CN1175899C - Medicine composition and preparation and use thereof - Google Patents
Medicine composition and preparation and use thereof Download PDFInfo
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- CN1175899C CN1175899C CNB031175783A CN03117578A CN1175899C CN 1175899 C CN1175899 C CN 1175899C CN B031175783 A CNB031175783 A CN B031175783A CN 03117578 A CN03117578 A CN 03117578A CN 1175899 C CN1175899 C CN 1175899C
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Abstract
The present invention provides a medicine composition which is a unit preparation, wherein each unit preparation contains 5 to 30 mg of mannosan peptide. The mannosan peptide of the present invention is extracted from a fermented product of streptococcus hemolyticus-alpha-hemolysis. The formulation of the medicine composition is a capsule. The present invention also provides a preparation method and an application for the medicine composition, and has the advantages of specific medicine component, definite therapeutic effect, low dosage, simple production technology, stable quality, easy control, convenient taking, transportation, storage and carrying, etc.
Description
Technical field
The invention provides a kind of pharmaceutical composition and its production and use, specifically, is pharmaceutical composition that contains mannatide and its production and use.
Background technology
Along with the change of environment, the incidence rate of human tumor improves constantly, and the generation age also passes by to reduce, and seeking safe and effective antitumor drug is the target of human effort always.
Carry out oncotherapy by the human body defense mechanism, be to advocate by national cancer institute the latter stage seventies, worldwide carry out gradually the eighties, Japan is being on the forefront aspect the research and development of employing immunoactivator antineoplastic, the listing in 1975 of the Saphlin of the Hemolytic streptococcus (OK-432) of approved production listing, the listing in 1977 of Intracellular Polysaccharide of Poly-stictus Versicolor PS-K (PSK is Krestin) fungus polysaccharide preparation, the sales volume of two kinds of medicines reaches 1,000 hundred million yen every year, distribute all over the world, sales volume accounts for half of Japanese antitumor medicine, the lentinan injection that in December, 1985 is ratified again to go on the market, the glucosan injection that extracts the Sohizop-hyllan of confirmation request (SPG) series wrinkle Pseudomonas just, ruburatin Nocard's bacillus thalline injection and bestatin (benzene fourth isoleucine) oral agents also has ten kinds of similar immunoactivators just in clinical research.This class medicine can activate body's immunological function, by changing the respond of tumor host to tumor cell, changes tumor cell and host's relation, to reach the effect of treatment tumor illness.The Hemolytic streptococcus SU (OK-432) of Japan is that the Hemolytic streptococcus thalline of attenuation is suspended in a kind of biological preparation in the penicillin, and side reaction is heavier, and main side reaction is heating, and injection site pain and cell reduce and use limited.
Chinese patent 98121898 discloses " a kind of composite mannosans peptides oral liquor ", contain multiple composition in this oral liquid, its mannatide is respectively from basidiomycete, heterobasidium fungus, porous order fungus, umbrella section fungus, composition is comparatively complicated, and oral dose is big.
The antitumor drug that seek a kind of new determined curative effect, becomes to distinguish one from the other, conveniently take is very necessary.
Summary of the invention
The purpose of this invention is to provide a kind of new pharmaceutical composition.
Another object of the present invention provides this preparation of drug combination method.
Pharmaceutical composition of the present invention is a kind of unit formulation, contains mannatide 5～30mg in the per unit preparation.
Mannatide of the present invention is to be prepared by following method:
A, (Streptococcus hemolyticus-α-Hemolysis) produces bacterium to ferment with alpha-Hemolytic streptococcus;
B, collection fermentation liquid extract the purification mannatide.
The pH value of the extraction overall process of step b remains 1.5 to 5.0, and employed solvent is the ethanol of 60%-99.9%.
Mannatide of the present invention is from alpha-Hemolytic streptococcus (Streptococcushemolyticus-α-hemolysis) extract the refining a kind of mannan peptide matters with pharmacologically active that forms the tunning.The relevant expert is in the research of Keshan disease; adopt Hemolytic streptococcus thalline preparation (OK-432) as proposing to adopt Hemolytic streptococcus metabolite antineoplastic imagination on the basis of antineoplastic immune reinforcing agent according to Japan; through strain improvement and discriminating; the research of fermentation manufacturing technique; discovery separates the alpha-Hemolytic streptococcus obtain from human mouth, and (α-mannatide that contains in the tunning of Streptococcushemolyticus-α-Hemolysis) has can enhancing body's immunological function; antiinflammatory; leukocyte increasing, multiple physiologically actives such as protection and enhancing bone marrow stem cell.
The preparation of pharmaceutical composition of the present invention is a capsule.
Described capsule is prepared by following method:
After getting mannatide dissolving, add adjuvant pharmaceutically commonly used and make soft material, granulate, drying, granulate, it is even to add mix lubricant pharmaceutically commonly used, filled capsules, packing makes every capsules contain mannatide 5～30mg.
It is the medicine that can strengthen the human immunologic function that the present invention also provides this pharmaceutical composition, and it is an adjuvant therapy medicaments of tumor specifically; It also is the medicine of treatment leukopenia, and it still treats the medicine of aplastic anemia.
The side reaction of mannatide of the present invention is slight, particularly oral administration has the drug effect identical with injection and uses wider, the Intracellular Polysaccharide of Poly-stictus Versicolor PS-K (PSK) of Japan is polysaccharose substance with mannatide, but composition complexity, dose for oral use big (be mannatide 200 times), its expense is higher, and mannatide dose for oral use is little, expense is lower, determined curative effect.
Capsule is compared with tablet, injection, lyophilized injectable powder, oral liquid etc., and it is simpler to have production technology, steady quality, is easy to advantages such as control.Compare with injection, lyophilized injectable powder, oral liquid, it takes, transports, stores and carry more convenient, so the present invention selects capsule for use.Capsule of the present invention is mainly used in immunologic hypofunction clinically, the treatment of leukopenia, and the auxiliary treatment of aplastic anemia and oncotherapy.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
The specific embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
The specific embodiment
The preparation of example 1 mannatide:
The preparation of sweat: i, strain: selecting strain for use is alpha-Hemolytic streptococcus (Streptococcushemolyticus-α-hemolysis);
Culture medium: bacterium culture medium: glucose 0.4-0.8%, sodium chloride 0.4-0.8%, peptone 0.4-1%, Carnis Bovis seu Bubali cream 0.1-0.5%, sheep blood 10%, agar 1.5-2%, PH7.2-7.8
Fermentation medium: glucose 0.1-0.5%, peptone 0.4-0.7%, yeast extract 0.4-0.6%, sodium chloride 0.4-0.8%, bubble enemy 0.01%
Extraction, the required raw and auxiliary material of subtractive process: ethanol, acetone, trichloroacetic acid, dehydrated alcohol
A, female bottle preparation: unpacking strain cryovial, with aseptic broth bouillon dilution, insert the blood inclined-plane under aseptic condition.18-45 ℃ of constant temperature culture 24h;
B, the preparation of son bottle: cultured blood slant strains is pressed the 1-8% inoculum concentration insert in the broth bouillon 18-45 ℃ of constant temperature culture 10-24h under aseptic condition;
C, bacterium culture medium sterilising temp are 120-124 ℃, sterilization time 30 minutes, sterilization steam pressure 0.12-0.14Mpa;
D, fermentation: good son bottle strain is inserted fermentation tank by 0.2% inoculum concentration under aseptic condition, overall process 18-45 ℃ of constant temperature culture, the filtrated air air inflow is advisable can stir culture fluid, and tank pressure is no more than 0.01Mpa, fermentation period 20-100 hour.The fermentation medium sterilising temp is 120-124 ℃, and steam pressure is 0.12-0.14Mpa, time 30min; Ferment to 20-100 hour, jar is put in deactivation, and deactivation is adopted to heat and made the fermentation liquid temperature reach 80 ℃, lowers the temperature after constant temperature 30-50 minute again and puts jar, concentrates, and the concentrated solution specific gravity control is got final product at 1.20-1.22;
Ii, extraction:
A, the concentrated solution of i step is added the ethanol of 1-5 times of volume, fully stir and leave standstill the centrifugal removal supernatant in back and promptly obtain precipitate;
B, with gained precipitate dissolved in distilled water, transfer PH, obtain lysate and lysate left standstill and control lysate pH value after leaving standstill;
C, the centrifugal removal impurity of lysate is obtained clear liquid, accurately measure the clear liquid volume, transfer pH value, calculate required ethanol amount, ethanol is slowly joined in the clear liquid, and fully stir, leave standstill the centrifugal removal supernatant in back and obtain precipitate by the clear liquid stereometer;
D, above extracting method by from b-c step repeatable operation to intermediate detection qualified till, the precipitate that promptly obtains is the mannatide semi-finished product;
Iii, refining: the ii step is extracted repeatedly the mannatide semi-finished product precipitate that obtains, after sampling detects and meets quality standard, carry out primary dewatering with dehydrated alcohol, fully grind to form powdery, obtain wet-milling one time, reuse acetone carries out second dehydration, fully stirs and obtains the secondary wet-milling, and the secondary wet-milling was promptly got mannatide in 4-10 hour 60-100 ℃ of baking.
The using method of active carbon in the leaching process of ii step wherein: use amount is the required active carbon 1-10g of 1L lysate, transfers the lysate pH value after adding active carbon, and this liquid is incubated 20-70min under 40-100 ℃ of temperature, and is centrifugal, and the control charcoal takes off the liquid pH value.
PH value in the said extracted overall process remains 1.5 to 5.0.
Concentration of alcohol in the leaching process of ii step is 60-99.9%.
The semi-finished product that example 2 said methods obtain and the evaluation of finished product
1, half-finished evaluation:
I, trap measure: it is an amount of to get the semi-finished product sample, adds water and makes the solution that contains 0.4mg among every 1ml, according to spectrophotography (two appendix VIA of Chinese Pharmacopoeia version in 2000), wavelength place at 260nm, its trap must not be greater than 0.25, and at the wavelength place of 280nm, its trap must not be greater than 0.20.
Ii, assay:
The preparation precision of a, reference substance solution takes by weighing the D-mannose reference substance 0.1g that is dried to constant weight through 105 ℃, puts in the 100ml measuring bottle, is dissolved in water and is diluted to scale, shakes up; Precision is measured 5ml, puts in the measuring bottle of 100ml, adds water to scale, shakes up.Contain mannose 50 μ g among every 1ml.
It is an amount of that this product is got in the preparation of b, test sample, and accurate the title decides, and is dissolved in water and makes the solution that contains 40 μ g among every 1ml.
The preparation precision of c, standard curve takes by weighing reference substance solution 0,0.2,0.4,0.6,0.8,1.0ml, put respectively in the tool plug test tube, respectively add water to 1.0m, add 3% phenol solution 1.0ml again, shake up, pour sulphuric acid 4.5ml, shake up, being positioned over room temperature, is blank with 0 pipe, measures trap according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A) at the wavelength place of 490nm.To corresponding trap, calculate regression equation with mannose μ g number.
D, algoscopy precision are measured offerings solution 1.0ml, and from " adding phenol solution 1.0ml again ", operation is in accordance with the law measured trap, by the content of regression equation calculation mannose under the sighting target directrix curve preparation.
2, the evaluation of finished product:
[character] this product is white or little yellow amorphous powder; Odorless, tasteless.
This product is easily molten in water, and is insoluble in ethanol, chloroform and acetone.
Specific optical rotation: get this product, accurate claim surely, be dissolved in water also, be dissolved in water and be diluted to the solution that contains 10mg among every 1ml approximately, measure (two appendix VI of Chinese Pharmacopoeia version in 2000 E) in accordance with the law, specific optical rotation is+70 ° to+80 °.
[discriminating]
I, get this product 10mg, add water 0.5ml and make dissolving, add a-naphthols alcoholic solution (5 → 100) 1ml, shake up, slowly add sulphuric acid 0.5ml along tube wall, after several minutes, the interface is aubergine.
Ii, get this product, add water and make the solution that contains 1mg among every 1ml, get about 10 μ l points on filter paper, dry, fix, put periodic acid solution and (get periodic acid 1.2g with dehydrated alcohol, after adding water 30ml dissolving, add 0.2mol/L sodium acetate solution 1.5ml and ethanol 100ml, mixing promptly.Put the dark place and preserve, can use the several months) the middle immersion 5 minutes, take out, wash with 70% alcoholic solution, (get potassium iodide 5g, sodium thiosulfate 5g adds water 100ml and makes dissolving to put reducing solution, add ethanol 150ml, 2mol/L hydrochloric acid solution 2.5ml again, with adding, face the time spent and join with stirring) the middle immersion 5 minutes, take out, wash with 70% alcoholic solution, put in the fuchsin sulfurous acid test solution and soaked about 30 minutes, take out, (get sodium pyrosulfite 0.4g with sodium metabisulfite solution, add hydrochloric acid 1ml, being dissolved in water makes into 100ml, promptly), and flushing, dry, the place should be aubergine at the filter paper point sample.
Iii, get test sample and reference substance is an amount of, add respectively the chlorination sodium injection make contain 1mg among every 1ml solution as need testing solution and reference substance solution, press the test of mannatide immunogenicity determining method, test sample and contrast QC should all not have haemolysis to be taken place.
[inspection]
I, acidity: get this product, add water and make the solution that contains 10mg among every 1ml, measure (two appendix VI of Chinese Pharmacopoeia version in 2000 H) in accordance with the law, pH value should be 3.0 ~ 5.0.
Ii, trap: get this product, add water and make the solution that contains 0.4mg among every 1ml, according to spectrophotography (two appendix VI of Chinese Pharmacopoeia version in 2000 A), wavelength place at 260nm, its trap must not be greater than 0.25, and at the wavelength place of 280nm, its trap must not be greater than 0.20.
Iii, total nitrogen are measured: get this product, measure according to N2 method (two appendix VII of Chinese Pharmacopoeia version in 2000 D, second method), press dry product and calculate, contain total nitrogen and should be 0.8-2.0%.
Iv, immunogenicity: get test sample and reference substance is an amount of, add the chlorination sodium injection respectively and make the solution that contains 10mg among every 1ml, make 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64,1: 128,1: 256 diluent as need testing solution and reference substance solution with phosphate buffer respectively again, check in accordance with the law (attached mannatide immunogenicity determining method) that the least concentration of the insoluble blood vessel of test sample should be higher than more than a times of reference substance respective concentration.
V, loss on drying are got this product, are dried to constant weight at 105 ℃, subtract weight loss and must not cross 5.0% (two appendix VIII L of team of Chinese Pharmacopoeia version in 2000).
Vi, heavy metal are got this product 1.0g, check to contain (Chinese Pharmacopoeia version VIII in 2000 H) heavy metal and must not cross 20/1000000ths in accordance with the law.
Vii, undue toxicity get this product, add the chlorination sodium injection and make the solution that contains 0.5mg among every 1ml, check in accordance with the law and (Chinese Pharmacopoeia version appendix in 2000 XI C) press the intravenous injection administration, should (injection) up to specification.
[assay]
The preparation precision of a, reference substance solution takes by weighing the D-mannose reference substance 0.1g that is dried to constant weight through 105 ℃, puts in the 100ml measuring bottle, is dissolved in water and is diluted to scale, shakes up; Precision is measured 5ml, puts in the measuring bottle of 100ml, adds water to scale, shakes up.Contain mannose 50 μ g among every 1ml.
5 parts of the mannatide test samples of different content are got in the preparation of b, test sample, accurately claim surely, are dissolved in water and make the solution that contains 40 μ g among every 1ml.
The preparation precision of c, standard curve takes by weighing reference substance solution 0,0.2,0.4,0.6,0.8,1.0ml, put respectively in the tool plug test tube, respectively add water to 1.0m, add 3% phenol solution 1.0ml again, shake up, pour sulphuric acid 4.5ml, shake up, being positioned over room temperature, is blank with 0 pipe, measures trap according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A) at the wavelength place of 490nm.To corresponding trap, calculate regression equation with mannose μ g number.
D, algoscopy precision are measured offerings solution 1.0ml, and from " adding phenol solution 1.0ml again ", operation is in accordance with the law measured trap, by the content of regression equation calculation mannose under the sighting target directrix curve preparation.
After measured, the product that said method obtains is a mannatide, and content is respectively 5mg, 10mg, 15mg, 20mg, 25mg, 30mg.
Mannatide immunogenicity determining method (complement combined techniques)
1, test solution
Phosphate buffer (pH7.2) is got sodium chloride 8.5g, sodium hydrogen phosphate 0.565g and potassium dihydrogen phosphate 0.135g, adds water to 1000ml and makes its dissolving, adds 10% Adlerika 1ml, shakes up.
1% sheep erythrocyte suspension
The preparation of sheeps blood erythrocyte by sheep jugular vein sterile blood sampling in filling equivalent A Shi liquid (get glucose 20.5g, sodium citrate 8.0g, sodium chloride 4.2g, citric acid 5.5g, add water to 1000ml and make dissolving, in 100 ℃ of sterilization containers, 4 ℃ of preservations).
It is an amount of that above-mentioned sheeps blood erythrocyte is got in the preparation of 1% sheeps blood erythrocyte suspension, and with sodium chloride injection washing three times, each centrifugal 5 minutes (2000 rev/mins), sheeps blood erythrocyte is amassed in pressure, makes 1% sheeps blood erythrocyte suspension with sodium chloride injection.Get suspension, make need testing solution for 20 times with the sodium chloride injection dilution, other gets 20 times of equivalent suspension thin ups as blank solution, according to spectrophotography (two appendix IV of Chinese Pharmacopoeia version in 2000 A), wavelength place at 541nm measures, its trap should be 0.65 ~ 0.70, should regulate the concentration of 1% sheep erythrocyte suspension as overrun.
Hemolysin and sensitization sheeps blood erythrocyte
Above-mentioned hematocrit sheeps blood erythrocyte is got in the preparation of hemolysin, makes 25% sheeps blood erythrocyte suspension with sodium chloride injection.Get 1 of rabbit, the above-mentioned sheeps blood erythrocyte suspension of intravenous injection, once a day, and totally seven times, first three time 3ml, back three 5ml, last inject blood sampling in back seven days.Separation of serum, deactivation in 56 ℃, 30 minutes, packing is preserved below 0 ℃.
It is an amount of that the mensuration of hemolytic unit is got hemolysin, add phosphate buffer and make 1: 1000,1: 2000,1: 3000,1: 4000,1: 5000,1: 6000,1: 7000,1: 8000,1: 9000,1: 10000 diluent respectively, respectively getting 0.1ml puts in the test tube, add 1% Sanguis caprae seu ovis cell suspension 0.1ml, shake up, add dilution factor and be 1: 30 guinea pig serum (complement) 0.2ml and phosphate buffer 0.2ml, shake up, 37 ℃ of insulations 30 minutes, the high dilution of complete hemolysis pipe is 1 unit hemolysin.
Before usefulness was faced in the preparation of sensitization sheeps blood erythrocyte, the sheeps blood erythrocyte suspension with 2% mixed with 2 unit hemolysin equal-volumes, and 37 ℃ are incubated 15 minutes promptly.
Complement is got the Cavia porcellus more than three, the heart blood sampling, and centrifugalize serum is preserved below 0 ℃.
It is an amount of that the mensuration of complement unit is got complement, add phosphate buffer, make 1: 40,1: 60,1: 80,1: 100,1: 120,1: 140,1: 160,1: 180 diluent respectively, respectively get 0.2ml and put in the test tube, add the 0.2ml phosphate buffer, shake up, 37 ℃ of insulations are after 30 minutes, add sensitization sheeps blood erythrocyte 0.2ml respectively, shake up, 37 ℃ are incubated 30 minutes again.The high dilution of complete hemolysis pipe is the complement of 1 unit.
It is an amount of that antibody is got the mannatide reference substance, add the chlorination sodium injection and make the reference substance solution immunizing rabbit that contains 10mg among every 1ml, the next day adopt ear vein injection reference substance solution once.Inject for the first time 0.2ml, for the second time respectively inject 0.5ml to the 5th time, respectively inject 1.0ml the 6th time to the tenth time, the tenth once respectively injects 2.0ml to the 15 time, and last is injected blood sampling in back three days, centrifugalize serum, 56 ℃ of deactivations in following 30 minutes, (before facing usefulness, needing 56 ℃ of sterilizations once more in 30 minutes) preserved in packing below 0 ℃.
It is an amount of that the mensuration of antibody unit is got antibody, add phosphate buffer and make 1: 2,1: 4,1: 8,1: 16,1: 32,1: 64,1: 128 diluent respectively, respectively getting 0.1ml puts in the test tube, add mannatide reference substance solution 0.1ml and the 2 complement 0.2ml of unit, shake up, place more than 4 hours in 4 ~ 8 ℃, put 37 ℃ of insulations 30 minutes, the high dilution of insoluble blood vessel is 1 unit antibody.Establish antigen simultaneously and (do not add antibody, replace with the 0.1ml phosphate buffer), antibody (do not add antigen, replace with the 0.1ml phosphate buffer), complement (do not add antigen, antibody, replace) control tube with the 0.1ml phosphate buffer, more than three kinds of control tube haemolysis entirely; The sensitization sheeps blood erythrocyte that other establishes (do not add antigen, antibody and complement, replace with the 0.4ml phosphate buffer) control tube is haemolysis not.
2, immunogenicity determining method
It is an amount of to get mannatide reference substance and test sample, make the reference substance solution and the need testing solution of variable concentrations according to the regulation under the medicine item, respectively getting 0.1ml puts in the test tube, add 2 antibody 0.1ml of unit and the 2 complement 0.2ml of unit, shake up, place more than 4 hours in 4 ~ 8 ℃, put 37 ℃ of insulations 30 minutes, add sensitization sheeps blood erythrocyte 0.2ml, shake up, 37 ℃ are incubated 30 minutes again.Observe the haemolysis situation of each pipe, the least concentration of insoluble blood vessel is represented the immunogenicity of mannatide.Establish antigen simultaneously and (do not add antibody, replace with the 0.1ml phosphate buffer), antibody (do not add antigen, replace with the 0.1ml phosphate buffer), complement (do not add antigen, antibody, replace) control tube with the 0.2ml phosphate buffer, more than three kinds of control tube haemolysis entirely; The sensitization sheeps blood erythrocyte that other establishes (do not add antigen, antibody and complement, replace with the 0.4ml phosphate buffer) control tube is haemolysis not.
The pharmacological evaluation that the mannatide of use method for preparing is correlated with.These tests comprise pharmacodynamics test, the clinical observation on the therapeutic effect test, and pharmacological toxicology is learned test.
Test example 1 pharmacology test
1, medicine of the present invention is to immunocompetent influence
1-1 is to the influence of the netted dermal system phagocytic function of mice
The 1-1-1 laboratory animal: Kunming mouse is provided by Sichuan Industrial Institute of Antibiotics's Animal House.
1-1-2 is subjected to the reagent thing: medicine of the present invention
The 1-1-3 experimental technique:
Normal mouse divides into groups, and irritates the medicine of the present invention of the different dosage of stomach respectively, once a day, continuous 5 days, next day after the last administration, each treated animal ivl:2-5 (Guangzhou Ink Factory drawing ink is with 0.5% gelatin dissolved dilution) presses the Biolli method and measures and calculate phagocytic index K value.
Mouse hypodermic inoculation S
180Next day after the sarcoma, irritate stomach or intramuscular injection medicine of the present invention respectively, once a day, continuous 10 days, next day after the last administration, measure and the calculating K value by last method.
The 1-1-4 experimental result
Normal mouse the results are shown in Table 1, table 2.
The oral medicine of the present invention of table 1 is to the influence of mice RES phagocytic function
| Dosage (MG/KG.DX5D) | N | The K value | The P value |
| ?400 | ?10 | ?0.1199±0.0240 | ＜0.05 |
| ?200 | ?10 | ?0.1427±0.0335 | ＜0.05 |
| ?100 | ?10 | ?0.0979±0.0223 | ＞0.05 |
| Contrast | 0.0993±0.0265 | ||
The oral medicine of the present invention of table 2 is to the influence of mice RES phagocytic function
| Dosage (MG/KG.DX5D) | Approach | N | The K value | The P value |
| 200 | Oral | 10 | ?0.1187±0.0166 | ＜0.05 |
| 100 | Intramuscular injection | 10 | ?0.1229±0.0131 | ＜0.05 |
| Contrast | 10 | ?0.960±0.0131 |
The result shows that normal mouse was irritated stomach medicine 200mg/kg of the present invention continuous five days, and medicine 100mg/kg of the present invention is the same with intramuscular injection, the also significantly enhancing of netted cortex system's phagocytic function.
The S180 tumor-bearing mice the results are shown in Table 3
The oral medicine of the present invention of table 3 is to the influence of S180 tumor-bearing mice RES phagocytic function
| Dosage (MG/KG.DX5D) | Approach | N | The K value | The P value |
| 200 | Oral | 10 | ?0.1205±0.0130 | ＜0.05 |
| 100 | Intramuscular injection | 10 | ?0.1056±0.260 | ＜0.05 |
| Contrast | 10 | ?0.0780±0.0170 |
By table five as seen, no matter medicine of the present invention is to irritate stomach or intramuscular injection, all can significantly improve S180 tumor-bearing mice reticuloendothelial system phagocytic function.
1-2 is to the influence of peritoneal macrophage (M ) phagocytic function
The 1-2-1 experimental technique
After the mice group, irritate stomach medicine 40 of the present invention and 200mg/kg respectively, once a day, continuous three days, after drug withdrawal, put to death each treated animal on the 1st, wash out peritoneal exudate cells with Hanks liquid, add chicken red blood cell (CRBC), drip on microscope slide behind the mixing, hatched 30 minutes for 37 ℃ in the wet basin, not adherent cell is removed in rinsing, and M φ then is bonded on the slide, Giemas dyeing back is number meter phagocytic rates (the M φ that engulfs CRBC accounts for the percentage rate of total M φ) and phagocytic index (on average each M φ engulf CRBC number) 1-2-2 experimental result under oily mirror, sees Table 4.
The oral medicine of the present invention of table 4 is to the influence of mice phagocytic function
| Dosage (MG/KG.DX3D) | N | Phagocytic rate (%) | Phagocytic index |
| Contrast | ?7 | ?17.1±4.7 | 0.217±0.080 |
| ?40 | ?8 | ?33.9±5.9 ** | 0.501±0.179 * |
| ?200 | ?8 | ?37.3±8.2 ** | 0.526±0.171 ** |
*: compare P＜0.001 with matched group;
*P＜0.01.
The result shows that mouse stomach medicine 40mg/kg of the present invention can significantly improve phagocytic rate and the phagocytic index of M φ to cRBC for three days on end.
The influence that 1-3 generates the mice hemolysin
1-3-1 laboratory animal: under the 1-2-1 item.
1-3-2 is subjected to the reagent thing: under the 1-2-2 item.
The 1-3-3 experimental technique:
Mice intramuscular injection sRBC4 * 10
8After/the immunity, be divided into two groups at random, one group gavages medicine 40mg/kg of the present invention immediately, once a day, and continuous three days; Another group is physiology saline control group.Two treated animals after immunity 3,5,7,10,15,20 days are got blood from the tail vein, press the hemolysin serial testing and measure anti-sRBC antibody-hemolysin level in the blood.
The 1-3-4 experimental result sees Table 5.
The oral medicine of the present invention of table 5 is to the influence of mice hemolysin level
| Post-immunized day | HC50 | The P value | |
| Matched group (N=8) | Medicine group of the present invention (N=9) | ||
| ?3 | ?1.8±1.7 | ?3.1±1.2 | ＞0.005 |
| ?5 | ?174.2±43.5 | ?479.4±57.4 | ＜0.001 |
| ?7 | ?108.1±45.0 | ?294.7±63.4 | ＜0.01 |
| ?10 | ?71.3±31.1 | ?114.4±33.5 | ＜0.05 |
| ?15 | ?68.6±42.1 | ?138.3±38.3 | ＜0.01 |
| ?20 | ?6.9±15.2 | ?18.1±21.1 | ＜0.01 |
Annotate: drug dose of the present invention: 40mg/kg d * 3d.
The result shows, mouse gavaging medicine 40mg/kg of the present invention, continuous three days, after immunity 3～20 days, hemolysin level is significantly higher than the normal control group always, and two groups all reached peak value in back 5 days in immunity, shows the humoral immune reaction that drug oral of the present invention also can enhancing body.
The influence that 1-4 generates mouse spleen lymphocyte interleukin-22 (IL-2)
The 1-4-1 laboratory animal: the C57BL/6J mice, body weight 20～24 grams in 14 ages in week, are provided by the anti-institute in river Animal House.
1-4-2 is subjected to the reagent thing: medicine of the present invention.
The 1-4-3 experimental technique:
Normal mouse is divided into double-dealing group, and one group gavages medicine 200mg/kg of the present invention, once a day, and continuous 10 days; Another group compares group to normal saline.Next day after the last administration, put to death animal, sterile preparation splenocyte 1 * 10
7/ ml adds ConA (10 μ g/ml), and mixing is put CO
2Incubator was cultivated 36 hours for 37 ℃, and is centrifugal, gets supernatant, places 96 hole microplates, adds ctll cell 1 * 10
4Cultivated 24 hours in/hole again, and the back adds 3H-TdR1 μ ci/ hole, cultivates after 16 hours, measures the amount (cpm value) that 3H-TdR mixes ctll cell, and calculates growth index GI.
Next day behind the C57BL/6J mouse hypodermic inoculation Lewis lung cancer, grouping as stated above, administration and measurement 3H-TdR mix the ctll cell amount, and calculate GI.
1-4-4 experimental result: see Table 6.
The oral medicine of the present invention of table 6 is to the pulmonary carcinoma growth of C57BL/6J Lewis and the influence of IL-2 generation
| Animal | Medicine | Dosage regimen | Tumor heavy (mg) | The IL-2 level | |
| GI | ?P | ||||
| Normal mouse | Medicine of the present invention | 200mg/kg.d×10d | ?33.47±5.3 | ＜0.01 | |
| Normal saline | ?19.15±1.2 | ||||
| Tumor-bearing mice | Medicine of the present invention | 200mg/kg.d×10d | ?43.2±17.9 * | ?20.05±2.1 | ＜0.01 |
| Normal saline | ?63.4±21.6 | ?13.17±2.7 | |||
*: compare P＜0.01 with matched group.
The result shows, gavages medicine 200mg/kg of the present invention ten days continuously, can obviously suppress the growth of Mice Bearing Lewis Lung Cancer; Can significantly improve the ability of normal mouse splenocyte generation IL-2; Behind the mouse inoculation Lewis lung cancer, the ability that produces IL-2 reduces greatly, gavage medicine of the present invention after, can make its ability that generates IL-2 return to normal level.
2, medicine of the present invention is to the effect of hematopoietic stem cell
2-1 laboratory animal: under the 1-2-1 item.
2-2 is subjected to the reagent thing: under the 1-2-2 item.
The 2-3 experimental technique:
Mice is divided donor group and receptor group, and the donor group is divided into administration group and matched group again, and two donor group mices gavage medicine (40mg/kg of the present invention respectively, once a day, continuous six days) and normal saline, took out each Mus femur bone marrow cell on the 7th day, the system single cell suspension, warp is given in intravenous injection respectively
60The receptor group mice of Co irradiation 750rad, counting receptor Mus spleen colony (CFU-S) number after 9 days.
2-4 experimental result: see Table 7.
The oral medicine of the present invention of table 7 is to the effect of mouse hematopoietic stem cell
| Group | N | CFU-S number/spleen | The P value |
| Matched group | 6 | ?20.8±9.6 | ＜0.01 |
| The administration group | 6 | ?40.3±6.4 |
Annotate: drug dose 40mg/kg of the present invention.
By table as seen, gavage three days donor group mice of medicine 40mg/kg of the present invention continuously, the average spleen CFU-S of its corresponding receptor Mus digital display work is higher than matched group, illustrates that oral medicine of the present invention also can promote the hypertrophy of hematopoietic stem cell.
3, conclusion
Oral medicine of the present invention has obvious inhibitory action to the growth of mice ehrlich carcinoma solid tumor;
Can enhancing normally reach tumor-bearing mice reticuloendothelial system phagocytic function, macrophage phagocytic function, increase mouse antibodies-hemolysin and generate, significantly improve immunocompetences such as normally reaching tumor-bearing mice splenocyte generation IL-2 ability; Also can promote the hypertrophy of mouse hematopoietic stem cell.
Experimental example two clinical drug verifying datas of the present invention are summed up
Much more comparatively leukopenia and immunologic hypofunction behind leukopenia and the immunologic hypofunction, particularly chemicotherapy are seen clinically.It is heavier that this class patient has disease, and the characteristics that are difficult to recover are comparatively thorny in the treatment.Thus, leukopenia and immunologic hypofunction behind the research treatment chemicotherapy have great importance clinically.This clinical verification Department of Pharmacy mannatide capsules, purpose are that the curative effect of its capsule preparations and safety are observed and evaluated.
By clinical treatment controlled observation to 200 routine leukopenia and immunologic hypofunction patient, use pharmaceutical composition of the present invention, drug administration with different content, select the treatment group to be respectively and contain mannatide 6mg, 10mg, 15mg, 24mg, 30mg in the unit formulation, matched group is mannan tablet (a Chengdu rel Pharmaceutical), the result shows: 1, each dosage group of medicine of the present invention is to leukopenia, has better curative effect, its clinical obvious effective rate 42.8%, effective percentage 49.5%, total effective rate 92.3%.Compare with matched group, there is significant difference P＜0.05; 2, behind two groups of patient treatments, T cell subsets (OKT4, OKT8, OKT4/OKT8) all improves, 68.4%, two group of curative effect of its effective percentage relatively, P＜0.05, there was no significant difference; 3, immunoglobulin (IgM, IgA, IgC) is relatively learned by statistics and is handled behind two groups of patient treatments, P＞0.05, and there was no significant difference, its effective percentage are 67.3%; 4, the comparison of two groups of patient NK cells, P＞0.05, there was no significant difference, its effective percentage are 79.3%.
According to above result, leukopenia and immunologic hypofunction behind the mannatide capsules treatment chemicotherapy of selecting various dose are described, clinically, have better curative effect.
In clinical observation on the therapeutic effect, the mannatide capsules of each dosage has carried out the observation of detection such as livers before and after the treatment, kidney merit and clinical symptoms respectively to the part patient, there is no toxic and side effects and exist.
Medicine of the present invention as the treatment chemicotherapy after a kind of preparation of leukopenia and immunologic hypofunction, its satisfactory effect, taking convenience, safety are the treatment of clinical putting leukopenia and immunologic hypofunction, and new treatment selection is provided.
Experimental example three medicines of the present invention toxicological study
1, acute toxicity
Behind the oral medicine 5g/Kg of the present invention of mice and rat, all have no adverse reaction; Behind mice and the rat intramuscular injection medicine 2g/Kg of the present invention, indivedual dead mouses only occur, show oral or this medicine of intramuscular injection, toxicity is all very low.
Mice and rat intravenous injection medicine of the present invention, its mortality rate and dosage size are not proportional.Dog intravenous injection medicine 5.2mg/Kg of the present invention (be equivalent to people's consumption 25 times), minor responses such as of short duration myasthenia of limbs only appear, after dosage was increased to 25mg/Kg, myasthenia of limbs, ground for sleeping in, vomiting, fecal incontinence, the last dead serious toxicity reaction of hypopnea then appearred.Should careful usefulness when prompting is done intravenous injection clinically.
2, long term toxicity
Rat intramuscular injection medicine 4mg/Kg of the present invention, once a day, continuous 2 months and 4 months; Dog intramuscular injection 10mg/Kg, once a day, continuous 3 months, all no abnormal change of the tissue morphology of the hepatic and renal function of animal, hemogram, routine urianlysis and each internal organs.The subacute toxicity that this medicine is described is also very little.
3, hypersensitive test and irritation test are all negative.
4, specific toxicity
(1) mutagenesis: medicine of the present invention does not have obvious influence to mice polychromatophilia micronucleus in erythrocytes incidence rate].The little dominant lethal result of intramuscular injection medicine 50mg/Kg of the present invention (be equivalent to people's consumption 250 times) is negative.
(2) teratogenesis tire:
Repeatedly intravenous injection medicine 100mg/Kg of the present invention does not all make significant difference to young skeleton of fertility, tire and the internal organs growth of the pregnant Mus of mice
[28]Continuous six days of the oral medicine 200mg/Kg of the present invention of mice (be equivalent to people's consumption more than 300 times) does not all make significant difference to female Mus average weight gain, fetal development, tire Mus average weight and tail length, outward appearance, internal organs and skeleton deformity recall rate.
Mode by embodiment further specifies the present invention, but does not therefore limit the present invention among the described scope of embodiments.
Embodiment 1
Mannatide 50g starch 720g
An amount of magnesium stearate 20g of distilled water
Get mannatide 50g, after an amount of dissolving of 50～60 ℃ of distilled water, add starch and make soft material, granulate with 14 order nylon mesh, 50 ℃ of dryings behind 12 mesh sieve granulate, add the magnesium stearate lubricant mix homogeneously, filled capsules, and packing is made 1000 promptly.
Embodiment 2
Mannatide 100g starch 682g
An amount of magnesium stearate 8g of distilled water
Get mannatide 100g, after an amount of dissolving of 50～60 ℃ of distilled water, add starch and make soft material, granulate with 14 order nylon mesh, 50 ℃ of dryings behind 12 mesh sieve granulate, add the magnesium stearate lubricant mix homogeneously, filled capsules, and packing is made 1000 promptly.
Embodiment 3
Mannatide 300g starch 480g
An amount of magnesium stearate 20g of distilled water
Get mannatide 300g, after an amount of dissolving of 50～60 ℃ of distilled water, add starch and make soft material, granulate with 14 order nylon mesh, 50 ℃ of dryings behind 12 mesh sieve granulate, add the magnesium stearate lubricant mix homogeneously, filled capsules, and packing is made 1000 promptly.
Claims (10)
1, a kind of pharmaceutical composition, it is characterized in that: contain mannatide 5～30mg in the per unit preparation, described mannatide is that (Streptococcus hemolyticus-α-hemolysis) is for producing the bacterium gained that ferments by alpha-Hemolytic streptococcus.
2, pharmaceutical composition according to claim 1 is characterized in that: described mannatide is to be prepared by following method:
A, (Streptococcus hemolyticus-α-hemolysis) produces bacterium to ferment with alpha-Hemolytic streptococcus;
B, collection fermentation liquid extract the purification mannatide.
3, pharmaceutical composition according to claim 2 is characterized in that: the pH value of the extraction overall process of the method step b of preparation mannatide remains 1.5 to 5.0.
4, pharmaceutical composition according to claim 2 is characterized in that: employed solvent is the ethanol of 60%-99.9% among the method step b of preparation mannatide.
5, pharmaceutical composition according to claim 1 is characterized in that: described preparation is a capsule.
6, pharmaceutical composition according to claim 5 is characterized in that: described capsule is prepared by following method:
After getting mannatide dissolving, add adjuvant pharmaceutically commonly used and make soft material, granulate, drying, granulate, it is even to add mix lubricant pharmaceutically commonly used, filled capsules, packing makes every capsules contain mannatide 5～30mg.
7, the described pharmaceutical composition of claim 1 is characterized in that: it is the medicine that can strengthen the human immunologic function.
8, pharmaceutical composition according to claim 5 is characterized in that: it is an adjuvant therapy medicaments of tumor.
9, pharmaceutical composition according to claim 5 is characterized in that: it is the medicine of treatment leukopenia.
10, pharmaceutical composition according to claim 5 is characterized in that: it is the medicine of treatment aplastic anemia.
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| CNB031175783A CN1175899C (en) | 2003-03-31 | 2003-03-31 | Medicine composition and preparation and use thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100592919C (en) * | 2003-12-31 | 2010-03-03 | 西安亨通光华制药有限公司 | Spray for treating chronic pharyngitis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1331531C (en) * | 2003-12-31 | 2007-08-15 | 赵恒� | Medicine for treating chronic gastritis |
| CN100408088C (en) * | 2003-12-31 | 2008-08-06 | 西安亨通光华制药有限公司 | Medicine for treating oral ulcer |
| CN100453063C (en) * | 2003-12-31 | 2009-01-21 | 西安亨通光华制药有限公司 | Frost-like powder possessing effects of beautification and nourishing face |
| CN100543129C (en) * | 2007-04-13 | 2009-09-23 | 孙卫 | Space efficient microbial strain of 18 th recoverable satellite, preparation and application |
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2003
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100592919C (en) * | 2003-12-31 | 2010-03-03 | 西安亨通光华制药有限公司 | Spray for treating chronic pharyngitis |
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