CN101077888A - Polyethylene glycol modified neurotoxin, preparation method and use thereof - Google Patents
Polyethylene glycol modified neurotoxin, preparation method and use thereof Download PDFInfo
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- CN101077888A CN101077888A CNA2006100268345A CN200610026834A CN101077888A CN 101077888 A CN101077888 A CN 101077888A CN A2006100268345 A CNA2006100268345 A CN A2006100268345A CN 200610026834 A CN200610026834 A CN 200610026834A CN 101077888 A CN101077888 A CN 101077888A
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Abstract
The present invention discloses one kind of polyglycol modified neurotoxin and its preparation process. The polyglycol modified neurotoxin features its amino radical with connected activated polyglycol or its derivative chain. The polyglycol modified neurotoxin has the bioactivity of neurotoxin basically maintained. Compared with un-modified neurotoxin, the polyglycol modified neurotoxin has relatively lower immunogenicity, lower toxicity, high stability, improved pharmacodynamic characteristic, raised tolerance and decreased administration times. The present invention also discloses the application of the polyglycol modified neurotoxin in preparing drug breaking medicine with high efficiency and low toxicity.
Description
Technical field
The present invention relates to field of biological pharmacy, specifically, relate to a kind of polyethyleneglycol modified neurotoxin, Preparation Method And The Use.
Background technology
Neurotoxin (neurotoxin, NT) be a class single chain polypeptide, can be from Gold-banded Krait (Bungarusfasciatus (Schneider)), coral snake (Bungarus multicinctus multicinctus (Blyth)), separating in the venom of Naja (Naja naja (Linnaeus)) and Ophiophagus hannan (Cantor) (Ophiophagushannah (Cantor)) purifies obtains.According to the difference of effect target site, neurotoxin can be divided into presynaptic neurotoxin and postsynaptic neurotoxin.Because the deadly specific activity postsynaptic neurotoxin of presynaptic neurotoxin is much higher, be postsynaptic neurotoxin so use more clinically.
The postsynaptic neurotoxin total order of actual measurement is shown more than 100 at present.Their molecular weight is approximately 6800, mostly is basic protein, and iso-electric point does not have enzymic activity between 9～10, and function singleness only acts on acetylcholine receptor.Short-chain neurotoxin wherein contains 60-62 amino-acid residue and 4 disulfide linkage, and long-chain neurotoxin contains 65-74 amino-acid residue and 5 disulfide linkage.Sequence comparison shows that the homology between the short-chain neurotoxin is up to more than 70～95%, and they all have tangible amino-acid residue multiple phenomenon, QQ for example, TT, GG etc.The short-chain neurotoxin N terminal sequence that derives from cobra venom mostly is LECHNNQQ.Short-chain neurotoxin is the strong antagonist of vagusstoff, and it stops the depolarize of muscle cell competitively in conjunction with the acetylcholine receptor (AChRs) that is positioned at the neuromuscular junction caudacoria, makes animal produce the slackness muscular paralysis, and is dead because of respiratory insufficiency at last.
Chinese patent CN104096A and CN1209998A disclose a kind of preparation method and clinical drug rehabilitation and lenitive medical usage that derives from neurotoxin in cobra venin.Secular clinical use shows that neurotoxin all has certain curative effect to the various physiology and the drug dependence symptom of inactive or back appearance such as abstinence heroine, morphine, and has the characteristics of no habituation, no tolerance.
But for human body, neurotoxin belongs to heterologous protein, thus in use have the common characteristics of pharmaceutical grade protein equally, as have immunogenicity, the transformation period is short, poor stability etc.Wherein the anaphylaxis that is caused by Cobratoxin has report.(Liu Wen; Chen Hui; Zheng Hui. section's Lip river knee-piece causes anaphylactic shock. adverse drug reaction magazine [J] .2001; (3): 196.) for this reason; be necessary to adopt the protein modification technology to obtain the Cobratoxin of reduced immunogenicity; thereby improve this proteinic pharmacokinetic properties, to improve its tolerance and to reduce administration number of times.
The method of the engineered protein molecule that generally adopts is that macromolecular chemistry is modified method at present.So-called proteinic macromolecular chemistry is modified, and is with respect to adopting modifier than small molecular weight to go for the modifying protein.The macromole modifier, be often referred to some artificial or natural polymkeric substance, as polyoxyethylene glycol (PEG), dextran (dextran), human serum albumin, heparin, polylysine, poly-L-Ala, (Lundblad such as acid polyethylene and polyethylene arsenic pyrrolidone, R.L.Bradshaw, R.A.Applications of Site-Specific Chemical Modification in the Manufacture ofBiopharmaceuticals:I.An Overview.Biotechnol.Appl.Biochem.1997,26:143-151.).What wherein application was maximum, success ratio is the highest is the PEG modification technique.
The PEG that is used for the modifying protein medicine has a variety of, comprises modifying amino alkylation PEG and acylations PEG, modifies the PEG maleimide of sulfydryl and PEG-neighbour-pyridine disulfide etc., and the PEG-hydrazides etc. of modifying carboxyl.Typical modification reaction is listed below:
A:PEG-aldehyde; The B:PEG-hydroxysuccinimide eater; The C:PEG-maleimide; D:
The PEG-hydrazides
Great majority are as decorating site with the amino in the peptide molecule in the modification research.
For overcoming the snake venom neurotoxin, adopt polyethyleneglycol modified technology that the amino of neurotoxin is modified, to obtain polyethyleneglycol modified neurotoxin as the existing deficiency of drug use.
Summary of the invention
Technical problem to be solved by this invention is, there is immunogenicity in neurotoxin at prior art, transformation period is short, defectives such as poor stability, adopt the protein modification technology to obtain the Cobratoxin of reduced immunogenicity, thereby improve this proteinic pharmacokinetic properties,, satisfy the demand of neurotoxin aspect clinical application better to improve its tolerance and to reduce administration number of times.
An object of the present invention is to provide a kind of polyethyleneglycol modified neurotoxin, it is characterized in that, be connected with activated polyglycol or derivatives thereof chain on the amino of neurotoxin.
Preferably, described polyethyleneglycol modified neurotoxin has following formula:
Wherein: m is the abbreviation of mono methoxy (monomethoxy), n=2～5;
An amino (NH is removed in the R representative
2) the neurotoxin molecule.
Said neurotoxin comprises from snake venom and to extract or with the various neurotoxins of genetic engineering means preparation; Preferably from cobra-venom, extract.
Said activated polyglycol or derivatives thereof is that persons skilled in the art are known, and for example is described in Robert MJ etc., advanced useful for drug delivery comment (Advanced DrugDelivery Reviews) (2002; 54:459-476).Preferably, use propionic acid methoxy poly (ethylene glycol) succinimide ester or butyric acid methoxy poly (ethylene glycol) succinimide ester, the product that can adopt U.S. Nektar Therapeutics company to produce.
The molecular weight of said polyglycol chain is 2000～100000.
Another object of the present invention provides the preparation method of polyethyleneglycol modified neurotoxin, comprises the steps:
In neurotoxin solution, add disodium phosphate soln, regulate its pH value at 4.5-9.5.Add the activated polyglycol or derivatives thereof then, preferably the mol ratio with neurotoxin is 0.1～100.Temperature of reaction is preferably 4-40 ℃, and the reaction times is preferably 5～120 minutes.After reaction finishes, preferably adopt ion exchange column to carry out chromatographic separation reaction mixture, with its balance, use the phosphate buffered saline buffer wash-out that contains NaCl, the neurotoxin that gets final product polyethyleneglycol modifiedly with phosphate buffered saline buffer.
For instance, the polyethyleneglycol modified neurotoxin of the present invention can be made by following formula:
R-NH wherein
2Be neurotoxin.MPEG is: CH
3O-(CH
2CH
2O)
t-CH
2CH
2OH
Wherein: the abbreviation of m=mono methoxy (monomethoxy), t=44～2200;
n＝2～5
An amino (NH is removed in R representative representative
2) the neurotoxin molecule.
The polyethyleneglycol modified neurotoxin of the present invention's preparation is single polyethyleneglycol modified neurotoxin, just only 1 amino of 1 molecule neurotoxin is modified, and this amino can be the amino of N-terminal certainly, also can be the epsilon-amino on the Methionin.
Another object of the present invention provides the application of polyethyleneglycol modified neurotoxin in the anti-additive medicament of preparation high-efficiency low-toxicity.
Polyethyleneglycol modified neurotoxin of the present invention can combine with any pharmaceutically acceptable carrier, is prepared into the anti-additive medicament of any pharmaceutically acceptable formulation.
Polyethyleneglycol modified neurotoxin of the present invention can keep the biological activity of neurotoxin substantially, it has lower immunogenicity, toxicity and better stable than the neurotoxin of unmodified simultaneously, therefore as anti-additive medicament, it has bigger superiority than the neurotoxin of unmodified.The preparation method of polyethyleneglycol modified neurotoxin of the present invention, simple.
Description of drawings
Fig. 1 is polyethyleneglycol modified Cobratoxin separation and purification collection of illustrative plates.
Fig. 2 is that polyethyleneglycol modified Cobratoxin HPLC analyzes collection of illustrative plates.
Fig. 3 is that Cobratoxin HPLC analyzes collection of illustrative plates.
Fig. 4 is the MALDI-TOF-MS figure of polyethyleneglycol modified Cobratoxin
Embodiment
In embodiment, only describe, but the present invention is not limited to this neurotoxin with a kind of neurotoxin that derives from cobra-venom.
Propionic acid methoxy poly (ethylene glycol) succinimide ester 5000 (mPEG-SPA-5000) is modified the selection of condition to Cobratoxin
The selection of temperature of reaction: the Cobratoxin solution 2ml that gets 1.5mg/ml, adding the pH value that the 2ml phosphate buffered saline buffer makes solution is 8.0, add mPEG-SPA-5000 solid 1.6mg again, dissolving, mixing, respectively get 0.3ml and place 4 test tube with ground stoppers, place termination reaction behind 4 ℃, 10 ℃, 25 ℃ and the 37 ℃ reaction 30min then respectively.Relatively modification rate is determined the modification condition.The result shows: can both obtain polyethyleneglycol modified Cobratoxin under these temperature, wherein 25 ℃ modification rate is the highest.
The selection in reaction times: the Cobratoxin solution 2ml that gets 1.5mg/ml, adding the pH value that the 2ml phosphate buffered saline buffer makes solution is 8.0, add mPEG-SPA-5000 solid 1.6mg again, dissolving, mixing, respectively get 0.3ml and place 4 test tube with ground stoppers, then in 25 ℃ react 5,10,15,30,60 respectively, termination reaction behind the 120min.Relatively modification rate is determined the modification condition.The result shows: can both obtain polyethyleneglycol modified Cobratoxin under these conditions, wherein the modification rate behind the 30min does not obviously improve.
MPEG-SPA-5000 is with the selection of the mol ratio of Cobratoxin: the Cobratoxin solution 2ml that gets 1.5mg/ml, adding the pH value that the 2ml phosphate buffered saline buffer makes solution is 8.0, respectively get 0.3ml and place 4 test tube with ground stoppers, add mPEG-SPA-5000 solid 0.016,0.16,1.6,16mg (be equivalent to mPEG-SPA-5000 with the mol ratio of Cobratoxin 0.1 ~ 100) then respectively, dissolving, mixing, termination reaction behind the reaction 30min.Relatively modification rate is determined the modification condition.The result shows: can both obtain polyethyleneglycol modified Cobratoxin under these conditions, when mPEG-SPA-5000 has reached the highest with the mol ratio of Cobratoxin modification rate 10 time, the amount modification rate that further increases mPEG-SPA-5000 also increases significantly.
The selection of pH value in reaction: the Cobratoxin solution 2ml that respectively gets 0.5mg/ml, place 6 test tube with ground stoppers, it is 4.5,5.5,6.5,7.5,8.5,9.5 that the phosphate buffered saline buffer that adds the different pH values of 2ml respectively makes the pH value of solution, add mPEG-SPA-5000 solid 7.0mg again, dissolving, mixing, termination reaction behind 25 ℃ of reaction 30min.Relatively modification rate is determined the modification condition.The result shows: can both obtain polyethyleneglycol modified Cobratoxin in these conditions, wherein the pH value is that 8.0 o'clock modification rates are the highest.
Embodiment 2
The separation and purification of modified outcome and evaluation
Get the Cobratoxin solution 2ml of 1.1mg/ml, adding the pH value that phosphate buffered saline buffer makes solution is 8.0, adds mPEG-SPA-5000 solid 15.7mg again, dissolving, and mixing in 25 ℃ of reaction 30min, adds 100mg glycine termination reaction.
Getting above-mentioned reaction solution, is that 3000 ultra-filtration membrane is concentrated into 15ml with the molecular weight that dams, and upper prop separates.Chromatographic condition is as follows:
Chromatographic stuffing: Source 30S
Post specification: 2.6 * 8cm
Moving phase: 0.02M Na
2HPO
4-NaH
2PO
4, pH8.0
Flow velocity: 3.0ml/min
Detect wavelength: 215nm
Collect: every pipe 5ml
The result obtains two elution peaks, be respectively polyethyleneglycol modified Cobratoxin and Cobratoxin, concrete outcome is seen Fig. 1, and the peak of retention volume 38.49ml is Pegylation Cobratoxin peak among the figure, and retention volume is that the peak of 68.96ml is the Cobratoxin peak.
Adopt RPLC (RP-HPLC) that the component peaks that obtains is analyzed, merge same composition.Chromatographic condition is as follows:
Chromatographic column: Symmetry300
TMC
45 μ m; 3.9 * 150mm
Detect wavelength: 215nm
Moving phase: A) water/trifluoroacetic acid (100/0.1)
B) water/trifluoroacetic acid/acetonitrile (10/0.1/90)
Gradient elution method: B (0～18min, 0%～50%; 18min ~ 23min, 50% ~ 0%)
Flow velocity: 1.0ml/min
Column temperature: 37 ℃
The RP-HPLC analytical results shows: the polyethyleneglycol modified Cobratoxin that obtains and the purity of Cobratoxin are all more than 95%, concrete outcome is seen Fig. 2 and Fig. 3, retention time is the polyethyleneglycol modified Cobratoxin that is of 12.079min among the figure, and retention time is the Cobratoxin that is of 8.789min.
Adopt MALDI-TOF that polyethyleneglycol modified Cobratoxin is carried out molecular weight determination, the result shows that the molecular weight of polyethyleneglycol modified Cobratoxin is in 12000 dalton's (see figure 4)s, and the molecular weight of Cobratoxin is 7000 dalton, the two differs about 5000, this shows that this polyethyleneglycol modified Cobratoxin is mono-modified, that is to say the polyoxyethylene glycol that combines a part.
Embodiment 3
Polyethyleneglycol modified Cobratoxin Study on Stability
Multigelation stability: get Cobratoxin and polyethyleneglycol modified Cobratoxin (aseptic liquid state does not have any protective material), put-28 ℃ freezing after, under room temperature, melt, detect its cracking situation with the HPLC method.So multigelation is three times.The result shows, behind Cobratoxin and the polyethyleneglycol modified Cobratoxin multigelation three times, peak area all remains unchanged substantially.Therefore, can think that polyethyleneglycol modified Cobratoxin has kept the stability of the anti-multigelation of Cobratoxin.
Thermostability: get Cobratoxin and polyethyleneglycol modified Cobratoxin (aseptic liquid state does not have any protective material), place respectively under 30 ℃, 40 ℃, the 60 ℃ conditions.In sampling in the 5th and the 10th day, detect its cracking situation with the HPLC method.The result shows, Cobratoxin has obvious minimizing at 60 ℃ of peak areas following 5 days the time, and polyethyleneglycol modified Cobratoxin peak area in the time of 10 days does not have obvious change.In the time of 30 ℃, 40 ℃, peak area all remains unchanged substantially in Cobratoxin and the polyethyleneglycol modified Cobratoxin 10 days.Therefore, can think tentatively that polyethyleneglycol modified Cobratoxin can obviously increase the thermostability of Cobratoxin.
Enzyme stability: get 0.1% trypsin solution (pH7.8) 2.7ml, add 0.3ml Cobratoxin and polyethyleneglycol modified Cobratoxin sample respectively, 37 ℃ of water-baths, in 0,0.5,1,2, the 4h 0.3ml that takes a sample, the biological activity of working sample.The result shows: Cobratoxin is active in 0.5h to be dropped to originally 20% rapidly, only drops to original 50% and polyethyleneglycol modified Cobratoxin is active in 1h.Therefore, polyethyleneglycol modified Cobratoxin can obviously increase the ability that Cobratoxin is resisted trypsin hydrolyzing.
The pharmacodynamics comparative studies of polyethyleneglycol modified Cobratoxin and Cobratoxin
Morphine is relied on the research that animal is given up effect:
(1) test method
1, mouse jump test:
Get 80 of mouse, be divided into 4 groups at random: promptly blank group, polyethyleneglycol modified Cobratoxin are 1,2,3 groups totally 4 groups.Each organizes equal abdominal injection morphine, treatment sequence sees Table 1, last to 50 minutes after the morphine, polyethyleneglycol modified cobra venom is pressed 55.32mg/kg, 110.64mg/kg, 221.28mg/kg dosage filling stomach respectively for 1,2,3 groups, after this respectively organized equal abdominal injection naloxone 15mg/kg in 30 minutes, observe mouse jump number of times and 1 hour body weight change in 15 minutes, the results are shown in Table 2.
2, rat is urged test:
Get 40 of rats, be divided into 4 groups at random: promptly blank group, polyethyleneglycol modified Cobratoxin are 1,2,3 groups totally 4 groups.Each organizes equal abdominal injection morphine, and every day 3 times, dosage is first day 10mg/kg/ time, 2-3 day 20mg/kg/ time, 4-10 day 30mg/kg/ time.1,2,3 groups of Cobratoxin group and polyethyleneglycol modified Cobratoxins are in the 8th day oral administration gavage of test, and once a day, continuous 3 times, treatment sequence sees Table 3.After the abdominal injection morphine 8 hours the last time, abdominal injection naloxone 10mg/kg, the Withrawal symptom and the body weight of observing in 1 hour change, and Withrawal symptom is marked.(seeing Table 3)
3, morphine relies on the monkey natural withdrawal test:
Get 6 of macaques, body weight 3-5kg is divided into 2 groups at random, be blank group and polyethyleneglycol modified Cobratoxin group, each treated animal equal every day of 2 subcutaneous injection morphines, dose of morphine is incremented to 25mg/kg by 2.5mg/kg in 21 days, after this pressed this dose maintenance 90 days, to off-test.
Reinstated in 81 days in test and to mix the food method add Cobratoxin and polyethyleneglycol modified Cobratoxin respectively in two groups of foods, dosage: 81-82 days is 11.79mg/kg/ days; 83-84 days is 23.58mg/kg/ days; 85-86 days is 47.16mg/kg/ days; 87-88 days is 94.32mg/kg/ days; The 89th day is 188.64mg/kg/ days.Observing each group situation after the drug withdrawal in the 90th day changes.
(2) test-results
1, mouse jump test:
Table 1 mouse jump test morphine treatment sequence
| Time (my god) | Morphine (mg/kg, ip) | |
| First day | 15:00 | 10.0 |
| 18:00 | 10.0 | |
| 21:00 | 10.0 | |
| 24:00 | 20.0 | |
| Second day | 9:00 | 20.0 |
| 12:00 | 20.0 | |
| 15:00 | 30.0 | |
| 18:00 | 30.0 | |
| 21:00 | 30.0 | |
| 24:00 | 30.0 | |
| The 3rd day | 9:00 | 30.0 |
| 12:00 | 30.0 | |
| 15:00 | 30.0 | |
| 18:00 | 30.0 | |
| 21:00 | 30.0 | |
| 24:00 | 30.0 | |
| The 4th day | 9:00 | 30.0 |
| 12:00 | 30.0 | |
| Amount to (mg/kg) | 450.0 | |
The polyethyleneglycol modified Cobratoxin of table 2 relies on the influence of mouse jump test to morphine
| Medicine (mg/kg, po) | Number of animals | Number of skips | (g) loses weight |
| Blank | 20 | 44.28±14.33 | 0.84±0.26 |
| Polyethyleneglycol modified Cobratoxin | |||
| 58.21 | 20 | 16.0±5.9 ** | 0.51±0.25 ** |
| 116.42 | 20 | 10.8±7.8 ** | 0.33±0.24 ** |
| 232.84 | 20 | 8.5±3.3 ** | 0.21±0.16 ** |
*The blank group in p＜0.01 relatively
As known from Table 4, polyethyleneglycol modified Cobratoxin group mouse is after naloxone is urged, with the Cobratoxin group comparison number of skips and the degree that loses weight significant difference (p＜0.05) is arranged all, point out polyethyleneglycol modified Cobratoxin that morphine-dependent mice is had and alleviate its Withrawal symptom effect.
2, rat is urged test:
The polyethyleneglycol modified Cobratoxin of table 3 relies on the influence that rat is urged test to morphine
| Group | Dosage (mg/kg/ time) | Administration number of times (inferior) | Number of animals (only) | Give up score value (X ± SD) | Lose weight (g) (X ± SD) |
| | 0 | 3 | 20 | 29.3±4.4 | 15.7±3.5 |
| Polyethyleneglycol modified Cobratoxin | 18.78 | 3 | 20 | 15.23±6.2 ** | 9.5±2.7 ** |
| Polyethyleneglycol modified Cobratoxin | 37.56 | 3 | 20 | 7.5±4.1 ** | 6.2±1.6 ** |
| Polyethyleneglycol modified Cobratoxin | 75.14 | 3 | 20 | 4.3±2.5 ** | 4.1±1.8 ** |
*Compare with the physiological saline group P＜0.01
In the test, after each is organized morphine and causes rat and rely on, urge with naloxone and found that Cobratoxin group rat shows as height excitation, diarrhoea, hydrostomia, grits one's teeth, abnormal posture and body weight obviously alleviate, its Withrawal symptom scoring (table 5) is apparently higher than each group (P＜0.01) of polyethyleneglycol modified Cobratoxin, each group of polyethyleneglycol modified Cobratoxin after the morphine of stopping using, take a favourable turn miniflow tear, diarrhoea, hydrostomia and lose weight.The polyethyleneglycol modified Cobratoxin of results suggest relies on rat to morphine to be had and alleviates its Withrawal symptom effect.
3, morphine relies on the monkey natural withdrawal test:
Each group is after the morphine of stopping using, the Cobratoxin group occurs manifest symptom at inactive morphine after 3 hours, mainly show as dysphoria, sting cage, sting chain, paroxysmal is trembled, hydrostomia, diarrhoea appears in 2 monkeys after 3 hours, symptom continued 2-3 hour, occurs repeatedly in later every 1-3 hour, drowsiness couching occurred by 72 hours, weight loss 0.7-0.9kg, a monkey death.Polyethyleneglycol modified Cobratoxin group then occurs slightly having the fidgets, shout, shedding tears after the morphine of stopping using, diarrhoea appears in 1 monkey, above symptom is all light than the Cobratoxin group, and symptom disappears substantially after 5 hours, weight loss 0.3-0.4kg after 72 hours.
Conclusion:
1, mouse jump test: when oral this product total amount is 1-4 times of mouse analgesia ED50, can obviously alleviate the number of skips that relies on the morphine mouse and the degree that loses weight, be starkly lower than Cobratoxin group (P＜0.05).
2, rat is urged test: when oral this product total amount was 4.5-18 times of rat analgesia ED50, its Withrawal symptom scoring and the degree of losing weight were starkly lower than Cobratoxin group (P＜0.01).
3, morphine relies on the monkey natural withdrawal test: when oral this product total amount was 26 times of monkey analgesia ED50, the Withrawal symptom of its appearance and the degree of losing weight were starkly lower than the Cobratoxin group.
In a word, urge test and morphine to rely on the monkey natural withdrawal test by mouse jump test, rat and confirm that all polyethyleneglycol modified Cobratoxin compares Cobratoxin, it alleviates morphine, and to rely on effect of animal Withrawal symptom more remarkable.
Embodiment 5
The acute toxicity test comparative studies of polyethyleneglycol modified Cobratoxin and Cobratoxin
The intraperitoneal that the polyethyleneglycol modified Cobratoxin and the Cobratoxin of four kinds of different concns is injected to the mouse of body weight 20-25g.8 mouse of every kind of concentration injection.Calculate the dosage (LD that polyethyleneglycol modified Cobratoxin and Cobratoxin cause 50% death
50).(LD
50Method of calculation are referring to Reed LJ and Muench H.Am.J.Hygiene, 1938; 27:493.)
The result determines Cobratoxin LD
50Be 0.20mg/kg, the LD of polyethyleneglycol modified Cobratoxin
50Be 10.0mg/kg.Therefore, Cobratoxin is after polyethyleneglycol modified, and its toxicity significantly reduces.
Embodiment 6
Polyethyleneglycol modified Cobratoxin is to the laboratory animal Studies on Immunogenicity
As the sero-fast laboratory animal of preparation, adopt the freund adjuvant immunization with rabbit, and respectively with Cobratoxin and polyethyleneglycol modified Cobratoxin as antigen, dosage is 20 μ g/kg/ time, 1 time weekly, totally 5 times.
Respectively with Cobratoxin and polyethyleneglycol modified Cobratoxin as antigen, measure their sero-fast separately tiring with double immunodiffusion, measurement result is: sero-fast the tiring of Cobratoxin group is 1: 16; Polyethyleneglycol modified sero-fast the tiring of Cobratoxin group can not surveyed.
Respectively with Cobratoxin and polyethyleneglycol modified Cobratoxin as antigen, then with the antiserum(antisera) of Cobratoxin group as first antibody, again with the goat anti-rabbit igg of horseradish peroxidase (HRP) mark as second antibody, measure their immunogenicities separately with enzyme-linked immunosorbent assay (ELISA), measurement result is: the Cobratoxin group is positive, and polyethyleneglycol modified Cobratoxin group is negative.
Above result shows: with Cobratoxin relatively, the immunogenicity of polyethyleneglycol modified Cobratoxin significantly reduces.
Embodiment 7
MPEG-SPA-5000 with among propionic acid methoxy poly (ethylene glycol) succinimide ester 2000 (mPEG-SPA-2000), propionic acid methoxy poly (ethylene glycol) succinimide ester 10000 (mPEG-SPA-10000), propionic acid methoxy poly (ethylene glycol) succinimide ester 20000 (mPEG-SPA-20000) or propionic acid methoxy poly (ethylene glycol) succinimide ester 30000 (mPEG-SPA-30000) the replacement embodiment 1 has obtained the similar result of 1-6 among the embodiment.
Embodiment 8
MPEG-SPA-5000 with among butyric acid methoxy poly (ethylene glycol) succinimide ester 2000 (mPEG-SBA-2000), butyric acid methoxy poly (ethylene glycol) succinimide ester 10000 (mPEG-SBA-10000), butyric acid methoxy poly (ethylene glycol) succinimide ester 20000 (mPEG-SBA-20000) or butyric acid methoxy poly (ethylene glycol) succinimide ester 30000 (mPEG-SBA-30000) the replacement embodiment 1 has obtained the similar result of 1-6 among the embodiment.
Claims (11)
1. a polyethyleneglycol modified neurotoxin is characterized in that, is connected with activated polyglycol or derivatives thereof chain on the amino of neurotoxin.
2. polyethyleneglycol modified neurotoxin as claimed in claim 1 is characterized in that, its structure is as follows:
Wherein: m is a mono methoxy, n=2～5;
The neurotoxin molecule of an amino (NH2) is removed in the R representative.
3. polyethyleneglycol modified neurotoxin as claimed in claim 1 is characterized in that, described neurotoxin comprises from snake venom and to extract or with the various neurotoxins of genetic engineering means preparation.
4. polyethyleneglycol modified neurotoxin as claimed in claim 3 is characterized in that, the neurotoxin of described neurotoxin for extracting from cobra-venom.
5. polyethyleneglycol modified neurotoxin as claimed in claim 1 is characterized in that, described activated polyglycol or derivatives thereof is propionic acid methoxy poly (ethylene glycol) succinimide ester or butyric acid methoxy poly (ethylene glycol) succinimide ester.
6. as each described polyethyleneglycol modified neurotoxin of claim 1～5, it is characterized in that the molecular weight of described polyglycol chain is 2000～100000.
7. prepare each described polyethyleneglycol modified neurotoxin of claim 1～5, it is characterized in that, comprise the steps: in neurotoxin solution, add disodium phosphate soln, regulate its pH value 4.5～9.5, add the activated polyglycol or derivatives thereof then, after reaction finishes, reaction mixture is carried out chromatographic separation, the neurotoxin that gets final product polyethyleneglycol modifiedly.
8. method as claimed in claim 7 is characterized in that, temperature of reaction is 4～40 ℃.
9. method as claimed in claim 7 is characterized in that, the reaction times is 5～120 minutes.
10. method as claimed in claim 7 is characterized in that, the mol ratio of activated polyglycol or derivatives thereof and neurotoxin is 0.1～100.
11. the described polyethyleneglycol modified application of neurotoxin in the preparation anti-additive medicament of claim 1～6.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101450215B (en) * | 2007-12-04 | 2013-08-07 | 上海抗体药物国家工程研究中心有限公司 | Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside |
| CN107108706A (en) * | 2016-05-03 | 2017-08-29 | 深圳市健元医药科技有限公司 | Snake venom C Fragment Polymorphism derivatives |
-
2006
- 2006-05-24 CN CNA2006100268345A patent/CN101077888A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101450215B (en) * | 2007-12-04 | 2013-08-07 | 上海抗体药物国家工程研究中心有限公司 | Polylactic acid-glycolic acid co-polymer nanometer granule with immunotoxin out-connection antibody inside |
| CN107108706A (en) * | 2016-05-03 | 2017-08-29 | 深圳市健元医药科技有限公司 | Snake venom C Fragment Polymorphism derivatives |
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