CN101067128A - Prepn process and medicine composition of natural arginine esterase - Google Patents
Prepn process and medicine composition of natural arginine esterase Download PDFInfo
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- CN101067128A CN101067128A CN 200710037286 CN200710037286A CN101067128A CN 101067128 A CN101067128 A CN 101067128A CN 200710037286 CN200710037286 CN 200710037286 CN 200710037286 A CN200710037286 A CN 200710037286A CN 101067128 A CN101067128 A CN 101067128A
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Abstract
The present invention is one kind of natural arginine esterase and its preparation process and medicine composition, and belongs to the field of biomedicine technology. The process of preparing natural arginine esterase includes the following steps: adsorbing natural arginine esterase from coarse snake venom material with affinity column mounted with triazozine fixed L-arginine separating material, eluting with alkaline buffering and collecting the eluted component to obtain coarse arginine esterase product, and final ion exchange chromatography to refine and to obtain refined natural arginine esterase product. The medicine composition of natural arginine esterase contains natural arginine esterase in 0.000083-0.129 wt% and medicine carrier and supplementary material in 99.871-99.999917 %. The natural arginine esterase and its medicine composition may be used in preventing and treating various kinds of thrombus and embolic diseases.
Description
Technical field
The present invention relates to a kind of preparation method and pharmaceutical composition thereof of enzyme, be specifically related to a kind of preparation method and pharmaceutical composition thereof of natural arginine esterase.Belong to the biological medicine technology field.
Background technology
Snake venom thrombin-like enzyme be in the snake venom with the general name of the class of enzymes of blood plasma zymoplasm similar performance, have the arginine ester enzymic activity usually, be called arginine esterase again.But fine proteinogen in its enzymolysis blood, but do not swash in vivo various thrombin.Therefore can make blood plasma or fibrin solution solidifies external, and the fibrin clotted texture that generates in vivo is loose, is easily removed by fibrinolytic system, and fine proteinogen concentration is significantly descended, and shows anticoagulation.This specific character makes arginine esterase become the important source of developing the thrombolytic-anticoagulant medicine, but there is multiple non-synonym coding mutation in arginine esterase molecular gene inside, and the isozyme of arginine esterase be there are differences on function.Contain arginine esterase isozyme more than 4 kinds in the viper venom, transformation period, Substratspezifitaet, specific activity are all had any different in the molecular weight of enzyme, degree of glycosylation, the body.Therefore as the clinical treatment medicine time, need separate, carry out structural confirmation, measure that amino acid is formed, the-terminal amino acid sequence, and accurately measure data such as relative molecular weight.Separation not exclusively can cause and contain multiple other echidnotoxin in the arginine esterase finished product, side reaction may occur in clinical application; The restive aborning quality product of product that while protein structure and character are fully identified can not ensure the security that medicine uses, harm patient life and health.Therefore, need development snake venom arginine esterase new preparation technology, and the arginine esterase of preparation is carried out sufficient structure and character evaluation, the security in the time of could ensureing as medicine.
Find through literature search prior art, Zhai Ning etc. have delivered the paper that is entitled as " a kind of separation and purification of new Thrombin-like enzyme in the ahylysantinfarctase " at " Chinese Journal of Pharmaceuticals " (2005 36 volume 601-603 pages or leaves), following technical scheme is proposed: at first use the anion-exchange chromatography preliminary purification, secondly make with extra care with high-efficient gel filtration chromatography, use the Thrombin-like enzyme purity of high-efficient gel filtration chromatography analyte preparation at last.Because this method uses gel filtration chromatography as one of preparation means, therefore exist applied sample amount low, yield poorly, shortcoming that production efficiency is difficult to improve.Simultaneously, because this research is not carried out sufficient protein structure and character evaluation to the Thrombin-like enzyme of preparation, be difficult to as curative drug.
Summary of the invention
The objective of the invention is to overcome deficiency of the prior art, a kind of preparation method and pharmaceutical composition thereof of natural arginine esterase are provided, make it can quick, easy preparation natural arginine esterase, and preparation contains the pharmaceutical composition of this natural arginine esterase, is used for the treatment of and prevents multiple thrombus and embolism class diseases.
The present invention is achieved by the following technical solutions, and the present invention at first prepares natural arginine esterase, and it is carried out structural confirmation, uses it for the pharmaceutical composition that preparation contains this natural arginine esterase then.
The preparation method of natural arginine esterase of the present invention specifically comprises the steps:
(1) under the buffer conditions of pH 6.0, adsorbs natural arginine esterase in the raw venin raw material with the affinity column that three nitrogen piperazine fixed L-arginine parting materials are housed, use the buffer solution elution of pH 10.3 again, collect elution fraction and obtain the natural arginine esterase crude product;
(2) with the weak anionic ion exchange chromatography above-mentioned natural arginine esterase crude product is made with extra care then, sample under pH 7.0 buffer conditions, concentration gradient wash-out with NaCl 0-0.3M is made with extra care, obtain the natural arginine esterase highly finished product, the natural arginine esterase highly finished product purity of RPLC and the preparation of denaturing polyacrylamide gel electrophoresis analysis revealed is higher than 98%.
Adopt the initial set method to survey the activity of above-mentioned natural arginine esterase highly finished product, the result shows that the specific activity of this natural arginine esterase is greater than every milligram of protein 10 000 fiber eliminating enzyme unit of activity, measure the N terminal amino acid sequence of this natural arginine esterase with the Edman edman degradation Edman, be Val Ile Gly Gly Val Glu Cys Asp Ile AsnGlu His Arg Phe Leu, with the ground substance assistant laser quality that mass spectrometry method measures the complete molecule of this natural arginine esterase of dissociating is 34.07 ± 0.20kDa, and sloughing contained sugar chain with TFMS (trifluoromethanesulfonic acid) is 28.01 ± 0.20kDa with the ground substance assistant laser quality that mass spectrometry method measures molecule of dissociating again.These structured datas of described natural arginine esterase help the bulk drug quality standard of the science of formulating, and stricter aborning control drug quality improves the security of clinical use.
Pharmaceutical composition of the present invention contains safety and treats above-mentioned natural arginine esterase 0.000083-0.129% and the pharmaceutically acceptable pharmaceutical carrier or the auxiliary material 99.871-99.999917% of significant quantity.Described pharmaceutical carrier or auxiliary material comprise weighting agent, tackiness agent, disintegrating agent or swelling property auxiliary material, lubricant, glidant, antitack agent, absorption enhancer.
Under the condition of the temperature of controlling production environment, humidity, moisture, with above-mentioned natural arginine esterase and various types of pharmaceutical carrier or auxiliary material uniform mixing, every dosage comprises above-mentioned natural arginine esterase (being calculated as 0.01-10 μ g with protein mass), weighting agent 0-200mg, tackiness agent 0-200mg, disintegrating agent or swelling property auxiliary material 0-300mg, lubricant 0-20mg, glidant 0-15mg, antitack agent 0-15mg, absorption enhancer 0-300mg etc., is prepared into the corresponding preparation form.
Described weighting agent is selected from starch, dextrin, cyclodextrin, Icing Sugar, lactose, N.F,USP MANNITOL, secondary calcium phosphate or Microcrystalline Cellulose, Mierocrystalline cellulose, also can be their mixture.
Described tackiness agent is selected from a kind of of starch slurry, hydroxypropylated starch, modified starch, pregelatinized Starch, dextrin, Icing Sugar, syrup, Microcrystalline Cellulose, derivatived cellulose (comprising Vltra tears, methylcellulose gum, carboxymethyl cellulose, ethyl cellulose), polyvinylpyrrolidone rubber cement, gelatine size, or their mixture.
Described disintegrating agent or swelling property auxiliary material are selected from a kind of in hydroxypropylated starch, modified starch, starch, carboxymethyl starch, Microcrystalline Cellulose, derivatived cellulose (comprising Vltra tears, methylcellulose gum, carboxymethyl cellulose, low-substituted hydroxypropyl cellulose, croscarmellose sodium, carboxy-propyl cellulose, calcium carboxymethylcellulose), cross-linked polyvinylpyrrolidone, tween-80, sodium lauryl sulphate, guar gum, Siberian cocklebur glue, the xanthan gum or their mixture.
Described lubricant, glidant, antitack agent can be selected a kind of in modified starch, Microcrystalline Cellulose, aluminium hydroxide, boric acid, Sodium Benzoate, polyoxyethylene glycol, stearic acid, Magnesium Stearate, calcium stearate, Zinic stearas, talcum powder, Stepanol MG, sodium lauryl sulphate or micropowder silica gel, glyceryl monostearate, rice-pudding paulownia acyl stearin, hydrogenant Viscotrol C, hydrogenated vegetable oil, light mineral oil, the fumaric acid octadecyl sodium or their mixture for use.
Described absorption enhancer can be selected a kind of in poloxamer, Yelkin TTS, sodium deoxycholate, sodium laurylsulfate, benzyl pool 7 (Bri j78), tween-80, sodium lauryl sulphate, polyoxyethylene glycol (PEG-6000), Sargassum polysaccharides, poly(lactic acid), gelatin, guar gum, Siberian cocklebur glue, the xanthan gum or their mixture for use.
The above-mentioned pharmaceutical composition that contains natural arginine esterase can be prepared as injection, oral preparation, sprays, patch, carry out administration by conventional route, comprising drug administration by injection (including, but are not limited to intramuscular, intravenously, subcutaneous, intracutaneous or topical), gastrointestinal administration, transdermal administration, intranasal administration and pulmonary administration.
Described injection is a freeze dried injection, every dosage contains natural arginine esterase 0.01-0.07% by mass percentage, and pharmaceutical carrier or auxiliary material 99.93-99.99%, described pharmaceutical carrier or auxiliary material are one or more the combination in low molecular dextran, poloxamer, Yelkin TTS, sodium deoxycholate, Sargassum polysaccharides, the poly(lactic acid).
Described oral preparation is enteric coated tablet, every dosage contains natural arginine esterase 0.013-0.129% and pharmaceutical carrier or auxiliary material 99.871-99.987% by mass percentage, described pharmaceutical carrier or auxiliary material are sodium deoxycholate, carboxymethyl cellulose salt, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, gelatin, cellulose acetate phthalate, Microcrystalline Cellulose, starch, croscarmellose sodium, Magnesium Stearate, the combination of one or more in the micropowder silica gel.
Described sprays, every dosage contains natural arginine esterase 0.000083-0.0012% and pharmaceutical carrier or auxiliary material 99.9988-99.999917% by mass percentage, and described pharmaceutical carrier or auxiliary material are one or more the combination in sodium deoxycholate, sodium laurylsulfate, the benzyl pool 7.
Described patch is a patch formulation, every dosage contains natural arginine esterase 0.0011-0.11% and pharmaceutical carrier or auxiliary material 99.89-99.9989% by mass percentage, and described pharmaceutical carrier or auxiliary material are one or more the combination in sodium deoxycholate, hydroxypropyl methyl fiber, polyvinylpyrrolidone, the polyoxyethylene glycol.
Find through comparative study, the preparation method of natural arginine esterase of the present invention, authentication method can be used for equally other venin-derived such as, come from the preparation and the evaluation of the natural arginine esterase of pallas pit viper, adder, green bamboo snake.
Pharmaceutical composition of the present invention can be directly used in treatment and pre-preventing thrombosis, the multiple embolism class diseases that causes by thrombus (as cerebral thrombosis, cerebral embolism, transient ischemic attack, and the cerebral infarction prevention of recurring again; Myocardial infarction, the prevention that unstable angina pectoris and myocardial infarction recur again; Limb angiopathy comprises femoral artery embolism, thromboangiitis obliterans, Raynaud disease; Blood is high viscosity and high coagulant state, hypercoagulative state, the preceding state of thrombus; Sudden deafness; Pulmonary infarction, or the like).Clinically for therapeutic purpose when using natural arginine esterase of the present invention, also can be simultaneously and the medication combined use of other treatment.
Pharmaceutical composition of the present invention can be made into injection formulations, for example with physiological saline or contain glucose, sodium-chlor etc., hydro-acupuncture preparation with the aqueous solution of other assistant agents is prepared by ordinary method also can add suitable auxiliary material and be prepared into powder injection or freeze-dried preparation; Also can be made into oral preparations,, can be prepared by characteristics on the basis of ordinary method at this proteolytic enzyme as tablet and capsule, dispersible tablet, enteric coated preparation by gastrointestinal administration.The preparation method of these preparations is to adopt on the basis of conventional formulation, needs the technical professional and in conjunction with the concrete physicochemical characteristics situation of this proteolytic enzyme described in the invention, could obtain in conjunction with different dosage forms and auxiliary material characteristics.
When using pharmaceutical composition of the present invention clinically, the natural arginine esterase of significant quantity in single-dose dosage of treatment be about 0.01 μ g/ kg body weight usually, and in most of the cases being no more than about 10 mg/kg body weight, this preferable dosage is about 0.001 μ g/ kg body weight-Yue 1 mg/kg body weight.Dosage as described herein is conventional, also should consider factors such as route of administration, patient symptom, healthy state at the dosage of clinical concrete use.In addition, natural arginine esterase of the present invention also can use with the other treatment agent.
According to method of the present invention and molecular structure information pharmaceutical compositions, have following advantage:
1, the molecular structure information of described natural arginine esterase can be used for carrying out in the production process material affirmation, formulates the more quality control standard of science, thereby guarantees the security of pharmaceutical composition in clinical use;
2, low, product purity quality of described natural arginine esterase preparation method's production efficiency height, product recovery rate height, production cost and specific activity height have been got rid of the toxic side effect that other snake venom component may cause.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
The experimental technique of unreceipted actual conditions among the following embodiment, usually outstanding according to normal condition such as Lip river (C.R.Lowe), Liu Yuxiu translates " affinity chromatography introduction " (Science Press, May nineteen eighty-three the 1st edition) and John M.Walker write " The Protein Protocols Handbook " (" protein method handbook (second edition) ", the Toronto, the New Jersey, Humana Press, 2002) condition described in.Biological activity determination method and condition (WS1-XG-031-2000) experimentize with reference to " Chinese Pharmacopoeia (version in 2005) " and " national drug standards-fiber eliminating enzyme ".
That is adopted in the invention process case is whole venin-derived, through identifying, for coming from Agkistrodon acutus (Guenther, agkistrodon acutus) and Agkistrodon halys (ussriensisEmelianor, Changbai Mountain agkistrodon halys ussuriensis).
The preparation of embodiment 1 agkistrodon acutus natural arginine esterase
Take by weighing agkistrodon acutus snake venom crude product 15g, be dissolved in 160ml sodium phosphate buffer (5mM sodium phosphate, 1mM EDTA, pH6.0), filter and remove insolubles, extremely use 10 times of volume sodium phosphate buffers (to contain 10mM sodium phosphate and 1mM EDTA in advance on the clarification sample, pH6.0) the usefulness three nitrogen piperazine fixed arginine affinity columns that balance is good are (in 5 * 10cm), use sodium phosphate buffer (contain 10mM sodium phosphate and 1mM EDTA, pH 6.0) to be washed till the 280nm photoabsorption to baseline, glycine-sodium hydrate buffer solution (10mM successively, pH10.3) wash-out, substep is collected, and merges to have the active component of Fibrinogen initial set, obtains this natural arginine esterase crude product.Measure total protein 150mg with forint phenol assay method; Measure with Fibrinogen initial set method, gross activity is about 800000-1200000 unit.
Regulate natural arginine esterase crude product amalgamation liquid pH to 7.0, go up to the sodium phosphate buffer (10mM that uses 5 times of volumes in advance, pH7.0) equilibrated weak anionic (DEAE) chromatography column (5 * 10cm), with sodium phosphate buffer (10mM, pH7.0) and contain the sodium phosphate buffer (10mM of 0.3M NaCl, pH7.0) gradient elution, substep is collected elution fraction, with denaturing polyacrylamide gel electrophoresis purity assay and apparent molecular weight, the merging apparent molecular weight is that the component of single band 40.5 ± 0.2KDa gets the natural arginine esterase highly finished product, and its purity is more than 98%.
Measure the total protein 75mg of natural arginine esterase highly finished product with forint phenol assay method; Measure with Fibrinogen initial set method, gross activity is about 850000 units, than living greater than 10000 units/mg albumen.
Arginine esterase experiment: get natural arginine esterase highly finished product 0.2ml, add 0.2ml L-BAPA liquid [Benzoyl-L-Arginyl-P-Nitroaniline Hydrocholoride (hydrochloric acid benzoyl-L-arginyl-P-N-methyl-p-nitroaniline) 5mg/5ml tris buffer (0.15M, pH 8.2)], put 37 ℃ of insulations, 20 minutes, it is faint yellow that solution has been, and 30 minutes, it is yellow that solution is.
Hemorrhage poison experiment: get the natural arginine esterase highly finished product, add the chlorination sodium injection make contain 25 units among every 1ml solution as need testing solution.Get 5 of the small white mouses of body weight 18-22g, back subcutaneous injection need testing solution 0.2ml injects and put to death animal in back 24 hours, and peeling is observed, and the mouse back injection site does not all have bleeding.
Neural poison experiment: get the natural arginine esterase highly finished product, with sodium chloride injection make contain 75 units among every 1ml solution as need testing solution, get body weight and be 3 of the doves of 300～500g, every 1kg body weight intravenous injection need testing solution 0.5ml, observed 24 hours, animal is not had a convulsion, death.
External thrombolysis experiment: get people-Citric Acid blood plasma [3.8%g liquor sodii citratis-human blood (1: 9) mixed, centrifugal 30 minutes of 3000 commentaries on classics/min] 0.3ml, add 400IU/ml heparin sodium aqua 0.05ml, mixing, add natural arginine esterase highly finished product 0.05ml, 37 ℃ of insulations, solution obvious retrogradation in the 20s left and right sides condenses into piece behind the 45s, add 1% monochloroacetic acid solution this moment, fast all dissolvings are coagulated in vibration, are clear solution.
The preparation of embodiment 2 agkistrodon halys ussuriensis natural arginine esterases
Take by weighing Changbai Mountain agkistrodon halys ussuriensis snake venom crude product 20g, be dissolved in the 200ml sodium phosphate buffer and (contain 5mM sodium phosphate and 1mMEDTA, pH 6.0), last sample is to using 10 times of volume sodium phosphate buffers (to contain 10mM sodium phosphate and 1mM EDTA in advance, pH 6.0) equilibrated is with three nitrogen piperazine fixed arginine affinity columns (in 2.5 * 20cm), use sodium phosphate buffer (to contain 10mM sodium phosphate and 1mM EDTA successively, pH 6.0) be washed till the 280nm photoabsorption to baseline, glycine-sodium hydrate buffer solution (10mM, pH 10.6) albumen of elution of bound, substep is collected, and merges to have the active component of Fibrinogen initial set, obtains the natural arginine esterase crude product.Measure total protein 130mg with forint phenol assay method; Measure with Fibrinogen initial set method, gross activity is in 280000 units.
Transfer natural arginine esterase crude product pH to 7.0, go up to the sodium phosphate buffer (10mM that uses 5 times of volumes in advance, pH 7.0) equilibrated weak anionic (DEAE) chromatography column (5 * 10cm), with sodium phosphate buffer (10mM, pH7.0) and contain the sodium phosphate buffer (10mM of 0.3M NaCl, pH7.0) gradient elution, substep is collected elution fraction, with SDS-polyacrylamide gel electrophoresis purity assay and apparent molecular weight, merging apparent molecular weight is the component of single band 40.1 ± 0.2KDalton, obtain the natural arginine esterase highly finished product of agkistrodon halys ussuriensis, its purity is more than 98%.
Measure the total protein 20mg of the natural arginine esterase goods of refining agkistrodon halys ussuriensis with forint phenol assay method; Measure with Fibrinogen initial set method, gross activity is about 210000 units, than living greater than 10000 units/mg albumen.
Arginine esterase experiment: the natural arginine esterase highly finished product 0.2ml that gets agkistrodon halys ussuriensis, add 0.2mlL-BAPA liquid [Benzoyl-L-Arginyl-P-Nitroaniline Hydrocholoride (hydrochloric acid benzoyl-L-arginyl-P-N-methyl-p-nitroaniline) 5mg/5ml tris buffer (0.15M, pH 8.2)], put 37 ℃ of insulations, 25 minutes, it is faint yellow that solution has been, and 30 minutes, it is yellow that solution is.
Hemorrhage poison experiment: the natural arginine esterase highly finished product of getting agkistrodon halys ussuriensis, add the chlorination sodium injection make contain 25 units among every 1ml solution as need testing solution, get 5 of the small white mouses of body weight 18-22g, back subcutaneous injection need testing solution 0.2ml, inject and put to death animal in back 24 hours, peeling is observed, and the mouse back injection site does not all have bleeding.
Neural poison experiment: get the natural arginine esterase highly finished product, with sodium chloride injection make contain 75 units among every 1ml solution as need testing solution, get body weight and be 3 of the doves of 300～500g, every 1kg body weight intravenous injection need testing solution 0.5ml, observed 24 hours, animal is not had a convulsion, death.
External thrombolysis experiment: [the 3.8%g liquor sodii citratis mixes by 1: 9 volume ratio with human blood to get people-Citric Acid blood plasma, centrifugal 30 minutes of 3000 commentaries on classics/min] 0.3ml, add 400IU/ml heparin sodium aqua 0.05ml, mixing, the natural arginine esterase highly finished product 0.05ml that adds agkistrodon halys ussuriensis, 37 ℃ of insulations, solution obvious retrogradation in the 30s left and right sides condenses into piece behind the 45s, add 1% monochloroacetic acid solution this moment, fast all dissolvings are coagulated in vibration, are clear solution.
Embodiment 3 measures the natural arginine esterase molecular mass
Get the natural arginine esterase highly finished product and mix, put about 1 μ l on target with sinapinic acid matrix, after the seasoning, the mass spectrograph of packing into (Brukerg company produces, model AutoFlex MALDI-TOF-TOF-MS), N
2Laser source (wavelength 337nm); Detect (flight pipe range 1.22M with the positive ion linear mode, acceleration voltage 20KV), obtain MALDI-TOP MS collection of illustrative plates, there is mass peak at m/z 17503,34069,68138, be respectively half point protonatomic mass, molecular mass and two times of molecular masses, so the natural arginine esterase molecular mass is 34.07 ± 0.20kDa.
Measure natural arginine esterase molecule sugar degree
It is freezing dry to get natural arginine esterase highly finished product solution (52 μ g/ml) desalination, add 20 μ l trifluoromethanesulfonic acids (TFMS),-70 ℃ of reactions are spent the night, the acetonitrile 1200 μ l that add-20 ℃ of precoolings then, mixing, under 12000rpm, 4 ℃ of conditions, acetonitrile and TFMS are removed in centrifugal 35min ultrafiltration with centrifugal ultrafiltration pipe (YM-10); Repeat once to add precooling acetonitrile-ultrafiltration again and remove acetonitrile and trifluoromethanesulfonic acid, add 600 μ l ultrapure waters at last again, mix, centrifugal ultrafiltration, remove residual acetonitrile and trifluoromethanesulfonic acid, collect last solution and find in electrophorogram, to occur altogether apparent molecular weight 36.3,34.9,33.8,30.4kDa totally 4 bands with the analysis of SDS-polyacrylamide gel electrophoresis, wherein 36.3,34.9,33.8kDa three bands content about 10% altogether, band 30.4kDa content is about 85%, is speculated as the agkistrodon acutus natural arginine esterase goods of complete desugar chain.Reclaim the albumen of band 30.4kDa, mix, put about 1 μ l on target with sinapinic acid matrix, after the seasoning, the mass spectrograph of packing into (Brukerg company produces, model AutoFlex MALDI-TOF-TOF-MS), N
2Laser source (wavelength 337nm) detects (flight pipe range 1.22M, acceleration voltage 20KV) with the positive ion linear mode, obtain mass-spectrogram, having a mass peak at m/z 28007 places, is the molecular mass after the natural arginine esterase desugar, is 28.01 ± 0.20kDa.Therefore sugar degree is that the complete molecular mass of natural arginine esterase deducts and is molecular mass 34.07-28.01 behind the desugar chain, and sugar degree is about 6.06 ± 0.20kDa.
Measure 15 aminoacid sequences of N end
Get the band 30.4kDa of the SDS-polyacrylamide gel electrophoresis of desugar natural arginine esterase, changeing the film system with half dry type albumen goes to albumen on polyvinylidene difluoride (PVDF) (PVDF) film, utilize the method for Edman degraded, measure the sequence of amino-acid residue from polypeptide chain free N-terminal, the N-terminal amino-acid residue is modified by PhNCS, downcut the residue of modifying from polypeptide chain then, identify through HPLC (high performance liquid chromatography) that again continuously tested has obtained preceding 17 round-robin amino acid high performance liquid chromatography and identified collection of illustrative plates.15 aminoacid sequences of N end are:
Val?Ile?Gly?Gly?Val?Glu?Cys?Asp?Ile?Asn?Glu?His?Arg?Phe?Leu
The pH stability test of natural arginine esterase
Getting natural arginine esterase highly finished product 40 μ l, to add 600 μ l pH respectively be 4,5,6 0.1M sodium-acetate buffer, pH is that 7,8 40mM Tris-HCl damping fluid and pH are in the Gly-NaOH damping fluid of 9,10,11,12 10nm, mix in back and 37 ℃ of water-baths and hatch a week, in 12% each sample of reduction SDS-PAGE electrophoretic analysis.Electrophoresis result shows that under pH 4,5,6 conditions, the natural arginine esterase of purifies and separates is that 40.3 ± 0.2KD place exists unique protein band at apparent molecular weight; Under pH 7,8 and 9 conditions, sample is that 40.3 ± 0.2KD and about 28.1 ± 0.2KD place exist two protein bands at apparent molecular weight; Under pH 10,11,12 conditions, almost do not seen at apparent molecular weight being the protein band at 40.3 ± 0.2KD place in the sample, all degradeds are described almost.Therefore, this natural arginine esterase is more stable under pH 4,5,6 conditions, keeps pH 4-6 as far as possible in production and storage process.
Embodiment 4 contains the enteric coated tablet of natural arginine esterase
The enteric coated tablet I that contains natural arginine esterase
The enteric coated tablet I that contains natural arginine esterase contains natural arginine esterase 0.013%, sodium deoxycholate 25.886%, carboxymethyl cellulose salt 16.179%, low-substituted hydroxypropyl cellulose 10.678%, cross-linked polyvinylpyrrolidone 8.090%, gelatin 9.707%, cellulose acetate phthalate 0.971%, Microcrystalline Cellulose 3.236%, starch 19.415%, croscarmellose sodium 5.177% by mass percentage, Magnesium Stearate 0.324%, micropowder silica gel 0.324%.
The preparation method of above-mentioned enteric coated tablet I is: get natural arginine esterase highly finished product 40mg and be dissolved in 10ml water for injection, after adding 990ml water for injection again, after adding sodium deoxycholate 80g stirring and dissolving, lyophilize, then with carboxymethyl cellulose salt 50g, low-substituted hydroxypropyl cellulose 25g, cross-linked polyvinylpyrrolidone 10g, gelatin 30g mixing granulation drying, with cellulose acetate phthalate 3g dressing, sieve and collect the particle of 18-24 order size, with Microcrystalline Cellulose 10g, starch 60g, cross-linked polyvinylpyrrolidone 15g, croscarmellose sodium 16g, low-substituted hydroxypropyl cellulose 8g, Magnesium Stearate 1g and micropowder silica gel 1g, mix and evenly be pressed into 2000, obtain containing the enteric coated tablet I of natural arginine esterase.
The enteric coated tablet II that contains natural arginine esterase
The enteric coated tablet II that contains natural arginine esterase contains natural arginine esterase 0.019%, sodium deoxycholate 25.884%, carboxymethyl cellulose salt 16.178%, low-substituted hydroxypropyl cellulose 10.678%, cross-linked polyvinylpyrrolidone 8.089%, gelatin 9.707%, cellulose acetate phthalate 0.971%, Microcrystalline Cellulose 3.236%, starch 19.413%, croscarmellose sodium 5.177% by mass percentage, Magnesium Stearate 0.324%, micropowder silica gel 0.324%.
The preparation method of above-mentioned enteric coated tablet II is: get natural arginine esterase highly finished product 60mg and be dissolved in 10ml water for injection, add 990ml water for injection again after, add sodium deoxycholate 80g stirring and dissolving after, lyophilize.Then with carboxymethyl cellulose salt 50g, low-substituted hydroxypropyl cellulose 25g, cross-linked polyvinylpyrrolidone 10g, gelatin 30g mixing granulation drying, with cellulose acetate phthalate 3g dressing, sieve and collect the particle of 18-24 order size, with Microcrystalline Cellulose 10g, starch 60g, cross-linked polyvinylpyrrolidone 15g, croscarmellose sodium 16g, low-substituted hydroxypropyl cellulose 8g, Magnesium Stearate 1g and micropowder silica gel 1g, mix and evenly be pressed into 2000, obtain containing the enteric coated tablet II of natural arginine esterase.
The enteric coated tablet III that contains natural arginine esterase
The enteric coated tablet III that contains natural arginine esterase contains natural arginine esterase 0.032%, sodium deoxycholate 25.881%, carboxymethyl cellulose salt 16.176%, low-substituted hydroxypropyl cellulose 10.676%, cross-linked polyvinylpyrrolidone 8.088%, gelatin 9.706%, cellulose acetate phthalate 0.971%, Microcrystalline Cellulose 3.235%, starch 19.411%, croscarmellose sodium 5.176% by mass percentage, Magnesium Stearate 0.324%, micropowder silica gel 0.324%.
The preparation method who contains the enteric coated tablet III of natural arginine esterase is: get natural arginine esterase highly finished product 100mg and be dissolved in 10ml water for injection, after adding 990ml water for injection again, after adding sodium deoxycholate 80g stirring and dissolving, lyophilize, then with carboxymethyl cellulose salt 50g, low-substituted hydroxypropyl cellulose 25g, cross-linked polyvinylpyrrolidone 10g, gelatin 30g mixing granulation drying, with cellulose acetate phthalate 3g dressing, sieve and collect the particle of 18-24 order size, with Microcrystalline Cellulose 10g, starch 60g, cross-linked polyvinylpyrrolidone 15g, croscarmellose sodium 16g, low-substituted hydroxypropyl cellulose 8g, Magnesium Stearate 1g and micropowder silica gel 1g, mix and evenly be pressed into 2000, obtain containing the enteric coated tablet III of natural arginine esterase.
The enteric coated tablet IV that contains natural arginine esterase
The enteric coated tablet IV that contains natural arginine esterase contains natural arginine esterase 0.065%, sodium deoxycholate 25.874%, carboxymethyl cellulose salt 16.171%, low-substituted hydroxypropyl cellulose 10.673%, cross-linked polyvinylpyrrolidone 8.085%, gelatin 9.702%, cellulose acetate phthalate 0.970%, Microcrystalline Cellulose 3.234%, starch 19.405%, croscarmellose sodium 5.175% by mass percentage, Magnesium Stearate 0.323%, micropowder silica gel 0.323%.
The preparation method who contains the enteric coated tablet IV of natural arginine esterase is: get natural arginine esterase highly finished product 200mg and be dissolved in 10ml water for injection, after adding 990ml water for injection again, after adding sodium deoxycholate 80g stirring and dissolving, lyophilize, then with carboxymethyl cellulose salt 50g, low-substituted hydroxypropyl cellulose 25g, cross-linked polyvinylpyrrolidone 10g, gelatin 30g mixing granulation drying, with cellulose acetate phthalate 3g dressing, sieve and collect the particle of 18-24 order size, with Microcrystalline Cellulose 10g, starch 60g, cross-linked polyvinylpyrrolidone 15g, croscarmellose sodium 16g, low-substituted hydroxypropyl cellulose 8g, Magnesium Stearate 1g and micropowder silica gel 1g, mix and evenly be pressed into 2000, obtain containing the enteric coated tablet IV of natural arginine esterase.
The enteric coated tablet V that contains natural arginine esterase
The enteric coated tablet V that contains natural arginine esterase contains natural arginine esterase 0.129%, sodium deoxycholate 25.857%, carboxymethyl cellulose salt 16.160%, low-substituted hydroxypropyl cellulose 10.666%, cross-linked polyvinylpyrrolidone 8.080%, gelatin 9.696%, cellulose acetate phthalate 0.970%, Microcrystalline Cellulose 3.232%, starch 19.393%, croscarmellose sodium 5.171% by mass percentage, Magnesium Stearate 0.323%, micropowder silica gel 0.323%.
The preparation method who contains the enteric coated tablet V of natural arginine esterase is: get natural arginine esterase highly finished product 400mg and be dissolved in 10ml water for injection, after adding 990ml water for injection again, after adding sodium deoxycholate 80g stirring and dissolving, lyophilize, then with carboxymethyl cellulose salt 50g, low-substituted hydroxypropyl cellulose 25g, cross-linked polyvinylpyrrolidone 10g, gelatin 30g mixing granulation drying, with cellulose acetate phthalate 3g dressing, sieve and collect the particle of 18-24 order size, with Microcrystalline Cellulose 10g, starch 60g, cross-linked polyvinylpyrrolidone 15g, croscarmellose sodium 16g, low-substituted hydroxypropyl cellulose 8g, Magnesium Stearate 1g and micropowder silica gel 1g, mix and evenly be pressed into 2000, obtain containing the enteric coated tablet V of natural arginine esterase.
The preparation and the check of embodiment 5 injection formulationss
1, freeze-drying injection formulations
The freeze-drying injection formulations I that contains natural arginine esterase
Freeze-drying injection formulations I contains natural arginine esterase 0.05%, low molecular dextran 99.95% for every bottle by mass percentage.
The preparation method who contains the freeze-drying injection formulations I of natural arginine esterase is: accurately take by weighing low molecular dextran 200 and restrain in water for injection, stirring and dissolving, drop among the embodiment 1 10 milligrams of gained natural arginine esterase highly finished product again, stirring and dissolving, regulating the pH value is 5.0～6.0, stir evenly, measure the pH value, after clarity and content are qualified, add water for injection to 20 and rise volume, filter to clear and bright with 0.22 μ m millipore filtration, every bottle of amount of pressing 1ml is aseptic subpackaged in 2ml control antibiotic bottle, add half plug, send in the freeze drying box, be cooled to-40 ℃, trading halt is cold, the open cold condenser when condenser temperature reaches-40 ℃, is opened vacuum system, open butterfly valve, with per hour 2-4 ℃ be warming up to 35 ℃ naturally; Shutdown, tamponade, outlet, roll lid, can obtain containing 20000 bottles of the freeze-drying injection formulationss " injection arginine esterase " of natural arginine esterase.
The freeze-drying injection formulations II that contains natural arginine esterase
The freeze-drying injection formulations II that contains natural arginine esterase contains natural arginine esterase 0.07%, low molecular dextran 99.93% for every bottle by mass percentage.
The preparation method who contains the freeze-drying injection formulations II of natural arginine esterase is: accurately take by weighing N.F,USP MANNITOL 300 and restrain in water for injection, stirring and dissolving, drop into 20 milligrams of embodiment 1 gained natural arginine esterase highly finished product again, stirring and dissolving, regulating the pH value is 5.0～6.0, stir evenly, measure the pH value, after clarity and content are qualified, add water for injection to 20 and rise volume, filter to clear and bright with 0.22 μ m millipore filtration, every bottle of amount of pressing lml is aseptic subpackaged in 2ml control antibiotic bottle, add half plug, send in the freeze drying box, be cooled to-40 ℃, trading halt is cold, the open cold condenser when condenser temperature reaches-40 ℃, is opened vacuum system, open butterfly valve, with per hour 2-4 ℃ be warming up to 35 ℃ naturally; Shutdown, tamponade, outlet, roll lid, can obtain containing 20000 bottles of the freeze-drying injection formulationss " injection arginine esterase " of natural arginine esterase.
The freeze-drying injection formulations III that contains natural arginine esterase
The freeze-drying injection formulations III that contains natural arginine esterase contains natural arginine esterase 0.01%, poloxamer 99.99% for every bottle by mass percentage.
The preparation method who contains the freeze-drying injection formulations III of natural arginine esterase is: accurately take by weighing poloxamer 400 and restrain in water for injection, stirring and dissolving, drop into 40 milligrams of embodiment 1 gained natural arginine esterase highly finished product again, stirring and dissolving, regulating the pH value is 5.0～6.0, stir evenly, measure the pH value, after clarity and content are qualified, add water for injection to 20 and rise volume, filter to clear and bright with 0.22 μ m millipore filtration, every bottle of amount of pressing 1ml is aseptic subpackaged in 2ml control antibiotic bottle, add half plug, send in the freeze drying box, be cooled to-40 ℃, trading halt is cold, the open cold condenser when condenser temperature reaches-40 ℃, is opened vacuum system, open butterfly valve, with per hour 2-4 ℃ be warming up to 35 ℃ naturally; Shutdown, tamponade, outlet, roll lid, can obtain containing 20000 bottles of the freeze-drying injection formulationss " injection arginine esterase " of natural arginine esterase.
The freeze-drying injection formulations IV that contains natural arginine esterase
The freeze-drying injection formulations IV that contains natural arginine esterase contains natural arginine esterase 0.011%, Yelkin TTS 55.549%, sodium deoxycholate 44.440% for every bottle by mass percentage.
The preparation method who contains the freeze-drying injection formulations IV of natural arginine esterase is: accurately take by weighing Yelkin TTS 1000 grams, sodium deoxycholate 800g is in water for injection, stirring and dissolving, drop into 200 milligrams of embodiment 1 gained natural arginine esterase highly finished product again, stirring and dissolving, regulating the pH value is 5.0～6.0, stir evenly, measure the pH value, after clarity and content are qualified, add water for injection to 20 and rise volume, filter to clear and bright with 0.22 μ m millipore filtration, every bottle of amount of pressing 1ml is aseptic subpackaged in 2ml control antibiotic bottle, add half plug, send in the freeze drying box, be cooled to-40 ℃, trading halt is cold, the open cold condenser when condenser temperature reaches-40 ℃, is opened vacuum system, open butterfly valve, with per hour 2-4 ℃ be warming up to 35 ℃ naturally; Shutdown, tamponade, outlet, roll lid, can obtain containing 20000 bottles of the freeze-drying injection formulationss " injection arginine esterase " of natural arginine esterase.
The freeze-drying injection formulations V that contains natural arginine esterase
The freeze-drying injection formulations V that contains natural arginine esterase contains natural arginine esterase 0.025%, Sargassum polysaccharides 99.975% for every bottle by mass percentage.
The preparation method who contains the freeze-drying injection formulations V of natural arginine esterase is: accurately claim Sargassum polysaccharides 800g in water for injection, stirring and dissolving, drop into 200 milligrams of embodiment 1 gained natural arginine esterase highly finished product again, stirring and dissolving, regulating the pH value is 5.0～6.0, stir evenly, measure the pH value, after clarity and content are qualified, add water for injection to 20 and rise volume, filter to clear and bright with 0.22 μ m millipore filtration, every bottle of amount of pressing 1ml is aseptic subpackaged in 2ml control antibiotic bottle, add half plug, send in the freeze drying box, be cooled to-40 ℃, trading halt is cold, the open cold condenser when condenser temperature reaches-40 ℃, is opened vacuum system, open butterfly valve, with per hour 2-4 ℃ be warming up to 35 ℃ naturally; Shutdown, tamponade, outlet, roll lid, can obtain 20000 bottles of freeze-dried preparation " injection arginine esterase ", obtain containing the injection of natural arginine esterase.
The freeze-drying injection formulations VI that contains natural arginine esterase
The freeze-drying injection formulations VI that contains natural arginine esterase contains natural arginine esterase 0.025%, poly(lactic acid) 99.975% for every bottle by mass percentage.
The preparation method who contains the freeze-drying injection formulations VI of natural arginine esterase is: accurately claim poly(lactic acid) 800g in water for injection, stirring and dissolving, drop into 200 milligrams of embodiment 1 gained natural arginine esterase highly finished product again, stirring and dissolving, regulating the pH value is 5.0～6.0, stir evenly, measure the pH value, after clarity and content are qualified, add water for injection to 20 and rise volume, filter to clear and bright with 0.22 μ m millipore filtration, every bottle of amount of pressing 1ml is aseptic subpackaged in 2ml control antibiotic bottle, add half plug, send in the freeze drying box, be cooled to-40 ℃, trading halt is cold, the open cold condenser when condenser temperature reaches-40 ℃, is opened vacuum system, open butterfly valve, with per hour 2-4 ℃ be warming up to 35 ℃ naturally; Shutdown, tamponade, outlet, roll lid, can obtain containing 20000 bottles of the freeze-drying injection formulationss " injection arginine esterase " of natural arginine esterase.
2, the inspection of injection formulations
Content uniformity: with reference to rules of preparations (" Chinese Pharmacopoeia (version in 2000) " two an appendix) relevant regulations inspection down, measure each 6 sample of 3 lot numbers altogether, the result shows that this product loading amount meets the relevant regulations of national standard preparation.Clarity test: get each five bottles of three lot number this product, according to clarity test detailed rules and regulations and judging criterion operation, every bottle with checking behind the entry needle injection 4ml water for injection, check every bottle of trichobothrium sum all＜5, wherein the color dot number is every bottle≤3, and the result shows that this product clarity meets the relevant regulations of national standard preparation; The color of solution and clarity: check according to " Chinese Pharmacopoeia (version in 2000) " two appendix methods; get five bottles of this product; every bottle adds water by labelled amount and makes the solution that contains 0.5mg among the 1ml; respectively with standard color solution yellow No. 1 and No. 1 comparison of turbidity standard; three batches of colors of this product all are lower than yellow No. 1 as a result; the clarity check result does not all surpass No. 0.5 standard turbidity solution, illustrates that color of this product and clarity are up to specification; The pH value: according to " Chinese Pharmacopoeia (version in 2000) " two appendix pH pH-value determination pH method tests, get one bottle of this product, add water 4ml and make it dissolving, measure the pH value of preparation, with reference to prescription, the technical study of this product, the pH value of determining this product is in scope; Moisture: get three batch samples and carry out moisture determination according to " Chinese Pharmacopoeia (version in 2000) " two appendix methods, the moisture content of control this product quality must not be 3.0%; It is aseptic: as, 11 every batch, to add the sterilization water respectively and make the solution that contains 0.5ug among every 1ml; check that in accordance with the law the result shows that this product sterility test meets the relevant regulations of national standard preparation with reference to " Chinese Pharmacopoeia (version in 2000) " two appendix methods; get three batches of this product; Pyrogen: get three batches of this product, add aseptic water respectively and make the solution that contains 0.2ug among every 1ml, according to " Chinese Pharmacopoeia (version in 2000) " two appendix tests, dosage is that the rabbit per kilogram of body weight is slowly injected 2ml (being equivalent to every 1kg body weight 0.4ug), and the result shows that this product pyrogen test meets the relevant regulations of national standard preparation; The assay of preparation: it is an amount of to get this product, it is fixed accurately to claim, water is made the solution that every 1ml contains arginine esterase 1mg approximately, with reference to " Chinese Pharmacopoeia (version in 2000) " two appendix high performance liquid chromatography tests, adopt gel chromatographic columns (as Biosep S2000,7.8mm * 300mm, 5 μ m) be moving phase with 0.2mol/L sodium phosphate buffer (pH6.8), flow velocity 1ml/min, be detected on 280nm wavelength place and measure absorption value, it is an amount of that other gets the arginine esterase reference substance, measures with method, with optical density calculation sample labelled amount, the result shows that the content of this product is all between 90.0-110.0% by external standard method.
To regulation, " Chinese Pharmacopoeia (2000 year version) " and the relevant ministerial standard of example " injection arginine esterase " quality of the pharmaceutical preparations research trial conclusion with reference to the relevant medicine preparation of country, the relevant regulations that meets national preparation quality standard of prepared " injection arginine esterase " preparation according to the present invention.
3, the stability test of injection formulations
High temperature test: " the injection arginine esterase " that will remove outer packaging is placed on respectively in 40 ℃, 60 ℃ the loft drier, and each detects in sampling in 5,10 days, with 0 day detected result relatively, the result shows that this product is at high temperature unstable.High wet test: " the injection arginine esterase " that will remove outer packaging is positioned in the RH92.5% environment and places, and detects in sampling in 5,10 days, compares with 0 day detected result, and the result shows that this product is more stable under super-humid conditions.Exposure experiments to light: " the injection arginine esterase " that will remove outer packaging is exposed to (with the luxmeter location) irradiation under the high light of 4500 ± 500Lx, detect respectively at sampling in 5,10 days, compare with 0 day detected result, the result shows that this product is more stable under illumination condition.Accelerated test: with this product simulation listing packing, put to place under 40 ℃, RH75% condition and detect respectively at sampling in 1,2,3,6 month, compare with 0 month detection result, accelerated test result shows, this product was placed 6 months under 40 ℃, RH75% condition, except that the outward appearance color slightly deepen and content slightly descend other index have no significant change.Test of long duration: with this product simulation listing packing, put long-term placement naturally under the room temperature condition (25 ± 2 ℃, RH60 ± 10%), detect respectively at sampling in 0,3,6,9,12,18,24,36 month, compare with 0 month detection result, the result indicates, this product room temperature keeps sample for a long time, and 12 months results are stable in investigation, and every detection index is all qualified.
Summary to the preparation stabilization Journal of Sex Research, with reference to regulation, " Chinese Pharmacopoeia (version in 2000) " and the relevant ministerial standard of the relevant medicine preparation of country, the relevant regulations that meets national preparation quality standard of prepared " injection arginine esterase " preparation according to the present invention.According to above-mentioned each test-results, and in conjunction with the characteristic of this product proteolytic enzyme, determine that this product answers shading, airtight, place low temperature, shady and cool dry place to save as suitable.
4, the external thrombolysis test of injection formulations
Adopt prepared " injection arginine esterase " injection formulations of present embodiment, according to country's " national drug standards-fiber eliminating enzyme " (WS1-XG-031-2000) method of indicating measure.Getting this product adds water and makes every ml and contain the solution of 1mg as need testing solution, get people-Citric Acid blood plasma 0.3ml, add 400IU/ml heparin sodium aqua 0.05ml, mixing adds need testing solution 0.05ml, 37 degree insulations, the clotting of plasma in 1 minute adds 1% monochloroacetic acid solution 1ml, vibration, grumeleuse all dissolves, and is clear solution.
Embodiment 6 contains the sprays of natural arginine esterase
The sprays I that contains natural arginine esterase
The sprays I that contains natural arginine esterase contains natural arginine esterase 0.000083%, sodium deoxycholate 99.999917% for every bottle by mass percentage.
The preparation method who contains the sprays I of natural arginine esterase is: take by weighing sodium deoxycholate 12kg and dissolve with water for injection 10L, taking by weighing natural arginine esterase highly finished product 10mg dissolves with water for injection 10ml, mix, regulating the pH value is 5.0～6.0, after mensuration pH value, clarity and content are qualified, add water for injection, to 150L, be sub-packed in quantitative atomizer (15ml volume, every spray 0.14ml, shared 100 sprays), obtain containing 10000 bottles of the sprayss of natural arginine esterase.
The sprays II that contains natural arginine esterase
The sprays II that contains natural arginine esterase contains natural arginine esterase 0.0005%, sodium laurylsulfate 99.9995% for every bottle by mass percentage.
The preparation method who contains the sprays II of natural arginine esterase is: take by weighing sodium laurylsulfate 10kg with water for injection 10L dissolving, take by weighing natural arginine esterase highly finished product 50mg with water for injection 10ml dissolving, mix, regulating the pH value is 5.0～6.0; Measure pH value, clarity and content qualified after, add water for injection, to 150L, be sub-packed in quantitative atomizer (15ml volume, every spray 0.14ml, shared 100 sprays), obtain containing 10000 bottles of the sprayss of natural arginine esterase.
The sprays III that contains natural arginine esterase
The sprays III that contains natural arginine esterase contains natural arginine esterase 0.0012%, benzyl pool 799.9988% for every bottle by mass percentage.
The preparation method who contains the sprays III of natural arginine esterase is: take by weighing benzyl pool 7 (Bri j78) 8kg with water for injection 10L dissolving, take by weighing natural arginine esterase highly finished product 100mg with water for injection 10ml dissolving, mix, regulating the pH value is 5.0～6.0; Measure pH value, clarity and content qualified after, add water for injection, to 150L, be sub-packed in quantitative atomizer (15ml volume, every spray 0.14ml, shared 100 sprays), obtain containing 10000 bottles of the sprayss of natural arginine esterase.
Embodiment 7 contains the patch formulation of natural arginine esterase
The patch formulation I that contains natural arginine esterase
The every square centimeter of paster of patch formulation I that contains natural arginine esterase contains natural arginine esterase 0.0011% by mass percentage, sodium deoxycholate 88.8879%, Vltra tears 3.3333%, polyvinylpyrrolidone (PVP, K30) 5.5555%, polyoxyethylene glycol (PEG-6000) 2.2222%.
The preparation method who contains the patch formulation I of natural arginine esterase is: take by weighing sodium deoxycholate 80g with water for injection 400ml dissolving, take by weighing natural arginine esterase highly finished product 1mg with water for injection 4ml dissolving, mix.Add the 3g Vltra tears, and the 5g polyvinylpyrrolidone (PVP, K30), 2g polyoxyethylene glycol (PEG-6000) mixes, and places and to go to pour in the mould behind the bubble, and the moulding of vacuum desolventizing obtains containing the paster 10000cm of described natural arginine esterase
2
The patch formulation II that contains natural arginine esterase
The every square centimeter of paster of patch formulation II that contains natural arginine esterase contains natural arginine esterase 0.011% by mass percentage, sodium deoxycholate 88.879%, Vltra tears 3.333%, polyvinylpyrrolidone (PVP, K30) 5.555%, polyoxyethylene glycol (PEG-6000) 2.222%.
The preparation method who contains the patch formulation II of natural arginine esterase is: take by weighing sodium deoxycholate 80g and dissolve with water for injection 400ml, taking by weighing natural arginine esterase highly finished product 10mg dissolves with water for injection 4ml, mix, add 3g Vltra tears, 5g polyvinylpyrrolidone (PVP, K30), 2g polyoxyethylene glycol (PEG-6000) mixes, placement goes to pour in the mould behind the bubble, and the moulding of vacuum desolventizing obtains containing the paster 10000cm of described natural arginine esterase
2
The patch formulation III that contains natural arginine esterase
The every square centimeter of paster of patch formulation III that contains natural arginine esterase contains natural arginine esterase 0.11% by mass percentage, sodium deoxycholate 88.79%, hydroxypropyl methyl fiber 3.33%, polyvinylpyrrolidone (PVP, K30) 5.55%, polyoxyethylene glycol (PEG-6000) 2.22%.
The preparation method who contains the patch formulation III of natural arginine esterase is: take by weighing sodium deoxycholate 80g and dissolve with water for injection 400ml, taking by weighing natural arginine esterase highly finished product 100mg dissolves with water for injection 4ml, mix, add 3g Vltra tears, 5g polyvinylpyrrolidone (PVP then, K30), 2g polyoxyethylene glycol (PEG-6000) mixes, placement goes to pour in the mould behind the bubble, and the moulding of vacuum desolventizing must contain the paster 10000cm of natural arginine esterase
2
Sequence that the present invention relates to and mark apportion are as follows:
The information of SEQ ID NO.1
＜110〉Shanghai Communications University
＜110〉Nisheng Biology Science and Technology Co Ltd, Shanghai
＜120〉preparation method of natural arginine esterase and pharmaceutical composition thereof
<160>1
<170>PatentIn?version?3.2
<210>1
<211>15
<212>PRT
＜213〉agkistrodon acutus (Guenther)
<400>1
Val?Ile?Gly?Gly?Val?Glu?Cys?Asp?Ile?Asn?Glu?Hi?s?Arg?Phe?Leu
1?5?10?15
Claims (8)
1, a kind of preparation method of natural arginine esterase is characterized in that, concrete steps comprise:
(1) under the buffer conditions of pH 6.0, adsorbs natural arginine esterase in the raw venin raw material with the affinity column that three nitrogen piperazine fixed L-arginine parting materials are housed, use the buffer solution elution of pH 10.3 again, collect elution fraction and obtain the natural arginine esterase crude product;
(2) with the weak anionic ion exchange chromatography above-mentioned natural arginine esterase crude product is made with extra care, sample under the pH7.0 buffer conditions is made with extra care with the concentration gradient wash-out of NaCl 0-0.3M, obtains the natural arginine esterase highly finished product.
2, the preparation method of natural arginine esterase according to claim 1 is characterized in that, described natural arginine esterase highly finished product, and its purity is higher than 98%.
3, the preparation method of natural arginine esterase according to claim 1, it is characterized in that, described natural arginine esterase, its complete molecular mass be 34.07 ± 0.20kDa, fully the molecular mass after the desugar be 28.01 ± 0.20kDa, specific activity greater than every milligram of protein 10 000 fiber eliminating enzyme unit of activity, the N terminal amino acid has the aminoacid sequence shown in the SEQ ID NO.1.
4, a kind of pharmaceutical composition that contains natural arginine esterase as claimed in claim 1, it is characterized in that, described pharmaceutical composition contains 0.000083-0.129% natural arginine esterase and pharmaceutical carrier or auxiliary material 99.871-99.999917% by mass percentage.
5, the pharmaceutical composition that contains natural arginine esterase according to claim 4, it is characterized in that, described pharmaceutical composition is a freeze dried injection, and every dosage contains natural arginine esterase 0.01-0.07% by mass percentage, and pharmaceutical carrier or auxiliary material 99.93-99.99%; Described pharmaceutical carrier or auxiliary material are one or more the combination in low molecular dextran, poloxamer, Yelkin TTS, sodium deoxycholate, Sargassum polysaccharides, the poly(lactic acid).
6, the pharmaceutical composition that contains natural arginine esterase according to claim 4, it is characterized in that, described pharmaceutical composition is enteric coated tablet, and every dosage contains natural arginine esterase 0.013-0.129% and pharmaceutical carrier or auxiliary material 99.871-99.987% by mass percentage; Described pharmaceutical carrier or auxiliary material are one or more the combination in sodium deoxycholate, carboxymethyl cellulose salt, low-substituted hydroxypropyl cellulose, cross-linked polyvinylpyrrolidone, gelatin, cellulose acetate phthalate, Microcrystalline Cellulose, starch, croscarmellose sodium, Magnesium Stearate, the micropowder silica gel.
7, the pharmaceutical composition that contains natural arginine esterase according to claim 4, it is characterized in that, described pharmaceutical composition is a sprays, and every dosage contains natural arginine esterase 0.000083-0.0012% and pharmaceutical carrier or auxiliary material 99.9988-99.999917% by mass percentage; Described pharmaceutical carrier or auxiliary material are one or more the combination in sodium deoxycholate, sodium laurylsulfate, the benzyl pool 7.
8, the pharmaceutical composition that contains natural arginine esterase according to claim 4, it is characterized in that, described pharmaceutical composition is a patch formulation, and every dosage contains natural arginine esterase 0.0011-0.11% and pharmaceutical carrier or auxiliary material 99.89-99.9989% by mass percentage; Described pharmaceutical carrier or auxiliary material are one or more the combination in sodium deoxycholate, hydroxypropyl methyl fiber, polyvinylpyrrolidone, the polyoxyethylene glycol.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880656B (en) * | 2009-05-08 | 2013-03-27 | 北京赛升药业股份有限公司 | Agkistrodon halys venom thrombin and preparation method and application thereof |
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2007
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101880656B (en) * | 2009-05-08 | 2013-03-27 | 北京赛升药业股份有限公司 | Agkistrodon halys venom thrombin and preparation method and application thereof |
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