL)
| Test tube numbering | No. 33 pipes | No. 34 pipes | No. 35 pipes | No. 36 pipes | No. 37 pipes |
| 37 DEG C of insulation 15min | +++ | +++ | ++ | - | - |
| 37 DEG C of insulation 30min | +++ | +++ | +++ | ++ | - |
| 37 DEG C of insulation 45min | +++ | +++ | +++ | ++ | - |
| 37 DEG C of insulation 1h | +++ | +++ | +++ | ++ | - |
| 37 DEG C of insulation 2h | +++ | +++ | +++ | ++ | - |
| 37 DEG C of insulation 3h | +++ | +++ | +++ | ++ | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 33～No. 35 pipe colors become dark brown.
Table 16 hydrochloride for injection gemcitabine hemolytic test result (commercially available thing 10mg/mL)
| Test tube numbering | No. 38 pipes | No. 39 pipes | No. 40 pipes | No. 41 pipes | No. 42 pipes |
| 37 DEG C of insulation 15min | - | - | - | - | - |
| 37 DEG C of insulation 30min | - | - | - | - | - |
| 37 DEG C of insulation 45min | - | - | - | - | - |
| 37 DEG C of insulation 1h | ++ | - | - | - | - |
| 37 DEG C of insulation 2h | ++ | ++ | - | - | - |
| 37 DEG C of insulation 3h | ++ | ++ | ++ | - | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 38～No. 40 pipe colors become light coffee color.
Table 17 hydrochloride for injection gemcitabine hemolytic test result (commercially available thing 7mg/mL)
| Test tube numbering | No. 43 pipes | No. 44 pipes | No. 45 pipes | No. 46 pipes | No. 47 pipes |
| 37 DEG C of insulation 15min | - | - | - | - | - |
| 37 DEG C of insulation 30min | - | - | - | - | - |
| 37 DEG C of insulation 45min | - | - | - | - | - |
| 37 DEG C of insulation 1h | - | - | - | - | - |
| 37 DEG C of insulation 2h | ++ | ++ | - | - | - |
| 37 DEG C of insulation 3h | ++ | ++ | - | - | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
In 15min, 43～No. 45 pipe colors become light coffee color.
Table 18 hydrochloride for injection gemcitabine hemolytic test result (commercially available thing 5mg/mL)
| Test tube numbering | No. 48 pipes | No. 49 pipes | No. 50 pipes | No. 51 pipes | No. 52 pipes |
| 37 DEG C of insulation 15min | - | - | - | - | - |
| 37 DEG C of insulation 30min | - | - | - | - | - |
| 37 DEG C of insulation 45min | - | - | - | - | - |
| 37 DEG C of insulation 1h | - | - | - | - | - |
| 37 DEG C of insulation 2h | - | - | - | - | - 13 --> |
| 37 DEG C of insulation 3h | - | - | - | - | - |
Note: +++ complete hemolysis; ++ part haemolysis; + aggegation;-not haemolysis, there is not aggegation yet
(injection) gemcitabine hydrochloride freeze-dried composition prepared by result: embodiment 1 is 40mg/mL, 20mg/ in concentrationWhen mL, there is hemolytic reaction in 3 hours. When concentration is 10mg/mL, 7mg/mL, 5mg/mL, in 3 hours without haemolysis and agglutinating reaction.Commercially available hydrochloride for injection gemcitabine except concentration be in 5mg/mL3 hour without hemolytic reaction, all the other concentration all have in various degreeHemolytic reaction.
By excitant, haemolysis and allergy etc. and specific safety test local, that whole body administration is relevant, result shows to adoptThe hydrochloride for injection gemcitabine of producing by this formula and preparation method, dosage be 5mg/kg, concentration while being 10mg/mL to rabbitVein blood vessel, without obvious irritation, does not all cause cavy allergic reaction, minimum in the time that high-concentration and low-concentration is 10mg/mL, 2mg/mLWhen being 10mg/mL, concentration do not cause haemolysis and agglutinating reaction in 3 hours, clinical use safety.
Three, the study on the stability of sample:
For present composition steady quality is described, by (injection) gemcitabine hydrochloride of preparing according to embodiment 1Freeze-dried composition is placed under room temperature condition, detects respectively at sampling in 3,6,9,12,18,24 months, measure its proterties, pH value,The project such as related substance and content, the results are shown in Table 19. By (injection) gemcitabine hydrochloride freeze-drying group of preparing according to embodiment 2Compound is placed under 40 DEG C of conditions, detects respectively at sampling in 1,2,3,6 month, measures its proterties, pH value, related substance and containsThe projects such as amount, the results are shown in Table 20.
The table 19 hydrochloride for injection gemcitabine room temperature investigation result that keeps sample
40 DEG C of investigation results that keep sample of table 20 hydrochloride for injection gemcitabine
As can be seen from the above table, hydrochloride for injection gemcitabine of the present invention is deposited 24 months and at ambient temperature 40Under DEG C condition, deposit after 6 months, proterties, pH value and content are without significant change, and related substance has increased slightly, and all meets USP34 versionThe regulation of middle hydrochloride for injection gemcitabine quality standard, shows that present composition quality stability is better.
Specific embodiment described in this description is only to the explanation for example of the present invention's spirit. Skill under the present inventionThe technical staff in art field can make various amendments or supplement or adopt similarly side described specific embodimentFormula substitutes, but can't depart from spirit of the present invention or surmount the defined scope of appended claims.
Claims (9)
1. a gemcitabine hydrochloride freeze-dried composition, it is prepared from by following raw material:
Gemcitabine hydrochloride is by gemcitabine 100 weight portions,
Sweet mellow wine 50 ~ 200 weight portions,
Malate 5 ~ 20 weight portions,
Water for injection adds to 2000-4000 parts by volume,
Unit=the g/mL of the unit/described parts by volume of described weight portion;
The pH value of described composition is 2.7 ~ 3.3.
2. gemcitabine hydrochloride freeze-dried composition according to claim 1, is characterized in that: described malate is solvableProperty salt, the metal ion of described malate is not heavy metal or iron, calcium, magnesium.
3. gemcitabine hydrochloride freeze-dried composition according to claim 1, is characterized in that: described malate is appleAcid sodium or potassium malate.
4. according to arbitrary described gemcitabine hydrochloride freeze-dried composition in claim 1-3, it is characterized in that: it is by former belowMaterial is prepared from:
Gemcitabine hydrochloride is by gemcitabine 100 weight portions,
Sweet mellow wine 75-150 weight portion,
Malate 5.5 ~ 15 weight portions,
Water for injection adds to 2000-4000 parts by volume.
5. gemcitabine hydrochloride freeze-dried composition according to claim 4, is characterized in that: it prepared by following raw material andBecome:
Gemcitabine hydrochloride is by gemcitabine 100 weight portions,
Sweet mellow wine 85-120 weight portion,
Malate 6-12 weight portion,
Water for injection adds to 2000-4000 parts by volume.
6. gemcitabine hydrochloride freeze-dried composition according to claim 5, is characterized in that: it prepared by following raw material andBecome:
Gemcitabine hydrochloride is by gemcitabine 100 weight portions,
Sweet mellow wine 90-110 weight portion,
Malate 6.5-10 weight portion,
Water for injection adds to 2000-4000 parts by volume.
7. gemcitabine hydrochloride freeze-dried composition according to claim 6, is characterized in that: it prepared by following raw material andBecome:
Gemcitabine hydrochloride is by gemcitabine 100 weight portions,
Sweet mellow wine 100 weight portions,
Malate 7 weight portions,
Water for injection adds to 2000-4000 parts by volume.
8. a preparation method for arbitrary described gemcitabine hydrochloride freeze-dried composition in claim 1-7, the steps include:
(1), get the water for injection of formula ratio 70-90%, temperature is 40 ± 5 DEG C, is accompanied by the sweet dew that stirring adds formula ratio successivelyAlcohol and malate are to dissolving; Take the gemcitabine hydrochloride of formula ratio, limit edged stirs and makes to dissolve completely, and sampling detects liquidPH value is in 2.7～3.3 scopes; According to formula cumulative volume, get 0.1% medicinal carbon, moistening with a small amount of water for injection afterAdd in preparation liquid, add to the full amount of water for injection, stir and evenly mix; To prepare liquid 40 ± 5 DEG C of insulated and stirred 30 minutes, then coldBut to room temperature, with 0.45 μ m filtering with microporous membrane decarburization, then use the filtering with microporous membrane degerming of 0.22 μ m, filtrate is collected into sealingSterile chamber in;
(2), by filled with solution in tubular injection bottle, after partly jumping a queue, send into freeze drying box;
(3), after freeze-drying process completes, tamponade, outlet, roll lid, lamp inspection, labeling, packaging after and get final product.
9. preparation method according to claim 8, is characterized in that, the program of described freeze-drying is as follows:
A, pre-freeze: first solution is cooled to-12 DEG C～-14 DEG C, then by conduction oil with 1 DEG C/(8-12) speed of min is fallenTo-18～-22 DEG C, insulation 2～3h, is down to after-16～-20 DEG C until products temperature, conduction oil is cooled in 30min to-40± 2 DEG C, insulation 4～5h;
B, lyophilization: in casing, be evacuated to 7～12Pa, with the speed of 1～2 DEG C/h, conduction oil heated up, work as heat conductionOil temperature is incubated after rising to 0 DEG C, and temperature retention time is no less than 30h, and the temperature difference of sample and conduction oil is less than 15～20 DEG C;
C, parsing-desiccation: the speed with every 1 DEG C/8 ~ 12min heats up to conduction oil, be warming up to 40 ± 2 DEG C by goods, insulation 3～5h。
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| CN101444478A (en) * | 2007-11-26 | 2009-06-03 | 天津市金圭谷木糖醇有限公司 | Injection preparation containing active constituent adefovir dipivoxil and preparation method thereof |
| CN102614137A (en) * | 2012-05-02 | 2012-08-01 | 南京臣功制药股份有限公司 | Gemcitabine hydrochloride freeze-dried powder injection and preparation method thereof |
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| CN101444478A (en) * | 2007-11-26 | 2009-06-03 | 天津市金圭谷木糖醇有限公司 | Injection preparation containing active constituent adefovir dipivoxil and preparation method thereof |
| CN102614137A (en) * | 2012-05-02 | 2012-08-01 | 南京臣功制药股份有限公司 | Gemcitabine hydrochloride freeze-dried powder injection and preparation method thereof |
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