CN100594930C - Medicinal composition using anterior pituitary adrenal cortical extract as main component, and its preparation method and use - Google Patents
Medicinal composition using anterior pituitary adrenal cortical extract as main component, and its preparation method and use Download PDFInfo
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- CN100594930C CN100594930C CN200510063218A CN200510063218A CN100594930C CN 100594930 C CN100594930 C CN 100594930C CN 200510063218 A CN200510063218 A CN 200510063218A CN 200510063218 A CN200510063218 A CN 200510063218A CN 100594930 C CN100594930 C CN 100594930C
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Abstract
A composite medicine for treating arthritis and viral cold and preventing cancer and injection pain contains corticotropic hormone, thymopeptide and immune ribonucleic acid, which are extracted from anterior pituitary adrenal cortex, and the process for preparing its liquid (or freeze-dried powder) injection are disclosed.
Description
Technical field:
The present invention relates to a kind of medicine, especially relating to a kind of is composition of medicine and preparation method thereof, quality determining method and its application in the medicine of diseases such as preparation treatment rheumatic arthritis, rheumatoid arthritis, viral influenza, hepatitis of Main Ingredients and Appearance with the anterior pituitary adrenal cortical extract.
Background technology:
The medicine that is used for the treatment of rheumatic arthritis, rheumatoid arthritis at present is NSAID (non-steroidal anti-inflammatory drug), glucocorticoids etc. such as aspirin.But glucocorticoid dosage causes easily when big that human immunity suppresses, protein part is separated and to adrenocortical negative feedback; NSAID (non-steroidal anti-inflammatory drug) such as aspirin can be strengthened the ulcer function that causes of glucocorticoid again.Though present existing anterior pituitary adrenal cortical extract injection does not have above-mentioned side effect, the injection pain patient can refuse to use.And medicine of the present invention is taken from the animal body, and avirulence does not have above-mentioned side effect, can not only treat inflammation, the more important thing is and can improve immune function of human body, the radical cure inflammation.
Summary of the invention:
The objective of the invention is to, solve the deficiencies in the prior art, provide two kinds to be the composition of medicine of Main Ingredients and Appearance with the anterior pituitary adrenal cortical extract.
Another object of the present invention provides the preparation method of combinations thereof medicine aqueous injection and lyophilized injection.
The invention still further relates to the application of combinations thereof medicine in the medicine of preparation treatment rheumatism, prevention and alleviation viral influenza, arthritis, tumor and hepatitis adjuvant drug.
Above-mentioned purpose of the present invention can realize by following technical scheme:
Of the present invention is that a kind of of composition of medicine of Main Ingredients and Appearance made by weight ratio by following raw material with the anterior pituitary adrenal cortical extract:
1. thyroliberin 8mg-12mg
2. immune ribonucleic acid 4mg-20mg
3. thymosin 8mg-50mg
4. reduced glutathion 0.5mg-1.0mg
Sterile water for injection 2000mg-5000mg
The preparation method of the aqueous injection of this composition of medicine comprises the steps:
(1) preparation thyroliberin:
1. raw material is handled: qualified pig pituitary and pig adrenal cortex are taken out, remove connective tissue, drop in the analytical pure acetone, slough moisture content after the immersion, till hypophysis and the hardening of pig adrenal cortex; Separately store in the acetone standby respectively adenohypophysis, neurohypophysis in the hypophysis; Take out adenohypophysis and adrenal cortex, wherein adrenal cortex accounts for 1/4 to 1/2 of the two gross weight, places vacuum drying under the room temperature, and pulverizing is crossed 80 mesh sieves, gets water content and is lower than 5% adenohypophysis dry powder;
2. extract: adenohypophysis dry powder 1Kg, add among the 0.5mol/L acetic acid 20L that is preheating to 50 ℃ and stir, slowly the citric acid solution of Dropwise 5 0% is 2.0-2.4 until transferring pH value, being milled to particle diameter in colloid mill is 0.5-1.5 μ m, add the 100g pepsin, be heated to 40-45 ℃, insulation 20min, reheat to 75 ℃ is kept 20min, after making enzyme deactivation, gradually be chilled to below 20 ℃, it is 2.0-2.4 that the citric acid solution with 50% is transferred the pH value value, adds injection-use activated carbon 100g-150g and helps filter, with 0.45 μ m membrane filtration 2 times, 2 filtrates are merged, and transferring pH value with 10% caustic lye of soda is 3.1-3.4, centrifugal 30 minutes in 4000 rev/mins with the 6L refrigerated centrifuger, centrifuging temperature is 0-2 ℃, supernatant molecular cut off is 100000 Hollow Fiber Ultrafiltration post ultrafiltration, and filtrate reuse molecular cut off is 10000 Hollow Fiber Ultrafiltration post ultrafiltration, and filtrate reuse molecular cut off is 8000 ultrafilter membrane ultrafiltration; The ultrafiltrate lyophilization becomes the thyroliberin dry product of moisture content≤3% standby;
(2) preparation thymosin:
1. raw material is handled: soak through refrigerated calf thymus with deionized water, back rejecting connective tissue and film and residual meat thaw, and clean repeatedly with deionized water, get rid of water purification, 10Kg thymus is blended in the meat grinder with 10Kg water for injection rubs, in colloid mill, grinding homogenate 2-3 time below 10 ℃, until particle diameter is 0.5-1.5 μ m, it is 1.5-2.0 that ground homogenate is transferred pH value with 50% citric acid solution, add pepsin 800g, be warming up to 40-50 ℃, behind the insulation 20-30min, rise to again 75 ℃ keep 20min and make enzyme deactivation after, reduce to room temperature rapidly, freezing 24 hours in the inherent sterilizing room of the stainless steel cask with cover of packing in-28 ℃, take out back room temperature negative catalysis 8 hours, again-28 ℃ of quick-freezings 24 hours, again in the room temperature negative catalysis after 8 hours, be heated to 60-70 ℃, reduce to below 30 ℃ more rapidly, put into the 6L refrigerated centrifuger in 0-2 ℃, 4000-8000 rev/min centrifugal 35 minutes, get supernatant, centrifuged supernatant is earlier 100000 Hollow Fiber Ultrafiltration post ultrafiltration through molecular cut off, the reuse molecular cut off is 10000 Hollow Fiber Ultrafiltration post ultrafiltration, the reuse molecular cut off is that 8000 ultrafilter membrane carries out fine straining, and it is standby that the thymosin dry product that is dried to moisture content≤3% is frozen in the fine straining liquid cooling;
(3) preparation immune ribonucleic acid:
1. raw material is handled: the sheep of selecting for 1 one full year of life, check free from infection, after Brucella and the cysticercus, 2-3mg/ml bacillus calmette-guerin vaccine dead or expire is added time-consuming adjuvant make Water-In-Oil shape emulsifying agent, in the sheep lymph node, inject, every sheep injection 1ml, massacre after 12-14 days, get liver, spleen, the heart, lung and kidney, make to extract the raw material of immune ribonucleic acid, clean with normal saline, be twisted into fragment, 1: 1: 1 by volume: 1 adds 1-2 doubly measured volume 0.1mol/L sodium chloride, 0.05mol/L trisodium citrate, 0.001% polyvinyl sulfuric acid ester, the 0.1%PH value is that (TritonX-100 is a surfactant for 9 TritonX-100, external trade name), with colloid mill homogenate 2-3 time, wearing into diameter is 0.5-1.5 μ m particle, in 0-2 ℃, 3500 rev/mins of centrifugal 20min, get the supernatant, the triethanolamine that in centrifugal clear liquid, adds equal-volume 0.2mol/L, the 10g/L sodium lauryl sulphate, 0.002mol/L pH value is 9 disodiumedetate, add equal-volume 90% phenol after stirring, the equal-volume chloroform, 30min vibrates under room temperature, 3500 rev/mins of centrifugal 20min, extract clear liquid, add the equal amounts of chloroform jolting again, till not having obvious albumin layer to the interface, intermediate layer, proteic extracting solution is removed, supernatant adds the 200g/L potassium acetate and the two volumes of 1/10 volume, be chilled to-20 ℃ dehydrated alcohol, placed 1 hour in 0 ℃, centrifugal, get white precipitate, in the sodium acetate of resolution of precipitate in the disodiumedetate that contains 0.001mol/L to 0.01mol/L, add equal-volume 2.5mol/L dipotassium hydrogen phosphate, under continuous stirring, add isopyknic ethylene glycol monomethyl ether again, place half an hour for 0 ℃, centrifugal, the extracting solution of glycogen is removed, clear liquid is under 0 ℃ of continuous stirring, add equal-volume 0.2mol/L sodium acetate liquid and 1/4 volume 10g/L cetyltrimethylammonium base amine, place half an hour for 0 ℃, centrifugal, get precipitation, the sodium acetate liquid that contains 0.001mol/L ethylenediamine tetraacetic ethanedioic acid disodium with 0.1mol/L, 70% ethanol cyclic washing, till non-foam, with resolution of precipitate in the 0.001mol/L disodiumedetate, elder generation is 8000 hollow fiber column ultrafilter with molecular cut off, reuse 0.22 μ m film aseptic filtration, measure content, aseptic canning, lyophilization, the immune ribonucleic acid dry product of moisture content≤3%;
(4) by number order 1.～4. mentioned above various raw materials are dissolved in sterilized water for injection fully, after transferring pH value to be 2.5-3.0 with 8% hydrochloric acid, with 0.22 μ m membrane filtration degerming, aseptic canning, with every bottle of fill 3-4ml of the inferior woods bottle of 7ml, and carry out the false add plug with butyl rubber plug, sending in the drying baker at vacuum pressure is lyophilization under the 40Pa-1.5Pa, making its residual moisture content≤2%, is the 95%-120% of antepituitary and adrenal cortex extract labelled amount by polypeptide, immune ribonucleic acid be the quality standard of the 95%-120% of labelled amount detect qualified after, packing is entered the storehouse.
Initial sum terminal point pH value is 2.5-3.0 in the preparation process of this aqueous injection.
Of the present invention is that the another kind of composition of medicine of Main Ingredients and Appearance is made by weight ratio by following raw material with the anterior pituitary adrenal cortical extract:
1. thyroliberin 8mg-12mg
2. immune ribonucleic acid 4mg-20mg
3. thymosin 8mg-50mg
4. reduced glutathion 0.5mg-2.0mg
5. low molecular dextran-40 2mg-4mg
6. mannitol 7mg-9mg
Sterile water for injection 3000mg-4000mg.
The preparation method of the lyophilized injection of this composition of medicine comprises the steps:
(1) preparation thyroliberin:
1. raw material is handled: qualified pig pituitary and pig adrenal cortex are taken out, remove connective tissue, drop in the analytical pure acetone, slough moisture content after the immersion, till hypophysis and the hardening of pig adrenal cortex; Separately store in the acetone standby respectively adenohypophysis, neurohypophysis in the hypophysis; Take out adenohypophysis and adrenal cortex, wherein adrenal cortex accounts for 1/4 to 1/2 of the two gross weight, places vacuum drying under the room temperature, and pulverizing is crossed 80 mesh sieves, gets water content and is lower than 5% adenohypophysis dry powder;
2. extract: adenohypophysis dry powder 1Kg, add among the 0.5mol/L acetic acid 20L that is preheating to 50 ℃ and stir, slowly the citric acid solution of Dropwise 5 0% is 2.0-2.4 until transferring pH value, being milled to particle diameter in colloid mill is 0.5-1.5 μ m, add the 100g pepsin, be heated to 40-45 ℃, insulation 20min, reheat to 75 ℃ is kept 20min, after making enzyme deactivation, gradually be chilled to below 20 ℃, it is 2.0-2.4 that the citric acid solution with 50% is transferred pH value, adds injection-use activated carbon 100g-150g and helps filter, with 0.45 μ m membrane filtration 2 times, 2 filtrates are merged, and transferring pH value with 10% caustic lye of soda is 3.1-3.4, centrifugal 30 minutes in 4000 rev/mins with the 6L refrigerated centrifuger, centrifuging temperature is 0-2 ℃, supernatant molecular cut off is 100000 Hollow Fiber Ultrafiltration post ultrafiltration, and filtrate reuse molecular cut off is 10000 Hollow Fiber Ultrafiltration post ultrafiltration, and filtrate reuse molecular cut off is 8000 ultrafilter membrane ultrafiltration; The ultrafiltrate lyophilization becomes the thyroliberin dry product of moisture content≤3% standby;
(2) preparation thymosin:
1. raw material is handled: soak through refrigerated calf thymus with deionized water, back rejecting connective tissue and film and residual meat thaw, and clean repeatedly with deionized water, get rid of water purification, 10Kg thymus is blended in the meat grinder with 10Kg water for injection rubs, in colloid mill, grinding homogenate 2-3 time below 10 ℃, until particle diameter is 0.5-1.5 μ m, it is 1.5-2.0 that ground homogenate is transferred pH value with 50% citric acid solution, add pepsin 800g, be warming up to 40-50 ℃, behind the insulation 20-30min, rise to again 75 ℃ keep 20min and make enzyme deactivation after, reduce to room temperature rapidly, freezing 24 hours in the inherent sterilizing room of the stainless steel cask with cover of packing in-28 ℃, take out back room temperature negative catalysis 8 hours, again-28 ℃ of quick-freezings 24 hours, again in the room temperature negative catalysis after 8 hours, be heated to 60-70 ℃, reduce to below 30 ℃ more rapidly, put into the 6L refrigerated centrifuger in 0-2 ℃, 4000-8000 rev/min centrifugal 35 minutes, get supernatant, centrifuged supernatant is earlier 100000 Hollow Fiber Ultrafiltration post ultrafiltration through molecular cut off, the reuse molecular cut off is 10000 Hollow Fiber Ultrafiltration post ultrafiltration, the reuse molecular cut off is that 8000 ultrafilter membrane carries out fine straining, and it is standby that the thymosin dry product that is dried to moisture content≤3% is frozen in the fine straining liquid cooling;
(3) preparation immune ribonucleic acid:
1. raw material is handled: the sheep of selecting for 1 one full year of life, check free from infection, after Brucella and the cysticercus, 2-3mg/ml bacillus calmette-guerin vaccine dead or expire is added time-consuming adjuvant make Water-In-Oil shape emulsifying agent, in the sheep lymph node, inject, every sheep injection 1ml, massacre after 12-14 days, get liver, spleen, the heart, lung and kidney, make to extract the raw material of immune ribonucleic acid, clean with normal saline, be twisted into fragment, 1: 1: 1 by volume: 1 adds 1-2 doubly measured volume 0.1mol/L sodium chloride, 0.05mol/L trisodium citrate, 0.001% polyvinyl sulfuric acid ester, the 0.1%PH value is 9 TritonX-100, with colloid mill homogenate 2-3 time, wearing into diameter is 0.5-1.5 μ ml particle, in 0-2 ℃, 3500 rev/mins of centrifugal 20min, get the supernatant, the triethanolamine that in centrifugal clear liquid, adds equal-volume 0.2mol/L, the 10g/L sodium lauryl sulphate, 0.002mol/L pH value is 9 disodiumedetate, add equal-volume 90% phenol after stirring, the equal-volume chloroform, 30min vibrates under room temperature, 3500 rev/mins of centrifugal 20min, extract clear liquid, add the equal amounts of chloroform jolting again, till not having obvious albumin layer to the interface, intermediate layer, proteic extracting solution is removed, supernatant adds the 200g/L potassium acetate and the two volumes of 1/10 volume, be chilled to-20 ℃ dehydrated alcohol, placed 1 hour in 0 ℃, centrifugal, get white precipitate, in the sodium acetate of resolution of precipitate in the disodiumedetate that contains 0.001mol/L to 0.01mol/L, add equal-volume 2.5mol/L dipotassium hydrogen phosphate, under continuous stirring, add isopyknic ethylene glycol monomethyl ether again, place half an hour for 0 ℃, centrifugal, the extracting solution of glycogen is removed, clear liquid is under 0 ℃ of continuous stirring, add equal-volume 0.2mol/L sodium acetate liquid and 1/4 volume 10g/L cetyltrimethylammonium base amine, place half an hour for 0 ℃, centrifugal, get precipitation, the sodium acetate liquid that contains 0.001mol/L ethylenediamine tetraacetic ethanedioic acid disodium with 0.1mol/L, 70% ethanol cyclic washing, till non-foam, with resolution of precipitate in the 0.001mol/L disodiumedetate, use earlier the 8000K hollow fiber column ultrafilter, aseptic filtration again, measure content, aseptic canning, lyophilization, the immune ribonucleic acid dry product of moisture content≤3%;
(4) by number order 1.～6. mentioned above various raw materials are dissolved in sterilized water for injection fully, after transferring pH value to be 2.5-3.0 with 8% hydrochloric acid, with 0.22 μ m membrane filtration degerming, aseptic canning, with every bottle of fill 3-4ml of the inferior woods bottle of 7ml, and carry out the false add plug with butyl rubber plug, sending in the drying baker at vacuum pressure is lyophilization under the 40Pa-1.5Pa, making its residual moisture content≤2%, is the 95%-120% of antepituitary and adrenal cortex extract labelled amount by polypeptide, immune ribonucleic acid be the quality standard of the 95%-120% of labelled amount detect qualified after, packing is entered the storehouse.
Initial sum terminal point pH value is 2.5-3.0 in the preparation process of this aqueous injection.
Thyroliberin, thymosin, immune ribonucleic acid are subject to photooxidation, easily be heated and prolong standing time and cause its active reduction, if in their solution, add the glutathion of reduced form, then can keep their activity constant (seeing Table) for a long time.
Table one: the active contrast table that keeps
| Room temperature 40W uviol lamp is according to 24h | 121 ℃ of heating 30min | Placed 1 year the airtight dark place of room temperature | |
| 8mg/ml concentration thyroliberin liquid | Active reduction is more than 20% | Active reduction by 25% | Active reduction by 10% |
| Add 0.5mg/ml glutathion liquid in the 8mg/ml concentration thyroliberin liquid | Active reduction by 10% | Active reduction by 15% | Active reduction by 25% |
| 4mg/ml immune ribonucleic acid solution | Active reduction by 15% | Active reduction by 10% | Active reduction by 10% |
| Add 0.5mg/ml glutathion liquid in the 4mg/ml immune ribonucleic acid liquid | Active reduction by 8% | Active reduction by 6% | Active reduction by 2% |
| The 12mg/ml thymus peptide solution | Active reduction by 16% | Active reduction by 20% | Active reduction by 12% |
| Add 0.5mg/ml glutathion liquid in the 12mg/ml thymosin liquid | Active reduction by 10% | Active reduction by 8% | Active reduction by 3% |
The character of this product aqueous injection is little yellow clear liquid, and the character of lyophilized injection is that white is block or Powdered.
Composition of medicine of the present invention can not only keep its activity for a long time, and remarkable to disease effects such as treatment of arthritis, viral influenza, to also very effective (seeing Table two) such as prophylaxis of cancer, protective inoculation pain.
Table two: clinical practice contrast test
| Arthritis effective percentage (totally 30 examples) | Treatment viral influenza remission rate (totally 60 examples) | Feel sick, vomit (totally 30 examples) | Injection pain (totally 60 examples) | |
| Aspirin-d1-lysine intravenous drip 2 each 0.9g every day | 87％ | 78％ | 21％ | Slight 19% |
| Anterior pituitary adrenal cortical extract injection 2 each 16mg every day | 89％ | ------ | Do not have | 65％ |
| Medicine aqueous injection intramuscular injection of the present invention every day contain active substance total amount 20mg 2 times at every turn | 92.5％ | 95％ | Do not have | Slight 8% |
| Medicine aqueous injection intramuscular injection of the present invention every day contain active substance total amount 45mg 1 time at every turn | 93％ | 95％ | Do not have | Slight 8.5% |
| Medicine aqueous injection intramuscular injection of the present invention every day contain active substance total amount 82mg 1 time at every turn | 94％ | 95％ | Do not have | Slight 9% |
| Medicament freeze-drying injection intramuscular injection of the present invention every day contain active substance total amount 20mg 2 times at every turn | 96％ | 97％ | Do not have | Slight 6% |
| Medicament freeze-drying injection intravenous drip of the present invention every day contain active substance total amount 45mg 1 time at every turn | 96.5％ | 97％ | Do not have | Slight 7% |
| Medicament freeze-drying injection intravenous drip of the present invention every day contain active substance total amount 82mg 1 time at every turn | 97％ | 98％ | Do not have | Slight 9.5% |
Quality of the pharmaceutical preparations detection method
This product is aseptic aqueous solution injection or the lyophilized injection that the immune ribonucleic acid that extracts in the liver, spleen, the heart, lung, kidney of pig pituitary frontal lobe and the adrenal cortex extract thymosin that adds calf thymus and extract, immune sheep is formed.Contain the 95%-120% that polypeptide should be antepituitary and adrenal cortex extract labelled amount; Contain the 95%-120% that immune ribonucleic acid should be labelled amount.
Differentiate
1. polypeptide is differentiated
(1) gets this product 2ml, add biuret reagent (get copper sulfate 0.75g and sodium potassium tartrate tetrahydrate 3g, add water 250ml and make its dissolving, under agitation add 10% sodium hydroxide solution 150ml, add water to 500ml) 4ml, place, should show purple or aubergine.
(2) get this product, measure according to spectrophotography (two appendix VI of Chinese Pharmacopoeia version in 2000 H), there is absorption maximum at the place at the 268nm wavelength.
2. immune ribonucleic acid is differentiated
Get this product and add water and make the solution that contains 100 μ g immune ribonucleic acids among every 1ml, get in right amount, add isopyknic 3,5-orcin test solution (get ferric chloride and 3, each 0.1g of 5-orcin adds hydrochloric acid 100ml dissolving), mixing is put in the boiling water bath and was heated 10 minutes, promptly shows yellow green.
Check
PH: should be 2.5-3.0 (two appendix VI of Chinese Pharmacopoeia version in 2000 H)
Trap: (1) polypeptide: precision is measured this product 2ml, puts in the 100ml bottle, adds water to scale, shakes up, and measures according to spectrophotography (two appendix VI of Chinese Pharmacopoeia version in 2000 H), and at 268nm wavelength place, trap is more than 0.288.
(2) immune ribonucleic acid: get 3 of this product, adding 0.9% sodium chloride liquid respectively makes and contains 20 μ g ribonucleic acid liquid in every every milliliter, measure according to spectrophotography (two appendix VI of Chinese Pharmacopoeia version in 2000 A), should there be absorption maximum at the place at (260 ± 2) nm wavelength, and 260nm and 280nm wavelength place absorbance ratio must not be less than 2.0.
Arginine and lysine:
Measure according to high performance liquid chromatography (two appendix V of Chinese Pharmacopoeia version in 2000 D), measure with suitable amino-acid analyzer, every 1ml contains arginine and should be 0.20-0.30mg, and lysine should be 0.40-0.60mg.
The preparation of DNA (deoxyribonucleic acid) reference substance solution:
It is an amount of to take by weighing the DNA reference substance, makes the solution that contains 0.1mg among every 1ml with the 0.005mol/L sodium hydroxide solution.
The preparation of need testing solution: get this product, make the solution of qiagen rnase 1.00mg among every 1ml with the 0.005mol/L sodium hydroxide solution.
Algoscopy: precision is measured DNA reference substance solution and each 2ml of need testing solution, put respectively in the tool plug test tube, respectively add the diphenylamines test solution and (get diphenylamines 1g, add glacial acetic acid 100ml and perchloric acid 10ml, dissolving, face acetaldehyde solution 1ml with preceding adding 1.6%) 4.0ml, put 60 ℃ of heated in water solution 50 minutes, cooling rapidly, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A), wavelength place at 600nm measures trap, and the solution absorption degree of test sample should be not more than the trap of reference substance solution.
The vitality test of immune ribonucleic acid: Hanks liquid, take by weighing sodium chloride 16g, sodium hydrogen phosphate 0.304g, potassium dihydrogen phosphate 0.136g, potassium chloride 0.8g, glucose 2g, being dissolved in water becomes 200ml, and sterilization is deposited for 4 ℃, face with 10 times of preceding dilutions, regulating PH with 10% sodium bicarbonate solution is 7.2-7.4.Antigenic solution is made into the solution that contains 5-10 μ g among every 1ml with Hanks liquid with hepatitis B antigen, freezing preservation.
Mouse boosting cell suspension preparation: gather fresh healthy mice spleen, be placed in the Hanks liquid, the lymphocyte of gently extruding out removes by filter thick piece through gauze, filtrate is added in the centrifuge tube upper strata that the 4ml lymphocyte separation medium is housed gently, makes the interface smooth, leave the heart after 15 minutes with per minute 2000, the leukocytic cream of absorption interface, reuse Hanks liquid is washed 2 times, counts with cell counting count board, makes among every 1ml with culture fluid to contain (1 * 10
1)～(2 * 10
2) individual leukocytic solution for later use.
Algoscopy: in 24 well culture plates, carry out, 3 holes are respectively done in test sample hole and reference substance hole, test sample is made the solution that contains ribonucleic acid 1mg among every 1ml with Hanks liquid, as need testing solution, every hole adds 150 μ l, adds Hanks liquid 150 μ l in the control wells, every then hole adds mouse boosting cell suspension 150 μ l, add antigen liquid 150 μ l again, in 87 ℃ of insulations 2 hours, cell is sticked in the culture plate bottom culture plate.Take out Tissue Culture Plate, under certain intensity, shake 5min with the constant-temperature shaking culture case, adherent cell has not shaken, the sucking-off supernatant, every hole adds Hanks liquid 450 μ l again, wash once by last method, washing liquid and supernatant are merged mixing, the trap value of measuring every hole at the wavelength place of 415nm is as the trap value of sticking the back cell.Other gets need testing solution 150 μ l, add Hanks liquid 450 μ l and mouse boosting cell suspension 150 μ l, add antigenic solution 150 μ l mixings again, measure the trap value as sticking precellular trap value at the wavelength place of 415nm, replace need testing solution 150 μ l with Hanks liquid 150 μ l in addition, with the method operation, stick precellular trap value in contrast.
Be calculated as follows:
Have only contrast adherence rate 〉=40%, trap value this result of the test between 0.1-0.4 is effective.The suppression ratio that sticks of test sample must not be less than 30%.
The determination of activity of polypeptide: it is an amount of to get this product, takes off the E receptor method according to T cytoactive algoscopy one and measures, and the difference of the percentage rate of the E rosette percentage rate of test sample and control tube E rosette must not be lower than 10.0%.
This medicine committee member of algoscopy country has special mensuration regulation.
Thymosin a
1: in the chromatogram that under the high molecular weight material item, writes down, press area normalization method and calculate, in the test sample corresponding to thymosin a
1The content at the peak of retention time must not be lower than 1.0%.
High molecular weight material: according to high effective liquid chromatography for measuring (two appendix V of Chinese Pharmacopoeia version in 2000 D).
Chromatographic condition and system suitability test: with gel chromatographic columns (as TSK GEL2000SWX17.8mm * 300mm, 5 μ m); Inferior trifluoracetic acid one acetonitrile, one water (0.05: 10: 90) is mobile phase; Flow velocity is per minute 0.7ml; The detection wavelength is 214nm.Theoretical cam curve is calculated by ribonuclease A and should be lower than 5000, separating degree by the ratio of adjacent peak height and peak valley calculate equal must not be less than 3.0.
The preparation of standard curve: get ribonic acid enzyme A (molecular weight 13700), insulin (molecular weight 5808), thymosin a respectively
1(molecular weight 3108), somatostatin (molecular weight 1521) are an amount of, make the solution that contains 1mg among every 1ml as molecular weight standard solution with mobile phase.Get molecular weight standard solution 20ml respectively and inject chromatograph of liquid, the record chromatogram repeats sample introduction, and its retention time relative deviation must not be greater than 2.0%.Retention time with each peak is an abscissa, and the molecular weight logarithm is that vertical coordinate is made standard curve, carries out linear regression, and coefficient must not be less than 0.99.
Algoscopy: it is an amount of to get test sample, adds mobile phase and makes the solution that contains polypeptide 1mg among every 1ml, gets 20 μ g and injects chromatograph of liquid, and the record chromatogram calculates with area normalization method, and molecular weight must not surpass 5.0% greater than 10000 daltonian components in the test sample.
Hypersensitive test: get this product, add the chlorination sodium injection, make the solution that contains the 1mg immune ribonucleic acid among every 1ml, as sensitization liquid and test sample liquid.Stipulate specially to check according to Chinese Pharmacopoeia Commission, should be up to specification.
The undue toxicity: get this product, add the chlorination sodium injection and make the solution that every 1ml contains immune ribonic acid 0.5mg, check in accordance with the law and (two appendix XI of Chinese Pharmacopoeia version in 2000 C) press the intraperitoneal injection administration, should be up to specification.
Pyrogen: get this product, make the solution that contains the 1mg immune ribonucleic acid among every 1mt with sodium chloride injection, check (two appendix XI of Chinese Pharmacopoeia version in 2000 D) in accordance with the law, dosage should be up to specification by the every 1kg injection of rabbit body weight 1ml.
Other: should meet every regulation relevant under the injection item (two appendix I of Chinese Pharmacopoeia version in 2000 B).
[assay]:
The immune ribonucleic acid assay:
Get 3 of this product, add 0.9% sodium chloride solution respectively and make the solution that contains 20 μ g ribonucleic acid among every 1ml,, measure trap, press the trap coefficient E of ribonucleic acid at the wavelength place of 260nm according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A)
1% 1cmBe 220 calculating, make content must not be lower than 90% of labelled amount.If any 1 against regulation, other gets 3, the result all should be up to specification.
Determining content of peptides: it is an amount of to get this product, adds water and makes the solution that contains polypeptide 0.15mg among every 1ml, and as need testing solution, precision is measured 1ml, measures according to forint phenol method.
There is special regulation in forint phenol method Chinese Pharmacopoeia Commission.
The specific embodiment
Below in conjunction with specific embodiment the present invention is further detailed, does not therefore limit the present invention among the described scope of embodiments.
3 kinds of active substances of preparation the present invention: thyroliberin, thymosin and immune ribonucleic acid:
Embodiment 1
The preparation of thyroliberin (ACTH):
(1) raw material is handled
Qualified pig pituitary and pig adrenal cortex are taken out, remove connective tissue (fascia) rapidly, drop into rapidly in the analytical pure acetone, soak dehydration 3 times, each about 24 hours, till hypophysis and the hardening of pig adrenal cortex.Separately store in the acetone standby respectively adenohypophysis, neurohypophysis in the hypophysis.This law is used adenohypophysis, takes out adenohypophysis and adrenal cortex (wherein adrenal cortex accounts for the 1/4-1/2 of the two gross weight), places vacuum drying under the room temperature, and pulverizing is crossed 80 mesh sieves, gets adenohypophysis dry powder, and water content should be lower than 5%.
(2) extract:
With adenohypophysis dry powder 1Kg (comprising that adrenal cortex accounts for part by weight 1/4-1/2), add and be preheating to 50 ℃ 0.5mol/L acetic acid 20L, stir, slowly Dropwise 5 0% citric acid solution is 2.0-2.4 until transferring pH value, being milled to particle diameter in colloid mill is to add the 100g pepsin about 1 μ m, heating makes temperature reach 40-45 ℃, insulation 20min, reheat to 75 ℃ is kept 20min, after making enzyme deactivation, gradually cold below 20 ℃, be 2.0-2.4 with 50% citric acid solution (citric acid is an analytical pure) accent pH value, add injection-use activated carbon 100g-150g and help filter, with 0.45 μ m membrane filtration.2 filtrates are merged, and transferring PH with 10% sodium hydroxide (analytical pure) liquid is 3.1-3.4.With the 6L refrigerated centrifuger in 4000 rev/mins centrifugal 30 minutes (centrifuging temperature is 0-2 ℃), the upper strata stillness of night is used the ultrafiltration of 100K Hollow Fiber Ultrafiltration post, the ultrafiltration of filtrate reuse 10K Hollow Fiber Ultrafiltration post, filtrate reuse molecular cut off is 8000 ultrafilter membrane ultrafiltration, and the ultrafiltrate lyophilization becomes dry product standby (moisture content≤3%).
Embodiment 2
The preparation of thymosin:
Soak through refrigerated calf thymus (weight ratio 2: 1) with purified water (deionized water), back rejecting connective tissue and film and residual meat thaw, and clean repeatedly 4 times with purified water, get rid of water purification, 10Kg thymus is blended in the meat grinder with 10Kg water for injection rubs, in colloid mill, grinding homogenate 2-3 time below 10 ℃, until about particle size 1 μ m, it is 1.5-2.0 that ground homogenate is transferred pH value with 50% citric acid solution, add pepsin 800g, be warming up to 40-50 ℃, behind the insulation 20-30min, rise to again 75 ℃ keep 20min and make enzyme deactivation after, reduce to room temperature rapidly, with 316 stainless steel casks (with cover) in sterilizing room in-28 ℃ freezing 24 hours, take out back room temperature negative catalysis 8 hours, again-28 ℃ of quick-freezings 24 hours, after 8 hours, be heated to 60-70 ℃ in the room temperature negative catalysis again, reduce to rapidly again below 30 ℃, put into the 6L refrigerated centrifuger in 0-2 ℃, 4000-8000 rev/min centrifugal 35 minutes, get supernatant.
Centrifugal supernatant is earlier 100000 Hollow Fiber Ultrafiltration post ultrafiltration through molecular cut off, the reuse molecular cut off is 10000 Hollow Fiber Ultrafiltration post ultrafiltration, the reuse molecular cut off is that 8000 ultrafilter membrane carries out fine straining, and the fine straining liquid cooling is frozen and is dried to dry product standby (moisture content≤3%).
Embodiment 3
The preparation of immune ribonucleic acid (iRNA):
Select the sheep at an age, inspection has free from infection, after Brucella and the cysticercus, 2-3mg/ml bacillus calmette-guerin vaccine dead or expire is added time-consuming adjuvant (1 part of lanoline, 1 part of liquid paraffin, mix, autoclaving, 4 ℃ of preservations are standby) make Water-In-Oil shape emulsifying agent, in the sheep lymph node, inject every sheep injection 1ml, massacre after 12-14 days, get liver, spleen, the heart, lung and kidney, work extracts the raw material of iRNA, cleans with normal saline, be twisted into fragment with meat grinder, add 1-2 and doubly measure volume 0.1mol/L sodium chloride-0.05mol/L trisodium citrate, 0.001% polyvinyl sulfuric acid ester (PVS), 0.1%TritonX-100 (PH7) with colloid mill homogenate 2-3 time, wears into 1 μ m left and right sides particle, in 0-2 ℃, 3500 rev/mins of centrifugal 20min get the upper strata stillness of night.
The triethanolamine of adding equal-volume 0.2mol/L, 10g/L (1%) sodium lauryl sulphate (SDS), 0.002mol/L disodiumedetate (EDTA) are (PH9) in the centrifugal stillness of night, add equal-volume 90% phenol (containing the 0.2%8-hydroxyquinoline), equal-volume chloroform after stirring, 30min vibrates under room temperature, 3500 rev/mins of centrifugal 20min, must extract the stillness of night, add the equal amounts of chloroform jolting again, 2-3 time repeatedly, till not having obvious albumin layer to the interface, intermediate layer, the centrifugal Deproteinization of removing, collect the stillness of night, proteic extracting solution is removed.
Supernatant add 1/10 volume 200g/L (20%) potassium acetate and two volumes, be chilled to-20 ℃ dehydrated alcohol, placed 1 hour in 0 ℃, centrifugal, white precipitate, resolution of precipitate is got disodiumedetate and gets in the sodium acetate to 0.01mol/L in containing 0.001mol/L, add equal-volume 2.5mol/L dipotassium hydrogen phosphate, under continuous stirring, add isopyknic ethylene glycol monomethyl ether again, place half an hour for 0 ℃, centrifugal, the extracting solution of the glycogen that is removed.
The stillness of night is under 0 ℃ of continuous stirring, add equal-volume 0.2mol/L sodium acetate liquid and 1/4 volume 10g/L (1%) cetyltrimethylammonium base amine (CTAB), place half an hour for 0 ℃, centrifugal, get precipitation, with 0.1mol/L sodium acetate liquid (containing 0.001mol/L ethylenediamine tetraacetic ethanedioic acid disodium), 70% ethanol cyclic washing 5 times, till non-foam.
With resolution of precipitate in 0.001mol/L disodiumedetate (containing 0.14mol/L sodium chloride).Earlier use the 8000K hollow fiber column ultrafilter, content is measured in aseptic filtration again (with 0.22 μ m membrane filtration), aseptic canning, and lyophilization gets iRNA dry product (moisture content≤3%).
Prepare aqueous injection of the present invention:
Embodiment 1
It is as follows to get raw material by weight ready:
1. thyroliberin 8mg * 1000
2. immune ribonucleic acid 4mg * 1000
3. thymosin 8mg * 1000
4. reduced glutathion 0.5mg * 1000
Sterile water for injection 2000mg * 1000
(initial sum terminal point PH is 2.5-3.0)
1000 of packing (every contains 3 kinds of active ingredient total amounts is 20mg)
Production operation:
1. the 8000mg thyroliberin is dissolved in the sterile water for injection that 1600mlPH is 2.5-3.0, stirring and dissolving is complete, adds (stirring down) immune ribonucleic acid 4000mg again, and stirring and dissolving is complete; Add (stirring down) thymosin 8000mg again, dissolving fully; Add reduced glutathion 500mg again, stirring and dissolving is complete; Adding PH again is that 2.5-3.0 sterile water for injection to total amount is 2000ml, is 2.5-3.0 with 8% hydrochloric acid solution readjustment PH;
With medicinal liquid with 0.22 μ m membrane filtration degerming;
3. under aseptic (100 grades) condition, with 2.0ml cillin bottle fill through the sterilization depyrogenation, every loading amount 2.0ml, and seal with the butyl rubber plug through the sterilization depyrogenation;
4. detect qualified back, pack the storehouse by quality standard.
Embodiment 2
It is as follows to get raw material by weight ready
1. thyroliberin 10mg * 1000
2. immune ribonucleic acid 15mg * 1000
3. thymosin 20mg * 1000
4. reduced glutathion 0.75mg * 1000
Sterile water for injection 5000mg * 1000
(initial sum terminal point PH is 2.5-3.0)
1000 of packing (every contains 3 kinds of active ingredient total amounts is 45mg)
The production operation step is identical with embodiment 1, but every fill amount of present embodiment is 5.0ml.
Embodiment 3
It is as follows to get raw material by weight ready
1. thyroliberin 12mg * 1000
2. immune ribonucleic acid 20mg * 1000
3. thymosin 50mg * 1000
4. reduced glutathion 1.0mg * 1000
Sterile water for injection 5000mg * 1000
(initial sum terminal point PH is 2.5-3.0)
1000 of packing (every contains 3 kinds of active ingredient total amounts is 82mg)
The production operation step is identical with embodiment 1, but every fill amount of present embodiment is 5.0ml
Prepare lyophilized injection of the present invention
Embodiment 1
It is as follows to get raw material by weight ready
1. thyroliberin 8mg * 1000
2. immune ribonucleic acid 4mg * 1000
3. thymosin 8mg * 1000
4. reduced glutathion 0.5mg * 1000
5. low molecular dextran-40 2mg * 1000
6. mannitol 7mg * 1000
Sterile water for injection 3000mg * 1000
(initial sum terminal point PH is 2.5-3.0)
1000 of packing (every contains 3 kinds of active ingredient total amounts is 20mg)
Production operation:
1. with the 8000mg thyroliberin under agitation, add in the sterile water for injection of 1600ml, it is 2.5-3.0 (transferring pH value with 8% hydrochloric acid) that sterile water for injection is transferred PH in advance, and stirring and dissolving is complete; Add (stirring down) immune ribonucleic acid 4000mg again, stirring and dissolving is complete; Add (stirring down) thymosin 8000mg again, dissolving fully; Add reduced glutathion 500mg again, stirring and dissolving is complete; Add 2000mg low molecular dextran-40 again, dissolving under agitation fully; Add 7000mg mannitol again, dissolving under agitation fully; It is 2.5-3.0 that reuse 8% hydrochloric acid solution is transferred medicinal liquid PH;
With medicinal liquid with 0.22 μ m membrane filtration degerming;
3. under aseptic (100 grades) condition, with 7.0ml cillin bottle fill through the sterilization depyrogenation, every loading amount 3.0ml, and carry out the false add plug with butyl rubber plug through the depyrogenation of sterilizing;
4. the fill and the medicine bottle of jumping a queue are sent in the freeze drying box in-40 ℃～+ 40 ℃, vacuum
Pressure is lyophilization under the 40Pa-1.5Pa, makes its residual moisture content≤2%, rolls lid;
5. detect qualified back, pack the storehouse by quality standard.
Embodiment 2
It is as follows to get raw material by weight ready
1. thyroliberin 10mg * 1000
2. immune ribonucleic acid 15mg * 1000
3. thymosin 20mg * 1000
4. reduced glutathion 1.5mg * 1000
5. low molecular dextran-40 3mg * 1000
6. mannitol 8mg * 1000
Sterile water for injection 3000mg * 1000
(initial sum terminal point PH is 2.5-3.0)
1000 of packing (every contains 3 kinds of active ingredient total amounts is 45mg)
The production operation step is identical with embodiment 1.
Embodiment 3
It is as follows to get raw material by weight ready:
1. thyroliberin 12mg * 1000
2. immune ribonucleic acid 20mg * 1000
3. thymosin 50mg * 1000
4. reduced glutathion 2.0mg * 1000
5. low molecular dextran-40 4mg * 1000
6. mannitol 9mg * 1000
Sterile water for injection 4000mg * 1000
(initial sum terminal point PH is 2.5-3.0)
1000 of packing (every contains 3 kinds of active ingredient total amounts is 82mg)
The production operation step is identical with embodiment 1, but every fill amount of present embodiment is 4.0ml.
Claims (3)
1. a composition of medicine is characterized in that, this composition of medicine is made by weight ratio by following raw material:
Thyroliberin 8mg-12mg;
Immune ribonucleic acid 4mg-20mg;
Thymosin 8mg-50mg;
Reduced glutathion 0.5mg-1.0mg;
Sterile water for injection 2000mg-5000mg.
2. a composition of medicine is characterized in that, this composition of medicine is made by weight ratio by following raw material:
Thyroliberin 8mg-12mg;
Immune ribonucleic acid 4mg-20mg;
Thymosin 8mg-50mg;
Reduced glutathion 0.5mg-2.0mg;
Low molecular dextran-40 2mg-4mg;
Mannitol 7mg-9mg;
Sterile water for injection 3000mg-4000mg.
3. claim 1 or the 2 described composition of medicine application in preparation treatment viral influenza and arthritic medicine.
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| CN200510063218A CN100594930C (en) | 2005-04-07 | 2005-04-07 | Medicinal composition using anterior pituitary adrenal cortical extract as main component, and its preparation method and use |
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| CN200510063218A CN100594930C (en) | 2005-04-07 | 2005-04-07 | Medicinal composition using anterior pituitary adrenal cortical extract as main component, and its preparation method and use |
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| CN103724427B (en) * | 2013-11-29 | 2016-09-28 | 青岛康原药业有限公司 | A kind of method of refined thyroliberin |
| CN105613936A (en) * | 2016-03-24 | 2016-06-01 | 刘冬明 | Preparation method for spleen polypeptides |
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| Title |
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| 小儿重症肌无力的免疫调节治疗. 姜桂英等.中风与神经疾病杂志，第12卷第2期. 1995 * |
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