CN104043129A - Lyophilized medicinal composition containing recombinant human granulocyte macrophage colony-stimulating factor and preparation method thereof - Google Patents
Lyophilized medicinal composition containing recombinant human granulocyte macrophage colony-stimulating factor and preparation method thereof Download PDFInfo
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- CN104043129A CN104043129A CN201410310593.1A CN201410310593A CN104043129A CN 104043129 A CN104043129 A CN 104043129A CN 201410310593 A CN201410310593 A CN 201410310593A CN 104043129 A CN104043129 A CN 104043129A
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Abstract
The invention relates to the field of medicinal preparations, and particularly relates to a lyophilized medicinal composition containing a recombinant human granulocyte macrophage colony-stimulating factor and a preparation method thereof. The medicinal composition comprises the components as shown in the specification.
Description
Technical field:
The present invention relates to field of pharmaceutical preparations, particularly a kind of freeze-drying medicinal composition that contains recombined human granulocyte-macrophage stimulating factors and preparation thereof.
Background technology:
Recombined human granulocyte-macrophage stimulating factors is a kind of medicine having gone on the market, and the basic condition of relevant this medicine is as follows:
1. pharmacological action: having the effect of hemopoietic cell proliferation and differentiation under rhGM-CSF effect, can differential stimulus grain be the huge propagation of biting CFU-GM of CFU-GM and monokaryon etc.
2. toxicological effect:
2.1 acute toxicities: select clinical people to intend observing 7 mice with dosage 1000-2000 rhGM-CSF doubly with it, result shows that after administration (iv and ip), animal is without death, without abnormal response.Dissect also no abnormality seen.The LD50 ﹥ 5000 μ g/kgs of rhGM-CSF to mice iv, the LD50 ﹥ 1000 μ g/kg of ip.
2.2 long term toxicities:
2.2.1 the long term toxicity of rat: have no overt toxicity reaction to 90 days rhGM-CSF40 μ KD of the continuous lumbar injection of wistar rat, can think that rhGM-CSF40 μ KD is non-toxic, this amount is clinical people's common dose 8 times.RhGM-CSF80 μ KD can produce slight toxic reaction.RhGM-CSF160 μ KD can cause rat loss of appetite, and body weight gain is suppressed.Hemoglobin declines, and urine is occulted blood and protein positive, and creatinine obviously raises, and kidney and liver have obvious pathological change, and prompting rhGM-CSF160 μ KD is the obvious toxic dose of rat, and poisoning target organ is kidney and liver.Above-mentioned variation has recovered or has seen recovery for 30 days after drug withdrawal.
2.2.2 dog long term toxicity test: give Beagle dog continuous intravenous injection 30 days, rhGM-CSF15 μ KD does not produce overt toxicity reaction, can think safe dose.This dosage is that clinical people intends 3 times with dosage and the clinical common dose of same kind of products at abroad.RhGM-CSF45 μ KD can produce slight toxic reaction, and its main manifestations is Some Animals heating, and medullary cell suppresses, and liver and kidney are produced to lighter reversibility pathology damage.RhGM-CSF135 μ KD can produce obvious toxic reaction, shows as intermittent fervescence, loss of appetite, and decreased heart rate, bone marrow nucleated cell number reduces, albuminuria and liver, nephridial tissue Drug pathological change.After drug withdrawal 30 days, Drugs damage was basic recovers for these.The target organ of pointing out its toxic action is the effect tissue of its pharmacological action, bone marrow, liver, kidney.
2.3 hypersensitive test: result shows that rhGM-CSF is without sensitization.(stabilizing agent human albumin also has no sensitization).
[pharmacokinetics]
With sensitive immunization experiment, carry out the pharmacokinetic of people rhGM-CSF, result iv0.3 μ g/kgrhGM-CSF is 14 ± 3 μ g/ml at very first time point maximum serum concentration, in 120 minutes, is eliminated rapidly.Sc0.3 μ g/kg rhGM-CSF, the highest blood drug level is latter 1 hour (30pg/ml) of injection.Iv1 μ g/kg rhGM-CSF blood drug level peak value is 54 ± 26ng/ml.Within 1 hour, drop to <5ng/ml, within 2 hours, be down to 1ng/ml.With 2 hours peakings of dosage Sc blood drug level, be 0.75 ± 0.3ng/ml.It is 1ng/ml that Sc3 μ g/kg just detects peak value for rhGM-CSF15-30 minute.Sc10 μ g/kg rhGM-CSF peak value 5ng/ml.Then maintain 3ng/ml, blood drug level >1ng/ml continues more than 12 hours.After mice Sc125I-GM-CSF, kidney content is the highest, is secondly in harmonization of the stomach blood.In the heart and bone, content is lower, and major part excretes by urine.
In prior art, recombined human granulocyte-macrophage stimulating factors injection is had to multiple report, the present invention finds, existing recombined human granulocyte-macrophage stimulating factors injection has blood vessel irritation when injection, even cause local pain, be unfavorable for the use of medicine, the present invention has carried out further research to formula for this reason, discovery joins valine and fructose in formula, to be prepared into lyophilized formulations together with recombined human granulocyte-macrophage stimulating factors, blood vessel irritation alleviates greatly, can accelerate the redissolution speed of medicine simultaneously, be conducive to the use of lyophilized formulations.
For this reason, the invention provides a kind of freeze-drying medicinal composition that contains recombined human granulocyte-macrophage stimulating factors and preparation method thereof, medicine of the present invention comprises following composition: recombined human granulocyte-macrophage stimulating factors, human albumin, mannitol, sodium hydrogen phosphate, sodium dihydrogen phosphate, valine and fructose.
Summary of the invention:
The invention provides a kind of freeze-drying medicinal composition that contains recombined human granulocyte-macrophage stimulating factors, its formula is composed as follows:
Prescription: every 1000 (1ml/ props up)
Preferably, prescription: every 1000 (1ml/ props up)
Preparation method
Get appropriate room temperature water for injection, add respectively GM-CSF, mannitol, human albumin, the Na of recipe quantity
2hPO
4, NaH
2pO
4h
2o, valine and fructose → add water standardize solution, the aseptic filtration of stirring and evenly mixing → survey pH value (6.7-7.3) → 0.22 μ m filter membrane is to clear and bright → fill → lyophilizing → gland → lamp inspection → packing → finished product.
The present invention finds in research injection process, valine and fructose can reduce blood vessel irritation and local pain, the use amount that the present invention simultaneously finds valine and fructose through screening is at 5mg, this dosage can not produce any side effect, but can play the effect of auxiliary recombined human granulocyte-macrophage stimulating factors.
Below data declaration beneficial effect of the present invention by experiment:
Contrast experiment:
Investigate embodiment 1 and do not add the comparative example of valine and fructose and the indices of the injection recombined human granulocyte-macrophage stimulating factors sample that gone on the market
In addition, experiment is found, adds separately valine or fructose, cannot obtain the effect that above-mentioned valine and fructose add simultaneously, and visible, both have brought into play synergism.
Vascular stimulation test method
9 of white big ear rabbits that select body weight 2.5～3.0kg, male and female half and half, divide three groups, 3 every group at random.The recombined human granulocyte-macrophage stimulating factors sample sets of be made as respectively embodiment 1 sample (adding valine and fructose) group, comparative example 1 sample (not adding valine and fructose) group, having gone on the market.Respectively at auricular vein, give the sample under clinical working concentration, administration in continuous 7 days, 48～96 hours perusal animals and injection site before administration every day and after administration.After last administration, 48～96 hour observation period finished, and got Some Animals medicine-feeding part tissue slice and carried out pathological examination.The animal staying, according to the feature of tested material and irritative response situation, continues to observe and within 14-21 days, carries out histopathological examination again.
Local pain experimental technique
From the Healthy People of voluntary participation experimental study, randomly draw 60 examples, men and women half and half, age 25-55 year.Be divided at random 3 groups, every group of 20 people.The recombined human granulocyte-macrophage stimulating factors sample sets of be made as respectively embodiment 1 sample (adding valine and fructose) group, comparative example 1 sample (not adding valine and fructose) group, having gone on the market.Vein gives after sample, observes experimenter's pain reaction.Adopt Wong-Banker facial expression scale (FPS-R) to assess, indicate the numeral of 0-5 under the types of facial makeup in Beijing operas, numeral is larger, represents that pain intensity is larger.After off-test, adopt SPSS11.5 version software to carry out statistical procedures, the P<0.05 of take has statistical significance as difference.
Dissolution velocity experimental technique
Get recombined human granulocyte-macrophage stimulating factors sample 0.75mg, be dissolved in 10ml sterilized water for injection.Observe sample dissolution situation.
Accelerate 3 months pharmaceutical biology determination of activity experimental techniques
The sample of getting at random 3 batches carries out accelerated test, is placed on and commercially produces in the same or analogous packing container of product, and experimental condition is 25 ℃ ± 2 ℃/RH75% ± 5%, and the investigation time is 3 months.Adopt the biologic activity of TF-1 cell/MTT colorimetric method for determining recombined human granulocyte-macrophage stimulating factors.Method And Principle: TF-1 cell strain is cultivated under 37 ℃, 5% carbon dioxide conditions with complete culture solution, controlling cell concentration is that every 1ml is containing 2.0*10
5～7.0*10
5individual cell, after going down to posterity 24～36 hours for Determination of biological activity, will test the pre-temperature of solution used to 37 ℃.The amount of taking fully TF-1 cell culture, centrifugal and collect TF-1 cell, with basic culture solution washing 3 times, be then resuspended in basic culture solution, be made into every 1ml containing 4.0*10
5the cell suspension of individual cell, put 37 ℃ standby.To being added with in 96 porocyte culture plates of standard solution and need testing solution, add cell suspension, every hole 50 μ l, under 37 ℃, 5% carbon dioxide conditions, cultivate after 48～52 hours, every hole adds people MTT solution 20 μ l, under 37 ℃, 5% carbon dioxide conditions, cultivates 5 hours, more than operate under aseptic condition and carry out, to above-mentioned each hole, add again lysate 100 μ l, after mixing. put people's microplate reader, take 630nm as reference wavelength, at wavelength 570nm place, measure absorbance, Ji Lu Measuring determines result.Test data adopts meter grate machine program or four parametric regression meter grate methods to process, and is calculated as follows result:
Test sample biologic activity (IU/ml)=P
t* (D
s* E%)/(D
t* E
t)
(biologic activity acceptability limit: 80.0%-150.0%)
The specific embodiment:
Embodiment 1
Every 1000 freeze dried injections are prepared from by following component:
Preparation method
Get appropriate room temperature water for injection, add respectively GM-CSF, mannitol, human albumin, the Na of recipe quantity
2hPO
4, NaH
2pO
4h
2o, valine and fructose → add water standardize solution, the aseptic filtration of stirring and evenly mixing → survey pH value (6.7-7.3) → 0.22 μ m filter membrane is to clear and bright → fill → lyophilizing → gland → lamp inspection → packing → finished product.
Comparative example 1
Every 1000 freeze dried injections are prepared from by following component:
Preparation method
Get appropriate room temperature water for injection, add respectively GM-CSF, mannitol, human albumin, the Na of recipe quantity
2hPO
4, NaH
2pO
4h
2o → add water standardize solution, the aseptic filtration of stirring and evenly mixing → survey pH value (6.7-7.3) → 0.22 μ m filter membrane is to clear and bright → fill → lyophilizing → gland → lamp inspection → packing → finished product.
Claims (3)
1. a freeze-drying medicinal composition that contains recombined human granulocyte-macrophage stimulating factors, is characterized in that, fills a prescription as follows:
2. a freeze-drying medicinal composition that contains recombined human granulocyte-macrophage stimulating factors, is characterized in that, fills a prescription as follows:
3. a freeze-drying medicinal composition that contains recombined human granulocyte-macrophage stimulating factors, is characterized in that, fills a prescription as follows:
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107773537A (en) * | 2016-08-18 | 2018-03-09 | 温州医科大学 | The method for preparing the SA hGM CSF bifunctional fusion proteins lyophilized formulations that can be preserved steadily in the long term on a large scale |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1435258A (en) * | 2002-11-26 | 2003-08-13 | 哈药集团技术中心 | Recombinant human granulocyte-macrophage colony stimulating factor gel |
| CN101125126A (en) * | 2006-08-16 | 2008-02-20 | 丛繁滋 | Method for preparing medical freeze-dried powder (injection) preparation |
| CN103720667A (en) * | 2014-01-09 | 2014-04-16 | 中国药科大学 | AP-25 polypeptide freeze-dried powder injection and preparation method and application thereof |
| CN103736078A (en) * | 2014-01-09 | 2014-04-23 | 南京安吉生物科技有限公司 | mPEG (Ethyl Glycolate)-HM-3 polypeptide lyophilized powder injection preparation as well as preparation method and application thereof |
-
2014
- 2014-07-01 CN CN201410310593.1A patent/CN104043129B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1435258A (en) * | 2002-11-26 | 2003-08-13 | 哈药集团技术中心 | Recombinant human granulocyte-macrophage colony stimulating factor gel |
| CN101125126A (en) * | 2006-08-16 | 2008-02-20 | 丛繁滋 | Method for preparing medical freeze-dried powder (injection) preparation |
| CN103720667A (en) * | 2014-01-09 | 2014-04-16 | 中国药科大学 | AP-25 polypeptide freeze-dried powder injection and preparation method and application thereof |
| CN103736078A (en) * | 2014-01-09 | 2014-04-23 | 南京安吉生物科技有限公司 | mPEG (Ethyl Glycolate)-HM-3 polypeptide lyophilized powder injection preparation as well as preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 谭奇凤等: "注射用重组人粒细胞巨噬细胞刺激因子中内毒素的定量检测", 《今日药学》, vol. 24, no. 2, 28 February 2014 (2014-02-28), pages 129 - 131 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107773537A (en) * | 2016-08-18 | 2018-03-09 | 温州医科大学 | The method for preparing the SA hGM CSF bifunctional fusion proteins lyophilized formulations that can be preserved steadily in the long term on a large scale |
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Inventor after: Li Bangdong Inventor after: Jin Xiaohui Inventor after: Sun Shidong Inventor after: He Jingqi Inventor after: Wang Qing Inventor after: Yu Junqing Inventor after: Ge Cunhui Inventor after: Pang Rui Inventor after: Chen Yujun Inventor after: Yang Zhongliang Inventor after: Song Chengjun Inventor after: Qin Weiying Inventor before: Li Bangdong Inventor before: Wang Qing Inventor before: Yu Junqing Inventor before: Ge Cunhui Inventor before: Chen Yujun Inventor before: Pang Rui Inventor before: Yang Zhongliang Inventor before: Song Chengjun Inventor before: Jin Xiaohui |
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Free format text: CORRECT: INVENTOR; FROM: LI BANGDONG WANG QING YU JUNQING GE CUNHUI CHEN YUJUN PANG RUI YANG ZHONGLIANG SONG CHENGJUN JIN XIAOHUI TO: LI BANGDONG WANG QING YU JUNQING GE CUNHUI PANG RUI CHEN YUJUN YANG ZHONGLIANG SONG CHENGJUN QIN WEIYING JIN XIAOHUI SUN SHIDONG HE JINGQI |
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