CN1465401A - Process for preparing acellular short corynebacteria preparation - Google Patents
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Abstract
The present method of non-cell corynebacterium parvum (NCP) preparation includes the following steps: continuous 2-4 times of passage of corynebacterium parvum (CP) working seed batch strain, then inoculating in large flask or nutrient tank, culturing for 5-7 days in proper culture medium, collecting thallus having no heterobacteria pollution, heating, boiling and inactivating to obtain bacetrial solution, breaking bacterial solution, centrifugation and collecting precipitate, washing to obtain suspension, heating said suspension and sterilizing so as to obtain the invented NCP. Said invention also provides its application function.
Description
Technical field
The present invention relates to a kind of preparation method of biological product, the preparation method of particularly a kind of acellular short corynebacteria preparation (NCP) belongs to field of biological product.
Background technology
Short corynebacteria vaccine (CPV, 1995 editions " Chinese biological goods rules ") or title short corynebacteria preparation (CPP, 2000 editions " Chinese biological goods rules ") are a kind of nonspecific immunity regulators.It is the bacterin of being made behind formalin-inactivated by short corynebacteria (CP), and its preparation process is disclosed in 1995 editions and 2000 editions " Chinese biological goods rules ".The CPP preparation of using also has the inactivated vaccine that French Merleux institute is produced at present; The inactivated vaccine that Britain Wellcome pharmaceutical factory produces etc.
CPP has the ability of activated mononuclear phagocyte system.CPP mainly shows immune effect: the activated mononuclear macrophage, make the NK cytoactive strengthen, promote body that multiple antigen is produced IgG and IgM, inducement interferon, interleukin II etc., therefore have antitumor and anti-infectious function.
The antitumor action of CPP is confirmed by many experiments and clinical research.To using CPP before and after the laboratory animal inoculated tumour, can significantly suppress some solid tumor (as breast carcinoma, melanoma etc.), ascitic type tumor and leukemic growth.The animal of tumor regression has specific resistance for the tumor cell of attacking once more.CPP also has inhibitory action to neoplasm metastasis.
CPP antitumor and anti-infective core are by activating macrophage, and its quality and quantity is improved greatly, and activated macrophage can kill the microorganism of tumor cell and infection.
CPP is used for the history of the existing many decades of tumor and other treatment of diseases as immunomodulator, and it is generally acknowledged by activated mononuclear macrophage system antitumor, anti-infective effect.CPP is as a kind of nonspecific immunity strengthening agent, and its effectiveness is one of best goods of finding up to now.But owing to there is certain side effect, easily produce as fever, chest pain, injection site swell and ache, side effect such as scleroma, limited its application.
Because CPP is a kind of good nonspecific immunity strengthening agent, if reduce its side effect by new technological means, will have a wide range of applications.
CPP is a gram positive bacteria, and no endotoxin itself also claims lipopolysaccharide (LPS), but CPP measures through limulus reagent test and contains more LPS at present, this mainly be because existing CPP makes and vertification regulation to due to its requirement of not limiting the quantity of.
CPP is the bacterin that CP makes through formalin-inactivated, and residual have a trace formaldehyde.Because formaldehyde has stronger zest to body, moreover formaldehyde also is the carcinogen of generally acknowledging in the world, though residual formaldehyde seldom but still can produce zest to the comparatively responsive pleura of human body among the CPP.
Studies show that medicine is worked into after the nanoscale, can significantly improve trap, diffusance, help reducing some side effect of medicine, improve drug effect.
Usually the crushing technology of microbial cell is two kinds of Mechanical Method, on-mechanical methods.Mechanical Method comprises: high pressure homogenizer, high speed ball mill, ultrasonic wave concussion device, on-mechanical method comprise chemical method, enzymatic isolation method, osmotic pressure ballistic method, freeze-thaw method and seasoning.Though cytoclastic method is a lot, all be difficult to the nanoscale of cell breakage to homogeneous grain diameter.
Summary of the invention
The object of the present invention is to provide the preparation method of a kind of acellular short corynebacteria preparation (NCP), adopt the culture medium of low pyrogen in the described preparation method, and in conjunction with strict Quality Control, in whole process of production, adopt aseptic low grade fever origin operation, guaranteed that final products hang down pyrogen, no living contaminants; Adopt heat inactivation to substitute traditional formalin-inactivated, formaldehydeless residual in the product, eliminated the zest of formaldehyde to body; CP is crushed to nanoscale, makes that product granularity reduces, the granule homogeneous, trap and diffusance improve, and help absorption of human body and improve drug effect; Reduce fever, chest pain simultaneously, side effect such as injection site redness, scleroma adopt preparation method of the present invention can obtain high-quality, fineness and all once good purpose product.
To achieve these goals, the technical solution used in the present invention is: the preparation method of a kind of NCP comprises the steps:
1) .CP work seed lot strain is continuous is inoculated in big bottle or nutrition jar after going down to posterity for 2-4 time, and cultivation is 5-7 days in culture medium;
2). collect the thalline of no living contaminants, obtained bacterium liquid in heated and boiled 15-60 minute;
3). the qualified bacterium liquid of sterility test is after washing, with the broken thalline of breaker;
4). the collecting precipitation after centrifugal of the suspension behind the bacterial cell disruption, suspension is made in washing precipitation;
5). with the suspension heat sterilization, obtain NCP.
Wherein the CP formal name used at school described in the step 1) is propionibacterium acne (propionibacterium acnes), and bacterium number is 65101 (76-27), 65102 (H-84) or 65103 (77-1); Wherein 76-27 also is called 7627, and 77-1 also is called 771.
Culture medium is to be selected from a kind of in THIOGLYCOLLIC ACID salt, peptone, liver or the yeast dialysis solution culture medium, and is low pyrogen culture medium.
Condition of culture is 36-37 ℃ of cultivation in the step 1).
Wherein said sulphur glycollate culture medium is not for containing the sulphur glycollate culture medium of agar.The preferred sulphur glycollate culture medium that does not contain agar.
Wherein the thalline described in the step 3) is an inactivated bacteria, and cell concentration is 50-500 hundred million/ml.
Described breaker is that bacterial cell disruption is arrived less than 100nm, and preferred size is the superhigh pressure water jet collider of 10-50nm.
Wherein said bacterial cell disruption is to carry out under aseptic condition.
Wherein the washing described in the step 3) is with precipitate physiological saline solution centrifuge washing at least 2 times; Washing described in the step 4) is with precipitate physiological saline solution centrifuge washing 0-5 time.
The centrifugal of suspension is 4 ℃-room temperature behind the step 4) bacterial cell disruption, the centrifugal 30-150 of 6000-12000rpm minute.
Being heated to be described in the step 5) 60-65 ℃ of heating 0.5-2 hour.
In the preparation method of the present invention, further comprise the NCP packing that step 5) is obtained after, ampoule 60 ± 2 ℃ the heating 30 minutes.
Culture medium described in the present invention is preferably the culture medium through 0.22 μ m or 0.45 μ m membrane filtration.
In the preparation method of the present invention, also comprise the NCP lyophilization that step 5) is obtained.
The NCP that preparation method of the present invention is prepared, pyrogen is less than 160EU/ml, protein content 10-40% (g/g), nucleic acid content 3-25% (g/g), all the other are polysaccharide.
Preferred pyrogen is less than 60EU/ml, protein content 20-30% (g/g), and nucleic acid content 3-10% (g/g), all the other are polysaccharide.
The prepared NCP granularity of preparation method of the present invention is less than 100nm, and preferred size is 10-50nm.
Hang down pyrogen culture medium, particularly THIOGLYCOLLIC ACID salt owing to adopting among the present invention, and in conjunction with strict Quality Control, and aseptic low grade fever origin operation in the whole process of production, guaranteed that final products hang down pyrogen and no living contaminants.
The present invention is crushed to nanoscale by the superhigh pressure water jet collider with CP, by selecting different centrifugal force (rotating speed), determined centrifugal extraction conditions, the precipitation that obtains partly is NCP, prove through animal experiment repeatedly, said preparation and whole cell have identical spleen and activate and press down biologic activity such as tumor, and supernatant part then alienia activates and tumor-inhibiting action, show adopt the inventive method preparation NCP have good biologic activity.
Removing residue formaldehyde especially to the zest of responsive pleura, adopts heat inactivation to substitute formalin-inactivated to the zest of body among the CPP among the present invention in order to eliminate.Zoopery shows that heat-killed NCP, CPP have identical spleen activation and but biologic activity such as tumor than the CPP of formalin-inactivated.
The operation principle of superhigh pressure water jet collider is that (operating pressure is 10-3000kgf/cm to the liquid that will be mixed with machined object with high-pressure pump
2) be divided into two strands after the pressurization, entering two diameters with high-speed jet is that flow out the head-on collision back in opposite directions in the passage formed of 8MM diamond wafer.Because the speed of liquid is very high, when clashing in opposite directions, fluid produces very strong shock wave, and make diamond wafer produce high frequency, high intense ultrasonic wave.The high-power high-frequency ultrasound wave makes the moment pulverizing of machined object granule, ultramicronising.
In the preparation process of the present invention, being broken thalline better, adopting the superhigh pressure water jet collider, is 50-500 hundred million/ml at cell concentration, and operating pressure is 800-1500kgf/cm
2Condition under broken, thalline suspension after the fragmentation is under 4 ℃-room temperature, the centrifugal 30-150 of 6000-12000rpm minute, abandon supernatant, precipitation is made suspension with behind physiological saline solution solution washing 0-5 time, heats 0.5-2 hour at 60-65 ℃, the qualified back of sterility test merges, through preparation, packing, ampoule obtains the good purpose product of high-quality, granularity and homogeneity 60 ± 2 ℃ of heating 15-60 minute after the packing.
NCP of the present invention be cell breakage with CP to nanoscale, remove that invalid supernatant partly prepares, product has low pyrogen, no living contaminants, formaldehydeless residual, granule has nanoscale, homogeneous grain diameter, trap and diffusance improve, and help absorption of human body and improve drug effect; Simultaneously can reduce fever, chest pain, side effect such as injection site redness, scleroma are a kind of promising immune formulations.
As required, NCP of the present invention can make the suspension formulation of physiological saline solution, and solids content is 1.0mg/ml or 2.0mg/ml, also can make freeze-dried powder or be fit to other medicinal dosage forms.
NCP of the present invention is an immunomodulator, can be used for cancerous hydrothorax, in conjunction with operative treatment early, mid-term pulmonary carcinoma, the treatment of breast carcinoma, nasopharyngeal carcinoma, advanced lung cancer, melanoma and cancer body surface metastasis also can be used for psoriasis, aplastic anemia, leukoplakia vulvae, infective asthma, treatment lungy or auxiliary treatment.
Describe the present invention in detail below in conjunction with the drawings and specific embodiments, described embodiment is used to describe the present invention rather than restriction the present invention.
Description of drawings:
Accompanying drawing 1 is the electromicroscopic photograph of the CPP of prior art.
Accompanying drawing 2 is electromicroscopic photographs of NCP of the present invention.
The specific embodiment
Be specific embodiments of the invention below, wherein the CP formal name used at school is propionibacterium acne (propionibacteriumacnes), bacterium number is that 65101 (76-27), 65102 (H-84) are disclosed in 1993 editions " Chinese medicine bacteria culture catalogues ", publishing house of Beijing Institute of Technology (ISBN7-81013-859-6/R.7), 65103 (77-1 or 771) are disclosed in 2000 editions " Chinese biological goods rules ", Chemical Industry Press.
Embodiment 1
Carry out foundation, calibrating and other calibrating of antibacterial seed lot with reference to the method for describing in 2000 editions " Chinese biological goods rules ".
The culture medium that adopts in the present embodiment is the sulphur glycollate culture medium (not containing agar) that Beijing three medicine scientific and technological development companies produce, and composition is: trypticase 15g/l, yeast soak powder 5g/l, glucose 5g/l, sodium chloride 2.5g/l, L-cystine 0.5g/l, sodium thioglycollate 0.5g/l.Before using with culture medium with 0.22 μ m membrane filtration.
Breakdown CP (formal name used at school propionibacterium acne, propionibacterium acnes) 771 (seeing " Chinese biological goods rules ") strain pipe, every pipe contains bacterium 6,000,000,000, draw about 0.2-0.3ml sulphur glycollate culture medium (not containing agar) with capillary tube, be added in strain pipe bottom and make it to dissolve fully the back sucking-off, in the pipe (capacity 38ml), mix in the adding, put 37 ℃ and cultivated 2 days with above-mentioned culture medium 8-10ml.Get bacterium liquid with above-mentioned culture medium (bacterium liquid: culture volume ratio=l: 9) mix, put 37 ℃ and cultivated 2 days.The bacterium liquid of getting above-mentioned cultivation mixes with above-mentioned culture medium (by 1: 9), puts 37 ℃ and cultivates 3 days, results bacterium liquid.By the bottle microscopy, the person abandons it the living contaminants.Merge to collect the thalline heated and boiled 15 minutes of no living contaminants, with superhigh pressure water jet to clashing into (Japanese Nanonizer Inc. product, model NMG-75-200) at 1000kgf/cm
2Broken thalline is 3 times under the pressure; Suspension after the fragmentation was centrifugal 90 minutes of room temperature, 6000rpm, collecting precipitation, with physiological saline solution washing precipitation 3 times, make the suspension that solids content is 0.5mg/ml after steriling test (2000 editions " Chinese biological goods rules ") and pyrogen test (2000 editions " The People's Republic of China's pharmacopeia ") are qualified, 60 ℃ were heated 1 hour, and obtained NCP.
CP wherein, formal name used at school is propionibacterium acne 65103 (77-1) or 771.
Detect (solids content 1mg/ml), NCP pyrogen 20EU/ml of the present invention through limulus reagent test (2000 editions Pharmacopoeias of the People's Republic of China); Coomassie brilliant blue method (" Biochemistry Experiment principle and method ", BJ University Press) is measured protein content 25% (g/g); Ultraviolet method (2000 editions " Chinese biological goods rules ") 260nm measures nucleic acid content 10% (g/g); It is 10% (g/g) that anthrone method (2000 editions " Chinese biological goods rules ") is measured polyoses content.The measurement result of considering protein and nucleic acid is more accurate, estimates that remaining polysaccharide do not measure.It is 60-100nm that Electronic Speculum is measured granularity.
After the packing, ampoule was 60 ± 2 ℃ of heating 20 minutes.
Embodiment 2
Method with reference to embodiment 1, with CP (formal name used at school propionibacterium acne, propionibacterium acnes) 65101 (76-27) are continuous is inoculated in the nutrition jar after going down to posterity for 4 times, in sulphur glycollate culture medium (not containing agar) (before using with culture medium with 0.45 μ m membrane filtration), 36-37 ℃ of cultivation 6 days; By the bottle microscopy, the person abandons it the living contaminants; The thalline heated and boiled of the no living contaminants of merging collection 60 minutes uses the superhigh pressure water jet collider at 1500kgf/cm
2Broken thalline is 5 times under the pressure; Centrifugal 1 hour of suspension 8000rpm after the fragmentation, with physiological saline solution washing precipitation 5 times, make the suspension that solids content is 2.0mg/ml after steriling test (2000 editions " Chinese biological goods rules ") and pyrogen test (2000 editions " The People's Republic of China's pharmacopeia ") are qualified, 65 ℃ were heated 60 minutes, and obtained NCP.
Detect (solids content 1mg/ml), NCP pyrogen 160EU/ml through limulus reagent test (2000 editions Pharmacopoeias of the People's Republic of China); Coomassie brilliant blue method (" Biochemistry Experiment principle and method ", BJ University Press) is measured protein content 40% (g/g); Ultraviolet method (2000 editions " Chinese biological goods rules ") 260nm measures nucleic acid content 5% (g/g); It is 20% (g/g) that anthrone method (2000 editions " Chinese biological goods rules ") is measured polyoses content.The measurement result of considering protein and nucleic acid is more accurate, estimates that remaining polysaccharide may not measure.It is 10-50nm that Electronic Speculum detects granularity.Referring to Fig. 2, epigranular.Fig. 1 is the electromicroscopic photograph of the CPP of prior art.
After the packing, ampoule was 60 ± 2 ℃ of heating 30 minutes.
Embodiment 3
With reference to the method for embodiment 1, be inoculated in the nutrition jar after going down to posterity for 4 times with CP (formal name used at school propionibacterium acne, propionibacterium acnes) 65102 (H-84) are continuous, in protein culture medium, cultivated 7 days for 36-37 ℃; By the bottle microscopy, the person abandons it the living contaminants; The thalline heated and boiled of the no living contaminants of merging collection 30 minutes is with superhigh pressure water jet collider (Japanese Nanonizer Inc. product, model AQUA300) 800kgf/cm
2Broken thalline is 10 times under the pressure; Suspension 12000rpm after the fragmentation is centrifugal, with physiological saline solution washing precipitation 4 times, make the suspension that solids content is 1.0mg/ml after steriling test (2000 editions " Chinese biological goods rules ") and pyrogen test (2000 editions Pharmacopoeias of the People's Republic of China) are qualified, 65 ℃ were heated 100 minutes, and obtained NCP.
Detect (solids content 1mg/ml) NCP pyrogen 60EU/ml through limulus reagent test (2000 editions Pharmacopoeias of the People's Republic of China), Coomassie brilliant blue method (" Biochemistry Experiment principle and method ", the BJ University Press) measures protein content 12% (g/g), ultraviolet method (2000 editions " Chinese biological goods rules ") 260nm measures nucleic acid content 25% (g/g), it is 16% (g/g) that anthrone method (2000 editions " Chinese biological goods rules ") is measured polyoses content, the measurement result of considering protein and nucleic acid is more accurate, estimates that remaining polysaccharide do not measure.It is 40-80nm that Electronic Speculum detects particle mean size, epigranular.After the packing, ampoule was 60 ± 2 ℃ of heating 30 minutes.
Embodiment 4
Other are with embodiment 1, and difference is that suspension after the fragmentation after 8000rpm is centrifugal 1 hour, uses the freeze dryer lyophilizing, obtains injectable powder.
Comparative example 1
Adopt the method for describing in 2000 editions " Chinese biological goods rules " with CP (formal name used at school propionibacterium acne, propionibacterium acnes) is inoculated in the nutrition jar after 771 continuous go down to posterity for 3 times, in sulphur glycollate culture medium (not containing agar), cultivated 5 days for 36-37 ℃; By the bottle microscopy, the person abandons it the living contaminants; The thalline of the no living contaminants of merging collection heated and boiled respectively carried out sterility test in 30,60 minutes, the qualified bacterium liquid of sterility test is after centrifugal, with fresh sterile normal saline solution washing precipitation 3 times and make suspension, 60-65 ℃ was heated 1 hour, the qualified back packing of aseptic detection, ampoule was 60 ± 2 ℃ of heating 30 minutes, and to contain bacterium for every bottle be 6.0 * 10 to two kinds of samples after testing
9
Comparative example 2
Other are with comparative example 1, and difference is that the thalline of the no living contaminants gathered adds formalin to ultimate density and is about 0.5% (ml/ml) sterilization 48 hours, after testing every bottle to contain bacterium be 6.0 * 10
9, free formaldehyde content is 0.06g/L.
Experimental example 1
Find out by comparative example 2, the residual formaldehyde that trace is arranged among the CPP, formaldehyde energy fixing protein, killing bacteria, but body is had stronger zest.Though residual formaldehyde seldom (should not be higher than 0.06g/l) among the CPP, but still can be to human body, especially the pleura to sensitivity produces zest.
Make according to CPP in the 240-241 page or leaf in 2000 editions " Chinese biological goods rules " among the present invention and vertification regulation in the method stipulated the spleen of the CPP of comparative example 1,2 preparations is activated and press down the tumor biologic activity and test, the results are shown in Table 1.
Simultaneously, according to following method the NCP spleen of embodiment of the invention 1-4 is activated among the present invention and press down the tumor biologic activity and test, the results are shown in Table 1.
Spleen activation experiment: with the purebred mice (C of body weight 18-20g
3H, 615 or C
57BL), each 10 of test group and matched groups, male and female half and half.Every mouse peritoneal of test group is injected CPP 0.5ml respectively and (is contained bacterium 1.5 * 10
9) and the NCP preparation 0.5ml (the NCP solids content of 1-3 is all adjusted to 1mg/ml) of embodiment 1-3, matched group is injected commensurability physiological sodium chloride solution.Put to death mice in 14 days, weigh respectively, spleen is heavy, calculate spleen index, its index should be not less than 2.0.
Press down tumor experiment (ehrlich ascites tumor): with the purebred mice (C of body weight 18-20g
3H, 615, C
57BL, K-LACA or Kunming mouse), each 10 of test group and matched groups, male and female half and half.Every oncocyte (5.0 * 10 that the mouse peritoneal injection is lived
6/ ml) 0.2ml (with the 5-8 days non-bloody ascites that go down to posterity).Test group is next day after preceding 1 day of oncocyte of injection and injection, and continuous abdominal cavity is injected CPP 0.25ml respectively and (contained bacterium 1.5 * 10
9) and the NCP 0.25ml (the NCP solids content of 1-3 is adjusted to 2mg/ml) of embodiment 1-3 5 times, each 1 day at interval.Matched group is injected commensurability physiological sodium chloride solution.Control group mice is all calculated test group mice survival rate because of cancer ascites is dead for starting point, and 30 days observation periods, its survival rate should be not less than 70%.
Table 1
| Spleen index | Press down the tumor experiment | |
| CPP (formalin-inactivated, comparative example 2) | ????＞2.0 | Qualified |
| CPP (heated and boiled deactivation 30 minutes, comparative example 1) | ????＞2.0 | Qualified |
| CPP (heated and boiled deactivation 60 minutes, comparative example 2) | ????＞2.0 | Qualified |
| NCP (embodiment 1) | ????＞2.0 | Qualified |
| NCP (embodiment 2) | ????＞2.0 | Qualified |
| NCP (embodiment 3) | ????＞2.0 | Qualified |
| NCP (embodiment 4) | ????＞2.0 | Qualified |
Spleen activates and presses down tumor and test biologic activity and the drug effect that CPP or NCP can be described.The result of table 1 shows, the CPP and the NCP of the present invention of heated and boiled 30 minutes, heated and boiled 60 minutes, 0.5% formalin deactivation preparation, its mice spleen activation reaches but the tumor experiment effect is identical, no significant difference illustrates that heated and boiled deactivation and broken the extraction do not have obvious influence to biologic activity and the drug effect of CPP and NCP.
Experimental example 2
This experimental example relate to NCP absorbent properties and with the comparison of CPP absorbent properties.
Content: rabbit CPP and NCP Intradermal absorption experiment.
Rabbit: 1500-2000 gram, male and female half and half.
Grouping: be divided into CPP group and NCP group, every group each 4.
Administration: every rabbit back intradermal injection, matched group CPP group 0.2ml/ is (15,000,000,000/ml is equivalent to 2.5mg/ml) only, and NCP group 0.2ml/ is (2.5mg/ml) only, observes and the test injection part, the results are shown in Table 2.
Table 2
| Time | Rabbit number | The NCP group | The CPP group |
| 24 hours results | ????1# | 0.2 * 0.2cm does not have redness, scleroma | 0.8 * 0.8cm redness, scleroma |
| ????2# | 0.2 * 0.2cm does not have redness, scleroma | 0.8 * 0.8cm redness, scleroma | |
| ????3# | 0.2 * 0.2cm does not have redness, scleroma | 0.8 * 0.8cm redness, scleroma | |
| ????4# | 0.2 * 0.2cm does not have redness, scleroma | 0.8 * 0.8cm redness, scleroma | |
| 48 hours results | ????1# | 0.2 * 0.2cm does not have redness, scleroma | 1.0 * 1.0cm redness, scleroma |
| ????2# | 0.2 * 0.2cm does not have redness, scleroma | 1.0 * 1.0cm redness, scleroma | |
| ????3# | 0.2 * 0.2cm does not have redness, scleroma | 1.0 * 1.0cm redness, scleroma | |
| ????4# | 0.2 * 0.2cm does not have redness, scleroma | 1.0 * 1.0cm redness, scleroma | |
| 72 hours results | ????1# | 0.2 * 0.2cm does not have redness, scleroma | 1.2 * 1.2cm redness, scleroma |
| ????2# | 0.3 * 0.3cm does not have redness, scleroma | 1.2 * 1.2cm redness, scleroma | |
| ????3# | 0.3 * 0.4cm does not have redness, scleroma | 1.5 * 1.5cm redness, scleroma | |
| ????4# | 0.4 * 0.4cm does not have redness, scleroma | 1.5 * 1.5cm redness, scleroma |
Conclusion: observed 72 hours, NCP is easy to be absorbed, and does not produce obviously red and swollen, scleroma, and CPP is difficult for being absorbed, and has produced tangible redness, scleroma reaction.This presentation of results more helps the absorption of body with the cell breakage of the CPP of comparative example 2 to the NCP of nanoscale preparation, and can reduce side effect.
Experimental example 3
This experimental example relates to the animal acute toxicity test of NCP.
Medicine: adopt the method preparation of embodiment 1, but NCP content 31.7mg/ml is provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute.
Animal: Kunming mouse, male and female half and half are provided by Nat'l Pharmaceutical ﹠ Biological Products Control Institute's animal center.
Maximum dosage-feeding is measured, get 40 of above-mentioned healthy mices, be divided into two groups at random, male and female half and half, an intramuscular injection Cmax of administration group mice (31.7mg/ml), the NCP of maximum volume (the every side of 0.1m/ * 2), an intramuscular injection equal-volume of matched group normal saline, the body weight and the clinical manifestation of the 3rd day and the 7th day the results are shown in Table 3 before and after the above two groups of administrations of record.
Table 3
| Group | Animal (only) | Dosage mg/Kg | Body weight g before the administration | ???? Body weight g after the administrationThe 3rd day the 7th day | Suitable clinical application multiple |
| The NCP group | 20 | ?31.7 | ?20.61±0.61 | ?20.80±1.50????26.67±2.19 | ?452.9 |
| Matched group | 20 | ?0 | ?20.10±0.48 | ?22.00±1.70????27.41±1.84 |
The above results shows, the NCP 31.7mg/ml of an intramuscular injection maximal dose of mice, death does not appear in mice, do not find the clinical manifestation relevant with administration, do not find the influence of administration to body weight yet, 452.9 times of the suitable clinical application amount of dosage illustrate that the clinical plan dosage of people is a safe dose.
Experimental example 4
Present embodiment relates to the pharmacodynamics evaluation of NCP to m tuberculosis infection Cavia porcellus experimental model.Cavia porcellus with m tuberculosis infection is an experimental animal model, has observed NCP of the present invention and contrast medicine immunostimulant mycobacterium phlei F.U.36 injection, and the chemotherapeutic rifampicin adds the therapeutic effect of isoniazid.
One, medicine:
Be subjected to reagent---with reference to the NCP of the method for embodiment 2 preparation, solids content 15mg/ml.
Contrast medicine---Utilins Injection (Mycobacterium phlei EU36) 17.2 μ g/ml, the healthy Pharma Inc. of Chengdu Venus packing (German Sanum-Kehlbe company original-pack), lot number 000321.
The pure powder of chemotherapeutic---isoniazid (INH), Yongkang, Beijing pharmaceutical factory provides, lot number 000313.
Rifampicin (RFP) capsule---Sichuan pharmaceutical Co. Ltd provides, lot number 000308.
Two, animal: regular grade pure white healthy guinea pig, body weight 350-400 gram, 15 every group, 8 of female 7 heros, Institute of Experimental Animals, Chinese Academy of Medical Sciences provides, the quality certification number: be betrothed to No. 071 in the capital.
Three, bacterial strain: mycobacterium tuberculosis H37RV, numbering 35810, Nat'l Pharmaceutical ﹠ Biological Products Control Institute's vaccine one Room provides.
Four, experimental technique:
1. attack the preparation of bacterium: picking is inoculated in the Luo Shi egg medium, the mycobacterium tuberculosis lawn in 37 ℃ of three weeks of cultivation is prepared into unicellular bacteria suspension with 8uM celluloid membrane filtration after grinding, and is sub-packed in small test tube,-20 ℃ of cold preservations, count plate is stand-by after two weeks of cold preservation.
2. the preparation (attacks) of model contrast and infection model: get the mycobacterium tuberculosis bacteria suspension after the cold preservation, melt the back and be diluted to 25000CFU/ml, in PPD skin test feminine gender Cavia porcellus pars inguinalis subcutaneous injection 0.2ml bacterium liquid by known viable count.
3. test is divided into groups and medication: will infect Cavia porcellus and be divided into following 6 groups at random
(1) model control group (normal saline) is 1ml/ time/
(2) chemotherapy group is INH20mg+RFP3.3mg/kg/ time
(3) mycobacterium phlei F.U.36 injection group 17.2 μ g/ time/
(4) the N.C.P group is 3mg/ time/
(5) the N.C.P group is 1mg/ time/
(6) the N.C.P group is 0.33mg/ time/
Five, medication:
(1), each dosage group of NCP and mycobacterium phlei F.U.36 injection group are in attacking back administered intramuscular in the time of 3,10,17 days, totally 3 times.
(2), chemotherapy group begins weekly to stop before oral administration 5 times was dissected to the 9th week in attacking the back next day, totally 8 all 40 times.
(3), tardy allergy carried out in attacking the back in 2,4,8 weeks, guinea pig back intradermal injection PPD10IU/0.2ml injects and measured the callosity size in back 24 hours.
Six, observation index and result:
(1) spleen bacterium separation number (CFU) is measured (this measures as the important indicator of estimating m tuberculosis infection and therapeutic effect): dissect animal when testing for the 5th, 7,9 weeks in three batches, get 1/2 spleen and put grinding in the dismembyator, add 2ml normal saline mixing and make tissue homogenate, the sulphuric acid that adds equivalent 4% before the inoculation makes 10 to kill assorted bacterium through 10 times of dilutions
-3-10
-8Inoculation 0.1ml in the tissue suspension of concentration, every pipe Russell medium, every kind of concentration inoculation two pipes.Cultivate 4-9 weeks for 37 ℃, carry out count plate in 5,7,9 weeks, viable count is to take the logarithm at the end then to carry out statistical procedures with the T method of inspection with (CFU) expression with 10.The results are shown in Table 5.
Table 5. GPS viable count (CFU) separating resulting
7 all 9 weeks of GPS viable count (CFU) logarithm value experiment grouping 5 weeks of dosage
N X SD N X SD N X SD model control group physiological saline 3 6.53 0.13 3 7.52 0.08 7 8.27 0.25 chemotherapy group INH20mg/kg/ time 3 cultivate had no in 6 weeks bacterium 3 cultivate had no in 6 weeks bacterium 6 0.48 1.18+RFP3.3mg/kg/ secondary growths grow black body woods this organize 3mg/ 3 3.23 0.14 3 3.07 0.10 9 3.08 0.19NCP of g/ 3 3.10 0.18 3 3.26 0.09 8 1.03 0.89NCP group of 1.72 μ and organize 1mg/ 3 3.80 0.05 3 1.27 1.10 9 2.99 0.19NCP and organize 0.33mg/ time 3 3.70 0.20 3 3.44 0.28 8 2.96 0.25
(2) result
1, relatively when testing for 5,7.9 weeks, CFU T checks equal P＜0.05, and significant difference is arranged for each dosage group of chemotherapy group, mycobacterium phlei F.U.36 injection group and NCP and model control group.
2, relatively, CFU T checks equal P＞0.05, there was no significant difference to each dosage group of NCP during 5,7,9 weeks.
3, each dosage group curative effect of NCP is close with the mycobacterium phlei F.U.36 injection group, but is lower than chemotherapy group.
(3) each dosage group of tardy allergy: measuring N CP callosity size of 24 hours behind PPD skin test during 2,4,8 weeks, with chemotherapy group and mycobacterium phlei F.U.36 injection group relatively, observe it to tardy allergic influence.The results are shown in Table 2.
4 all 8 weeks of the tardy allergy result of table 6. Cavia porcellus (mm) experiment grouping 2 weeks of dosage
This organizes N X SD N X SD N X SD model control group physiological saline 15 5.13 0.23 14 9.58 0.97 7 14.49 0.96 chemotherapy group INH20mg 15 4.71 1.32 13 7.02 1.25 6 10.70 0.96+RFP3.3mg/kg/ black body woods 1mg/ 15 5.21 1.02 15 7.96 2.60 9 11.87 2.08NCP of 3mg/ 15 5.52 1.31 15 8.01 2.12 9 12.68 4.25NCP group of g/ 15 4.96 1.19 15 10.92 1.39 8 10.93 1.43NCP group of 17.2 μ and organizes 0.33mg/ time 15 5.78 1.07 14 8.26 2.17 8 12.17 2.04
Skin test result (measured value)=transverse diameter+perpendicular footpath/2
(4) result
1.2 each experimental group is not seen obvious tardy allergy during week.
4 weeks of each dosage group of chemotherapy group and NCP and 8 when all tardy allergy intensity all be lower than model control group, there is significant difference P＜0.05.
3.4 the tardy allergy intensity of each dosage group of NCP all is lower than the mycobacterium phlei F.U.36 injection group during week, there is significant difference P＜0.05, and both response strengties are close during to 8 weeks, P＞0.05.
(5) pathological section: spleen is done to observe under the tissue slice mirror behind the zootomy, the results are shown in Table 7.
(6) result
1. model control group is at whole experimental session, and the mirror undertissue of spleen learns the pathological change degree and increases the weight of in time, and is the most serious during 9 weeks.
2. when testing for 7 weeks, tuberculosis specificity pathological change does not all appear in each dosage group of NCP, and the mycobacterium phlei F.U.36 injection group is 66% appearance then +++tuberculosis specificity pathological change.
3. tested for 9 whens week, all occur under each dosage group of NCP and the mycobacterium phlei F.U.36 injection arrangement of mirrors+--+++pathological change, but except that individual groups, all be lighter than model control group, chemotherapy group takes a turn for the better to some extent with the prolongation pathological change of the course of treatment.
By above-mentioned effect experiment as can be seen:
1. after each dosage group of immunomodulator NCP was treated through 9 weeks the guinea pig model of m tuberculosis infection, GPS bacterium separation number is starkly lower than model control group, but is higher than chemotherapy group, and was close with contrast medicine mycobacterium phlei F.U.36 injection group.Show that NCP has the good curing effect to the guinea pig model of m tuberculosis infection.
2. when testing for 7 weeks, the pathological change comparison is excellent according to medicine mycobacterium phlei F.U.36 injection group under each dosage group Cavia porcellus spleen mirror of NCP, tuberculosis specificity pathological change all do not occur, and tuberculosis specificity pathological change appears in mycobacterium phlei F.U.36 injection group 66%.But when testing for 9 weeks, the two is close.
3. when testing for 4 weeks, the tardy allergy intensity of each dosage group of NCP all is lower than contrast medicine mycobacterium phlei F.U.36 injection group, and the response strength of the two is close during to 8 weeks.
The credit of table 7. GPS disease of ZANG-organs reason section mirror undertissue is analysed
Classification percentage ratio (%) experiment grouping 7 all 9 weeks (dosage) of 5 weeks under the spleen pathological changes mirror
Classification N (%) N (%) N (%) model control group+3 3/3 100 3 2/3 66.6 7 2/7 28.5 (normal saline) ++ 28.5
+++1/3 33.4 43.0 chemotherapy group+3 3/3 100 3 1/3 33.3 6 6/6 100 (INH20mg ++ 1/3 33.3+RFP3.3mg/kg/ time) +++1/3 33.3 black this group of body woods+3 3/3 100 3 1/3 33.3 8 4/8 50.0 (17.2 μ g/ time) ++ 2/8 25.0
+++2/3 66.7 2/8 25.0NCP organizes+3 3/3 100 3 3/3 100 9 4/9 44.4 (3mg/ time) ++ and 2/9 22.2
+++3/9 33.4NCP organizes+3 3/3 100 3 3/3 100 9 1/9 11.1 (1mg/ time) ++ and 5/9 55.5
+++3/9 33.3NCP organizes+3 3/3 100 3 3/3 100 8 4/8 50.0 (0.33mg/ time) ++ and 3/8 37.5
+++???????????????????????????????????????????????????????????????????????1/8????12.5
Note: pathological grading standard :+--exudative change is arranged, ++--there is tubercle to form, +++--extensive cheesy pathological change is arranged.Wherein ++--+++be tuberculosis specificity pathological change.
Claims (10)
1. acellular short corynebacteria preparation is called for short the preparation method of NCP, comprises the steps:
1). short corynebacteria, be inoculated in big bottle or nutrition jar after being called for short that CP work seed lot strain is continuous and going down to posterity for 2-4 time, cultivation is 5-7 days in culture medium;
2). collect the thalline of no living contaminants, obtained bacterium liquid in heated and boiled 15-60 minute;
3). the qualified bacterium liquid of sterility test is after washing, with the broken thalline of breaker;
4). the collecting precipitation after centrifugal of the suspension behind the bacterial cell disruption, suspension is made in washing precipitation;
5). with the suspension heat sterilization, obtain NCP.
2. the preparation method of NCP according to claim 1, wherein the CP formal name used at school described in the step 1) is a propionibacterium acne, bacterium number is 76-27, H-84 or 77-1, and culture medium is to be selected from a kind of in THIOGLYCOLLIC ACID salt, peptone, liver or the yeast dialysis solution culture medium, and is low pyrogen culture medium.
3. NCP according to claim 1, wherein condition of culture is 36-37 ℃ of cultivation in the step 1).
4. the preparation method of NCP according to claim 2, wherein said sulphur glycollate culture medium is not for containing the sulphur glycollate culture medium of agar.
5. the preparation method of NCP according to claim 1, wherein the thalline described in the step 3) is an inactivated bacteria, described breaker be with bacterial cell disruption to less than 100nm, preferred size is the superhigh pressure water jet collider of 10-50nm.
6. the preparation method of NCP according to claim 1 or 5, wherein said bacterial cell disruption carries out under aseptic condition.
7. the preparation method of NCP according to claim 1, wherein the washing described in the step 3) is with physiological saline solution centrifuge washing at least 2 times with precipitate; Washing described in the step 4) is with precipitate physiological saline solution centrifuge washing 0-5 time;
The centrifugal of suspension is 4 ℃-room temperature behind the step 4) bacterial cell disruption, the centrifugal 30-150 of 6000-12000rpm minute;
Being heated to be described in the step 5) 60-65 ℃ of heating 0.5-2 hour.
8. the preparation method of NCP according to claim 1, further comprise the NCP packing that step 5) is obtained after, ampoule was 60 ± 2 ℃ of heating 30 minutes.
9. according to the preparation method of claim 1 or 2 or 4 described NCP, wherein said culture medium is the culture medium through 0.22 μ m or 0.45 μ m membrane filtration.
10. according to the preparation method of any one described NCP of claim 1-8, the pyrogen of wherein said NCP is less than 160EU/ml, protein content 10-40% (g/g), and nucleic acid content 3-25% (g/g), all the other are polysaccharide.
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| CN103505475A (en) * | 2012-06-19 | 2014-01-15 | 熊慧 | Application of Propionibacterium acne cell-free preparation |
| CN103505476A (en) * | 2012-06-19 | 2014-01-15 | 熊慧 | Cell-free preparations of immunopotentiators, and preparation methods and uses thereof |
| CN109486741A (en) * | 2018-12-20 | 2019-03-19 | 天津瑞普生物技术股份有限公司 | A kind of ablation method of pair chicken poultry bacillus |
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2002
- 2002-07-03 CN CN 02123572 patent/CN1199691C/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103505475A (en) * | 2012-06-19 | 2014-01-15 | 熊慧 | Application of Propionibacterium acne cell-free preparation |
| CN103505476A (en) * | 2012-06-19 | 2014-01-15 | 熊慧 | Cell-free preparations of immunopotentiators, and preparation methods and uses thereof |
| CN103505475B (en) * | 2012-06-19 | 2015-11-25 | 熊慧 | The application of Propionibacterium acne cell-free preparation |
| CN103082090A (en) * | 2012-09-10 | 2013-05-08 | 湖南圣雅凯生物科技有限公司 | Mycobacterium phlei Sq-1 feed additive and preparation method thereof |
| CN109486741A (en) * | 2018-12-20 | 2019-03-19 | 天津瑞普生物技术股份有限公司 | A kind of ablation method of pair chicken poultry bacillus |
| CN109486741B (en) * | 2018-12-20 | 2021-10-29 | 天津瑞普生物技术股份有限公司 | Method for inactivating avibacterium paragallinarum |
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| Publication number | Publication date |
|---|---|
| CN1199691C (en) | 2005-05-04 |
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