CN103285372B - Cerebroprotein hydrolysate and injection preparation thereof - Google Patents
Cerebroprotein hydrolysate and injection preparation thereof Download PDFInfo
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Abstract
The invention belongs to the field of medicinal preparations, specifically relates to an injection preparation prepared from cerebroprotein hydrolysate, and comprises a cerebroprotein hydrolysate for injection, and cerebroprotein hydrolysate injection, and a preparation method thereof. The new technical problem is discovered and solved by the invention, and a high-quality, safe and effective cerebroprotein hydrolysate and the preparation thereof are provided. The technical scheme adopted by the invention is to provide a cerebroprotein hydrolysate with low sodium chloride content and undue toxicity and capable of reducing the dosage of other auxiliary materials, and the preparation of the cerebroprotein hydrolysate. Compared with the similar product on the market, the obvious improvement is realized on the product provided by the invention.
Description
Technical field
The invention belongs to field of pharmaceutical preparations, be specifically related to injection preparation prepared by Cerebrolysin Vial, comprise brain protein hydrolysate for injection, brain protein hydrolysate injection etc., and preparation method.
The seventies in last century, Austria is than the large pharmaceutical factory of prestige cattle, sheep, Medulla sus domestica, a kind of preparation that contains aminoacid and polypeptide (trade name: Cerebrolysin) of making through enzymolysis, have that treatment development in children is complete, the effect of various diseases or the effect of auxiliary treatment such as after senile dementia, cerebrovascular, and obtained good effect, obtained affirming of clinician.Subsequently, said preparation enters Chinese market with the form of injection, has received equally good therapeutic effect.Yet in all data in literature, all openly how said preparation is not specifically prepared, it is also unclear how it being carried out to quality control.
At home, the beginning of the nineties in last century, Hua Kang institute of biological products, Hainan (Hainan Tongyong Tongmeng Pharmaceutical Co., Ltd), associating The Fourth Military Medical University, Xian Medical Univ (predecessor of Xi'an Jiaotong University Medical College), and as Yuan Yan enterprise stand-alone development, go out domestic Cerebrolysin Vial and injection thereof, become domestic first successfully develop the pharmacy corporation of Cerebrolysin Vial.Through contrast verification, Cerebrolysin Vial has the aminoacid similar with Cerebrolysin and peptide spectrum, and has equivalent clinical therapeutic efficacy.
From October 30 2002, promulgate that, after medicine registration management way (trying), domestic each pharmacy corporation rises the upsurge of imitated and dosage changing form.Wherein, pharmaceutics of hydrolysate of brain protein, due to its sure curative effect, obtains everybody approval, and becomes and fall over each other imitated object.At that time, the main main points of the preparation technology of Cerebrolysin Vial are also little by little disclosed.Basically, Cerebrolysin Vial obtains through pepsin and trypsin digestion.Although technical process seems simply,, in fact so not simple.No matter, from selection, initial processing, defibrination, enzymolysis, the post-treating and other steps of the raw material of animal brain, technical process is still very complicated, and technique is relatively difficult to control.Wherein, the link of most critical is exactly enzymolysis, and the pH of enzymolysis is very crucial.Especially, for pepsin, the pH of best performance hydrolysis is 2.0, should in the scope of 1.2-3.0, select, preferably 1.5-2.8; The temperature of hydrolysis is also important, and for example 35-60 ℃, preferably goes in the scope of 40-50 ℃ to select.Reason is very simple, if can not carry out enzymolysis in above-mentioned technological parameter, not only makes hydrolysis not exclusively, the albumen of a large amount of residual xenogenesis or polypeptide, and this is breakneck for biological preparation.Again for example, if tryptic enzymolysis time is long, in the environment of nutrition, can produce rapidly growing of microorganism, cause product variation, even thoroughly corrupt, this is also breakneck.
Certainly, various documents are also reported the research of Cerebrolysin Vial in succession, and this series of research comprises: CN93103863A 19930410 discloses a kind of preparation method of cerebrolysin, and Medulla sus domestica adds water homogenate, with Ca (OH)
2adjust pH7-9, then add pig or bovine trypsin, toluene, distilled water to stir at 37-50 ℃, with phosphoric acid, adjust pH5.7-5.8, then through a series of adjust pH that boils, activated carbon decolorizing.Yet, do not adopt pepsin, not Cerebrolysin Vial truly.
CN01130523A 20011124 discloses a kind of process for preparing injection liquid of brain protein hydrolyzate, it is characterized in that: process for preparing injection liquid of brain protein hydrolyzate is: fresh Medulla sus domestica is added to the water homogenate of 2 times of volumes, filter, filtrate is 1.5-1.6 with salt acid for adjusting pH value, 40-45 degree is first used pepsin hydrolysis 6-8 hour, with sodium hydroxide, regulate pH value to 7.6-7.7 afterwards, pancreatin hydrolysis 4-6 hour for 40-45 degree, adjust pH 2.5-3.0 again, standing, at ambient temperature, filter, add again water, adjust pH is to neutral, with filtering with microporous membrane, clarify, successively with the difference molecular weight ultrafilter membrane ultrafiltration of damming, obtain concentrated solution, heating, then cooling, pass through successively again ultrafilter membrane, obtain the proteoclastic mixed polypeptide of brain, Freamine Ⅲ highly finished product.Obviously, related to repeatedly and regulated acidity with hydrochloric acid and sodium hydroxide.
CN03117554A 20030325 discloses a kind of cerebroprotein hydrolysate freeze-dried injection for injection and preparation method thereof.It is characterized in that take white or off-white color lyophilizing block or powder that Cerebrolysin Vial is main component, effective ingredient is the mixture of micromolecule brain polypeptide and free amino acid.Freeze-dried formulation of the present invention is mainly used in biological preparation, vaccine and some unsettled product formulation, to the effect of increasing significantly of the stability of product.Overcome the limitation that brain protein hydrolysate injection can not be frozen, guaranteed the stability of product.Because the technological design of innovating makes not change pharmaceutical properties after Cerebrolysin Vial product change dosage form, in pharmacological action, indication, contraindication, usage and dosage, untoward reaction etc., be all same as brain protein hydrolysate injection.
CN200410022091A 20040319 discloses a kind of preparation method of pharmaceutics of hydrolysate of brain protein.It is characterized in that: with fresh or frozen Medulla sus domestica, add the purified water homogenate of 1～2 times of volume, be heated to 85 ℃, after cooling, with hydrochloric acid, adjust pH to 1.5～2.0, with pepsin enzymolysis 3～12 hours, after homoiothermic to 42 ℃, with sodium hydroxid, adjust pH to 7.7～8.0, with pancreatin multienzyme under 38～42 ℃ of conditions, enzymolysis 2～4 hours, then adjust pH 2.5-3.0, standing freezing at-20～-10 ℃; Filter at ambient temperature, hydro-oxidation sodium adjust pH, to neutral, is got clear liquor, with the molecular weight that dams, be 10,000 ultrafilter membrane ultrafiltration, collect permeate, nanofiltration is concentrated, the concentrated solution obtaining, through 121 ℃ of steam sterilizations 30 minutes, obtains degerming solution and is called Cerebrolysin Vial highly finished product; Allotment L-Trp, TYR, each seed amino acid of interpolation, allotment peptide figure, becomes concentrated wiring liquid with above-mentioned solution preparation, then rarely joins, fill finished product, and steam sterilization, makes sterile preparation.The present invention has shortened the production cycle, output raising, easy to operation, reduces production link, has avoided the contaminated chance of medicine.
In addition, the document of this type has also been reported some preparation methoies about Cerebrolysin Vial: CN200610150892A 20061013, CN200710055421 20070316, CN200710072680A 20070820, CN200910071802A 20090417, CN200910072481A 20090709, CN201110092244A 20110412 etc.
Yet very different in these documents, some meets the condition of preparing Cerebrolysin Vial substantially, some document " is done things creatively " for document itself purely, but not for the consideration of innovating, inadvisable.For example, the pepsic optimum pH in this area is 2 left and right, also can suitably extend to 1.2-3.0 or 1.5-2.8, yet, the pH value that Many researchers is used carries out pepsic hydrolysis 3.5,4.0, even 6.0, obviously, for a person skilled in the art, such acidity environment can not be realized pepsic hydrolysis.Again for example, the disclosed Medulla sus domestica 60KG of CN201110270154A 20110904 adds 5 times of purified water (300 kilograms), then with 37% hydrochloric acid 1.5L, regulates PH2.2-2.7.Yet, any technical staff of general knowledge this area and saying slightly, consumption is few hydrochloric acid so, can not make the Medulla sus domestica of 60 kilograms that comprises a large amount of protein, polypeptide, aminoacid, electrolyte, other alkaline matter, realizes the hydrolysis under the condition of pH2.2-2.7.
These research reports also relate to the research to pharmaceutics of hydrolysate of brain protein.For example CN200910042359A 20090901 discloses a kind of preparation method of cerebroprotein hydrolysate freeze-dried injection for injection, this preparation method comprises the following steps: take Cerebrolysin Vial solution 5000 volumes, under aseptic condition, in solution, add skeleton agent 4～10 mass/volume %, adjust pH scope is 6.9～7.5; After 0.45 μ m aseptic filtration, collect filtrate; Fill, lyophilizing, wherein, it is 5 hours that the time of described lyophilizing is followed successively by-40 ℃, and-40 ℃～-15 ℃ is 4 hours, and-15 ℃～-5 ℃ is 4 hours, and-5 ℃～5 ℃ is 2 hours, and 5 ℃～40 ℃ is 3 hours, and 40 ℃ is 10 hours, 28 hours total used times of lyophilizing.Preparation method of the present invention, the constant product quality preparing is controlled, and operating procedure is simple.Obviously, in order to ensure the molding of preparation, in this patent application, used a large amount of mannitol.
About the research of Cerebrolysin Vial and preparation thereof, be only exemplary above.But, because different enterprises does not have unified manufacturing condition, cause product very different., there is the event that increasing harmful said preparation is applied in companion's appearance, these are mainly reflected in untoward reaction and increase.Such example can be with reference to " Cerebrolysin Vial caused untoward reaction document analysis in 2000 to 2010 " of the reports such as Pang Jialian, Chinese Medicine guide, in December, 2012, the 10th volume, the 35th phase, 510-512 page.So, how to go to overcome these bad reactions, everybody does not have detailed research report, the more reliable solution of neither one at present.
In the face of the problem day by day occurring, state supervision department has to start to strengthen the management of brain protein injection liquid, has really also found a lot " artificial " factors, yet for " technology " factor.Specifically, state supervision department gives notice continuously: its content is, about strengthening the notice (2008-12-15) of brain protein hydrolysate injection supervision and check, point out, in the whole nation, carry out in parenteralia pharmaceutical production technique and prescription verification work, finding brain protein hydrolysate injection kind at drug standard and carry out the aspects such as technology preparation to exist comparatively distinct issues, is mainly the quality standard imperfection that enterprise selects Medulla sus domestica raw material; Between enterprise, existing production technology difference is larger; It is inconsistent that Medulla sus domestica is hydrolyzed protease kind used, enzyme amount and hydrolysis temperature, time etc., even adds amino acid whose behavior.For above-mentioned outstanding problem, some areas have been taked control measure.For guaranteeing public's drug safety, now just further strengthen the supervision and check notice of brain protein hydrolysate injection as follows: one, will be fully recognized that brain protein hydrolysate injection is in the security risk existing aspect product quality, various places should be on the basis of parenteralia pharmaceutical production technique and prescription verification work, and positive tissue strength is conscientiously carried out supervision and inspection work.Advise that local brain protein hydrolysate injection manufacturing enterprise initiatively stops the production of this kind, and require brain protein hydrolysate injection manufacturing enterprise by correlation technique requirement, organize the research work that improves technique and method of quality control, related process improve and quality standard without approval before, wouldn't resume production.Two, for manufacturing enterprise, think that its process for preparing injection liquid of brain protein hydrolyzate is reasonable, quality controllable, proceed to produce, location provincial food Drug Administration tackles its production overall process and gives follow-up investigations, and the product of work under supervision is carried out to scene sampling, by provincial institute for drug control, checked.All manufacturing enterprises do not exist and do not produce prescription explained hereafter by approval change, or in manufactured goods, add the unlawful practices such as aminoacid, and on-the-spot sampling inspection is underproof, should sternly investigate and prosecute in accordance with the law.Three, State Bureau will organize relevant expert to carry out brain protein hydrolysate injection effectiveness, safety evaluatio work, the raising work of tissue to the improvement of process for preparing injection liquid of brain protein hydrolyzate, quality control standard, and Supervision Measures and improvement idea are proposed on this basis.
State Food and Drug Administration strengthens brain protein hydrolysate injection supervision and check (2008-12-22) and points out, in the whole nation, carry out in parenteralia pharmaceutical production technique and prescription verification work, part province office finds brain protein hydrolysate injection kind at drug standard and carries out the aspects such as technology preparation to exist comparatively distinct issues.For guaranteeing public's drug safety, a few days ago, State Food and Drug Administration gives notice, and requires various places on the basis of parenteralia pharmaceutical production technique and prescription verification work, further strengthen brain protein hydrolysate injection supervision and check, positive tissue strength is conscientiously carried out supervision and inspection work.Advise that local brain protein hydrolysate injection manufacturing enterprise initiatively stops the production of this kind, and require brain protein hydrolysate injection manufacturing enterprise by correlation technique requirement, organize the research work that improves technique and method of quality control, related process improve and quality standard without approval before, wouldn't resume production.For manufacturing enterprise, think that its process for preparing injection liquid of brain protein hydrolyzate is reasonable, quality controllable, proceed to produce, location provincial food Drug Administration tackles its production overall process and gives follow-up investigations, and the product of work under supervision is carried out to scene sampling.Notice emphasizes, all manufacturing enterprises exist and by approval change, do not produce the unlawful practices such as prescription explained hereafter, and on-the-spot sampling inspection is underproof, and each province office should sternly investigate and prosecute in accordance with the law.
Under such background and context, as we of Yuan Yan enterprise, also in the method for constantly seeking to improve Cerebrolysin Vial and the quality of the pharmaceutical preparations thereof.Unceasingly, Cerebrolysin Vial and the preparation of Dui Ben manufacturing enterprise are further furtherd investigate, thereby, obtain the present invention.
Summary of the invention
It should be understood that due to Cerebrolysin Vial complicated component, how non-single component preparation, so, improve Cerebrolysin Vial and preparation thereof, and have few precedent and can follow.For example, these improve, and no matter are the process modification to Cerebrolysin Vial stock solution, or the preparation to Cerebrolysin Vial, as the improvement of liquid drugs injection or powder pin, are difficult to obtain the improved reference of other raw material of this area and preparation.Therefore, applicant is on the basis of the test through a large amount of, unexpectedly obtains the present invention, specific as follows.
one, the discovery of technical problem
The first, the discovery of undue toxicity's technical problem
In order to improve the quality of Cerebrolysin Vial and preparation thereof, applicant has carried out measuring and evaluating (undue toxicity is normally by an index of everybody ignorance) to its undue toxicity, and find that existing brain protein hydrolysate for injection exists potential undue toxicity's risk, and all do not relate to or mention in the research all about Cerebrolysin Vial before.Discovery procedure is as follows.
1, experimental technique undue toxicity's experimental technique: with reference to 2010 editions second appendix 98(XI C of Chinese Pharmacopoeia).Particularly, on approbation mice health is qualified, and body weight 17-20 gram raises 7 days before test, normally raises.Inspection technique: injection volume: 0.5ml/, injection speed: 4-5s fast injection 0.5ml sample, complete from the injection of mouse tail passages through which vital energy circulates.All mice in 48 hours, is observed after administration.Every a collection of 5 mices, if there is death, then get 10 and test.
Source and preparation from the sample of two different manufacturers:
Commercially available sample 1: brain protein hydrolysate for injection (labelled amount: total nitrogen 30mg).
Commercially available sample 2: brain protein hydrolysate for injection (labelled amount: total nitrogen 30mg).
Take brain protein hydrolysate for injection content, with normal saline, be mixed with 6.0mg(content) sample solution of/ml, for above-mentioned undue toxicity's detection.
2, undue toxicity's experimental result (table 1):
Table 1 undue toxicity screening experiment
3, the analysis of result
Obviously, according to the above results, may there is comparison severely subnormal toxicity problem in brain protein injection liquid.Yet this problem, is normally ignored by everybody.In the face of this technical problem, next, should how to go to solve? normally, those skilled in the art easily expect, also will inevitably expect, what in experiment, adopt is the fast injection mode of injection speed 4-5s standard, causes easily occurring undue toxicity.So, use injection system slowly instead, should solve " apparent " undue toxicity problem.Experimenter of the present invention also attempts, if adopt slowly injection, for example, injection speed is changed into 10 seconds, 20 seconds, even within 30 seconds, really can solve.But, for a person skilled in the art, although solved " apparent " abnormal toxicity test problem, just really there is not undue toxicity's risk? obviously, this and be not equal to Cerebrolysin Vial and preparation has not existed the objective fact of undue toxicity's potential risk.On the contrary, the interest that this apparent phenomenon has excited research worker of the present invention to attempt to solve this technical problem more.
The second, the technical problem of freeze-drying preparation for injection molding
As everyone knows, for the molding of preparation, for brain protein hydrolysate for injection (powder pin), for example, described in above-mentioned CN200910042359A 20090901, enough proppant adjuvants must be added, molding could be realized.Otherwise the Cerebrolysin Vial that lyophilizing obtains is difficult to obtain qualified products, for example, outward appearance is defective, moisture is defective, visible foreign matters is defective, it is defective etc. to dissolve.Normally, what the selection of these adjuvants was the most frequently used is mannitol, as lyophilizing excipient and freeze drying protectant; Other can also use sorbitol, dextran etc.Yet adding too much these lyophilizing adjuvants is very disadvantageous for the safety of the medicine of injection type, the factor of these safeties is also obvious for those skilled in the art.Therefore, certainly, the supplementary product consumption that reduces pharmaceutics of hydrolysate of brain protein should become the technical problem that those skilled in the art face.
Research worker of the present invention further finds, above-mentioned first, second two the uncorrelated technical problems of original milli, on this medicine of Cerebrolysin Vial, but exist association.In our experience, to carry out the sample of two batches of experiments of undue toxicity above, commercially available sample 1: brain protein hydrolysate for injection (labelled amount: total nitrogen 30mg), through weighing, calculate 0.65 gram of average loading amount; Commercially available sample 2: brain protein hydrolysate for injection (labelled amount: total nitrogen 30mg), through weighing, calculating average loading amount is 0.67 gram.With the mean value calculation of two batches of products, average loading amount is 0.66 gram, 660 milligrams.So, according to labelled amount total nitrogen, being 30mg is basic calculation, and total amino acids and total peptide weight sum are about 188 milligrams.So 472 milligrams of remaining quality of brain protein hydrolysate for injection (Zong be approximately total amino acids and peptide 2.5 times), have been exactly the content of adjuvant and other composition.After experiment and calculating above, experimenter of the present invention, further thinks deeply and studies.Object is just to locate the way solving the problems of the technologies described above.Speculatively, undue toxicity likely appears on the adjuvant that makes preparations shaping.
two, the content of sodium chloride and the solution of technical problem
In order to solve the problems of the technologies described above, research worker of the present invention, different angles, has been done a large amount of experiments, is specifically not repeated herein.But wonderful discovery is that problem does not have not as usual, be present on the adjuvant of preparation, but research worker of the present invention finds that the content that reduces the sodium chloride in Cerebrolysin Vial is only the key technology means that solve the problems of the technologies described above.Concrete prescreen experimental result is as follows.According to the preparation method of Cerebrolysin Vial routine, prepared the brain protein hydrolysate for injection of specification (total nitrogen content labelled amount is that 30mg/ props up), and detected undue toxicity with above-mentioned method.
Sample 3: adopt the preparation method of conventional Cerebrolysin Vial, prepare Cerebrolysin Vial, be then prepared into the dosage form of brain protein hydrolysate for injection, total nitrogen content labelled amount is that 30mg/ props up.
Sample 4: conventional brain Proteolytic enzyme without preparation method in, add the processing step that reduces sodium chloride content, prepare Cerebrolysin Vial, be then prepared into the dosage form of brain protein hydrolysate for injection, total nitrogen content labelled amount is that 30mg/ props up; Wherein, the content of sodium chloride props up lower than 45mg/.
Abnormal toxicity test (table 2):
Table 2 undue toxicity screening experiment
Further, experimenter, after reducing sodium chloride content, has further done the experiment that reduces freeze drying protectant and excipient mannitol, finds, also can obtain good result.After lyophilizing, indices all can meet the requirements.
As can be seen here, research worker has been surprised to find, and the content of sodium chloride is to solve brain abnormal protein toxicity and reduce adjuvant as the key parameter of mannitol technical problem.This point, in any one research before, is not all found or is directly enlightened.
So, how does sodium chloride in Cerebrolysin Vial come? careful research worker is found, sodium chloride has two important sources: (1) is a large amount of protein because animal brain contains, polypeptide, aminoacid, electrolyte and other alkaline matter, in preparing the process of Cerebrolysin Vial, in order to realize pepsic hydrolysis, must adopt hydrochloric acid to adjust pH to selecting in the scope of 1.2-3.0, preferred 1.5-2.8, existence due to above-mentioned substance, in hydrolytic process, exist powerful buffering right, so, must use a large amount of hydrochloric acid just can make acidity meet above-mentioned requirements.Obviously, in this step, a large amount of hydrions and chloride ion must have been introduced.Then, in order to carry out tryptic hydrolysis, must neutralize with sodium hydroxide again, approach neutrality or weak basic condition hydrolysis.Obviously, in this step, introduce a large amount of sodium ion.So, after above two committed steps, produced a large amount of sodium chloride.And through follow-up Cerebrolysin Vial and preparation process thereof, sodium chloride is brought in final preparation.(2) electrolyte of animal brain, itself contains abundant sodium chloride.The total amount of above sodium chloride, can make the sodium chloride (by weight) in Cerebrolysin Vial, many times (especially in the halfway situations of brain Proteolytic enzyme) that can surpass total nitrogen (by quality ratio), for example, 2 times of above, 3 times of above, 4 times of clothing, even more than 5 times.
three, the utilization of technical scheme and technological means
1, a Cerebrolysin Vial, its preparation process comprises take animal brain as raw material, and the step through pepsin hydrolysis and trypsin hydrolyzing, is characterized in that,
In described Cerebrolysin Vial, by quality ratio, the percentage composition of sodium chloride is less than 1.0 times of percentage composition of total nitrogen, is preferably less than 0.8 times, 0.6 times, 0.4 times, 0.2 times, 0.1 times;
Described animal brain is selected from Medulla sus domestica, Medulla Bovis seu Bubali or Medulla caprae seuovis;
Described pepsin hydrolysis and the step of trypsin hydrolyzing can be both first to carry out carrying out trypsin hydrolyzing after pepsin hydrolysis again; Also can be first to carry out carrying out again pepsin hydrolysis after trypsin hydrolyzing;
Described preparation process, must not comprise the step that adopts chromatographic process separation.
Further, described Cerebrolysin Vial, can be Cerebrolysin Vial stock solution, can be also Cerebrolysin Vial stock solution through processing, for example, filters, concentrated, the dry intermediate products that obtain.
Further, described preparation process, before pepsin hydrolysis, regulating pH value is 1.2-3.0, preferably 1.5-2.8; Before trypsin hydrolyzing, regulating pH value is 6.5-9, preferably 6.8-8.0.
Described preparation process, must not comprise and adopt the step of chromatographic process separation to refer to, adopt the method for chromatographic isolation, chromatography methods such as ion exchange resin, gel chromatography, macroporous resin, complex process not only, and can introduce impurity residual on resin, more worthlessly be, according to the general knowledge of this area, the intrinsic defect of these method existence itself, can cause the variation of the chemical composition of Cerebrolysin Vial.
2, a preparation formulation for described Cerebrolysin Vial, described dosage form is selected from, peroral dosage form, injection (liquid drugs injection, powder pin, transfusion).
3, the compound preparation that the Cerebrolysin Vial described in a kind of comprising and this area other medicines carry out use in conjunction.
4, a kind of method of preparing described Cerebrolysin Vial, its preparation process comprises take animal brain as raw material, step through pepsin hydrolysis and trypsin hydrolyzing, it is characterized in that, in described preparation process, add the processing step that removes sodium chloride, or adopt the processing step that does not produce or produce less sodium chloride; And make in the described Cerebrolysin Vial of preparation, by quality ratio, the percentage composition of sodium chloride is less than 1.0 times of percentage composition of total nitrogen, is preferably less than 0.8 times, 0.6 times, 0.4 times, 0.2 times, 0.1 times;
Described preparation process, must not comprise the step that adopts chromatographic process separation.
The scheme that realizes above-mentioned preparation method is:
The first, the preparation method that adds electrodialytic processing step, specifically comprises the steps:
(1) animal brain ﹢ water, defibrination, is heated to 90-100 ℃, insulation 10-20 minute;
(2) cooling, adjusts pH1.2-3.0 with hydrochloric acid, adds pepsin reaction, obtains pepsin enzyme hydrolyzate;
(3) homoiothermic, sodium hydroxide is adjusted PH7.5-8, adds trypsin reaction;
(4) filter ultrafiltration, fill, sterilizing;
It is characterized in that, in described preparation process, after step (2) and in step (3) before, also comprise the electrodialytic processing step of step (5), step (5) is specific as follows; The pepsin hydrolysis liquid of step (2) is added in the cathode pool of electrodialytic cell, between cathode pool and anode pool, with anion exchange membrane, isolate; In anode pool, add the electrolyte solution of equivalent 0.01-1.0mo1/L, put into electrode, regulation voltage steady statue, electrodialysis is carried out in energising continuously, controls the temperature of electrodialytic cell, until cathode pool liquid pH value reaches suitable value.Take out pepsin enzyme hydrolyzate, carry out step (3).
The electrolyte solution of described step (5) is selected from: sodium chloride, calcium chloride;
The voltage of described step (5) is preferably 7 ± 5V;
The temperature of the electrodialytic cell of described step (5) is below 25 ℃, preferably below 20 ℃;
The cathode pool liquid pH value of described step (5) reaches suitable value and is selected from: 3.2,3.5,4.0,4.5,5.0,5.4, or any intermediate value of these values;
Further, the animal brain ﹢ water of described step (1), adds with 1:1;
The cooling of described step (2) is to 40-50 ℃, preferably to 45-48 ℃;
Described step (2) with hydrochloric acid, adjust pH to be preferably pH1.5-2.8;
The time of the pepsin enzyme reaction of described step (2) is 4-12 hour;
The homoiothermic of described step (3) is to 45-58 ℃, preferably to 48-54 ℃;
The time that adds trypsin reaction of described step (3) is 3.0-8.0 hour;
Described step (4), if desired, can adjust pH6.9-7.4 according to the actual needs, can also, according to electrodialytic result, supplement in right amount glutamic acid.
Schematic diagram as shown in Figure 1.
The second, there being the preparation method of carrying out the technique of pepsin hydrolysis under sulphuric acid environment, specifically comprise the steps:
(1) animal brain ﹢ water, defibrination, is heated to 90-100 ℃, insulation 10-20 minute;
(2) cooling, adjusts pH1.2-3.0 with acid, adds pepsin reaction, obtains pepsin enzyme hydrolyzate;
(3) homoiothermic, adjusts PH7.5-8 with alkali, adds trypsin reaction;
(4) filter ultrafiltration, fill, sterilizing;
It is characterized in that, in described preparation process, the acid of described step (2) is selected from: sulphuric acid, or the mixed acid of sulphuric acid and hydrochloric acid; The alkali of described step (3) is selected from: calcium hydroxide, or calcium hydroxide and sodium hydroxide are combined use;
Further, the animal brain ﹢ water of described step (1), adds with 1:1;
The cooling of described step (2) is to 40-50 ℃, preferably to 45-48 ℃;
Described step (2) with acid, adjust pH to be preferably pH1.5-2.8;
The time of the pepsin enzyme reaction of described step (2) is 4-12 hour;
The homoiothermic of described step (3) is to 45-58 ℃, preferably to 48-54 ℃;
The time that adds trypsin reaction of described step (3) is 3.0-8.0 hour;
Described step (4), if desired, also can adjust pH6.9-7.4 according to the actual needs.
explanation of nouns:
Cerebrolysin Vial, is that preparation process comprises and take animal brain as raw material, through steps such as pepsin hydrolysis and trypsin hydrolyzings, and the hydrolyzate obtaining.It can be Cerebrolysin Vial stock solution, can be also Cerebrolysin Vial stock solution through processing, for example, filters, concentrated, the dry intermediate products that obtain.About enzymolysis, Cerebrolysin Vial of the present invention obtains through transmission from one meridian to another pepsin and trypsin hydrolyzing, the hydrolysis of other enzyme to brain albumen, or only adopt a kind of mode of carrying out enzymolysis in above-mentioned enzyme, all can not be referred to as Cerebrolysin Vial of the present invention.In addition, selecting pepsin hydrolysis to regulate pH value is before 1.2-3.0, preferably 1.5-2.8; Before trypsin hydrolyzing, regulating pH value is 6.5-9, and preferably 6.8-8.0 is also necessary.Some document is for the consideration studied or for " doing things creatively " but not above-mentioned process conditions are not used in the consideration of innovation, and the Cerebrolysin Vial of this type can not be referred to as Cerebrolysin Vial of the present invention.
Animal brain: the brain that is selected from Medulla sus domestica, Medulla Bovis seu Bubali or Medulla caprae seuovis.It can be fresh, also through simple processing, for example, defat or decontamination or through freezing, stock, the brain that is selected from Medulla sus domestica, Medulla Bovis seu Bubali or Medulla caprae seuovis of all this area proper form all can.
The step of chromatographic process separation: for the consideration of scientific research, some document attempts adopting the method for chromatographic isolation, and Cerebrolysin Vial is carried out to separation, however these separation methods are only suitable for research, are not suitable for Cerebrolysin Vial of the present invention.These adopt the method for chromatographic isolation, chromatography methods such as ion exchange resin, gel chromatography, macroporous resin, complex process not only, and can introduce impurity residual on resin, more worthlessly be, according to the general knowledge of this area, the intrinsic defect of these method existence itself, can cause the variation of the chemical composition of Cerebrolysin Vial.Therefore, Cerebrolysin Vial prepared by the step of the above-mentioned conventional chromatogram separation of every employing, can not be called Cerebrolysin Vial of the present invention.
Total nitrogen: the total amount that refers to nitrogen element in Cerebrolysin Vial.
Total chlorine: the total amount that refers to chlorine element in Cerebrolysin Vial.
Sodium chloride: be that content by total chlorine converts and obtains.
accompanying drawing explanation:
Fig. 1: electrodialytic processing step schematic diagram
1. be anode pool, in-built sodium chloride solution; 2. be cathode pool, in-built pepsin hydrolysis liquid; 3. be anion exchange membrane.
The specific embodiment
In order to further illustrate the present invention, be below specific embodiment, should be noted that these embodiment are only representational.
The mensuration of sodium chloride content: adopt this area to measure the ultimate principle of sodium chloride, measure.These methods can be selected from: by measuring the mode of chloride ion content, or adopt chlorine ion electrode or adopt the assay method of total chlorine, measure and calculate by total chlorine the content of sodium chloride, and the method for the various Accurate Determining sodium chloride that can know of those of ordinary skills.
embodiment 1:the technique of contrast Cerebrolysin Vial
Cerebrolysin Vial stock solution technique: 2 kilograms of ﹢ water of (1) Medulla sus domestica (1:1), defibrination, is heated to 90-95 ℃, insulation 10-20 minute; (2) be cooled to 45-48 ℃, with 4.5% hydrochloric acid 440mL left and right, adjust pH2.5-3.0, add pepsin reaction, reaction is spent the night, and obtains pepsin enzyme hydrolyzate; (3) homoiothermic 48-54 ℃, adjusts PH7.5-8 with 2.5mol/L sodium hydroxide 220mL left and right, adds trypsin reaction 4 hours; (4) standing, filtration, ultrafiltration, fill, sterilizing; Obtain Cerebrolysin Vial stock solution 2.6L.
Embodiment 2: electrodialysis deacidification to 4.0
Cerebrolysin Vial stock solution technique: 2 kilograms of ﹢ water of (1) Medulla sus domestica (1:1), defibrination, is heated to 90-95 ℃, insulation 10-20 minute; (2) be cooled to 45-48 ℃, with 4.5% hydrochloric acid 440mL, adjust pH2.5-3.0, add pepsin reaction, reaction is spent the night, and obtains pepsin enzyme hydrolyzate;
The pepsin hydrolysis liquid of step (2) is added in the cathode pool of electrodialytic cell, between cathode pool and anode pool, with anion exchange membrane, isolate; In anode pool, add the sodium chloride solution of equivalent 0.1mo1/L, put into electrode, regulation voltage steady statue 10V left and right, electrodialysis is carried out in energising continuously, and the temperature of controlling electrodialytic cell is less than 25 ℃, until cathode pool liquid pH value reaches 3.6.Take out pepsin enzyme hydrolyzate, carry out step (3);
(3) homoiothermic 48-54 ℃, adjusts PH7.5-8 and adds suitable quantity of water with 2.5mol/L sodium hydroxide, adds trypsin reaction 4 hours; (4) filter ultrafiltration, fill, sterilizing; Obtain Cerebrolysin Vial stock solution 2.6L.
Embodiment 3: electrodialysis deacidification to 4.5
Cerebrolysin Vial stock solution technique: 2 kilograms of ﹢ water of (1) Medulla sus domestica (1:1), defibrination, is heated to 90-95 ℃, insulation 10-20 minute; (2) be cooled to 45-48 ℃, with 4.5% hydrochloric acid 440mL, adjust pH2.5-3.0, add pepsin reaction, reaction is spent the night, and obtains pepsin enzyme hydrolyzate;
The pepsin hydrolysis liquid of step (2) is added in the cathode pool of electrodialytic cell, between cathode pool and anode pool, with anion exchange membrane, isolate; In anode pool, add the sodium chloride solution of equivalent 0.1mo1/L, put into electrode, regulation voltage steady statue 10V left and right, electrodialysis is carried out in energising continuously, and the temperature of controlling electrodialytic cell is less than 25 ℃, until cathode pool liquid pH value reaches 4.5.Take out pepsin enzyme hydrolyzate, carry out step (3);
(3) homoiothermic 48-54 ℃, adjusts PH7.5-8 and adds suitable quantity of water with 2.5mol/L sodium hydroxide, adds trypsin reaction 4 hours; (4) filter ultrafiltration, fill, sterilizing; Obtain Cerebrolysin Vial stock solution 2.6L.
Embodiment 4: sulphuric acid replaces 50% hydrochloric acid
Cerebrolysin Vial stock solution technique: 2 kilograms of ﹢ water of (1) Medulla sus domestica (1:1), defibrination, is heated to 90-95 ℃, insulation 10-20 minute; (2) be cooled to 45-48 ℃, add after 4.5% hydrochloric acid 220mL, and adjust pH2.5-3.0 with 2.5% sulphuric acid, add pepsin reaction, reaction is spent the night, and obtains pepsin enzyme hydrolyzate; (3) homoiothermic 48-54 ℃, adjusts PH7.5-8 with the water of 2.5mol/L sodium hydroxide 110mL and calcium hydroxide and 110mL, adds trypsin reaction 4 hours; (4) filter ultrafiltration, fill, sterilizing; Obtain Cerebrolysin Vial stock solution 2.6L.
Embodiment 5: sulphuric acid replaces 75% hydrochloric acid
Cerebrolysin Vial stock solution technique: 10 kilograms of ﹢ water of (1) Medulla sus domestica (1:1), defibrination, is heated to 90-95 ℃, insulation 10-20 minute; (2) be cooled to 45-48 ℃, add after 4.5% hydrochloric acid 110mL, and adjust pH2.5-3.0 with 2.5% sulphuric acid, add pepsin reaction, reaction is spent the night, and obtains pepsin enzyme hydrolyzate; (3) homoiothermic 48-54 ℃, adjusts PH7.5-8 with the water of 2.5mol/L sodium hydroxide 55mL and calcium hydroxide and 165mL, adds trypsin reaction 4 hours; (4) filter ultrafiltration, fill, sterilizing; Obtain Cerebrolysin Vial stock solution 2.6L.
Embodiment 6: content and the undue toxicity of sodium chloride test comparison
Experimental technique: first that the brain albumen stock solution of above-described embodiment 1-5 is appropriate, take mannitol as adjuvant, through dilution, activated carbon, remove the steps such as thermal source, filtration, ultrafiltration, subpackage, lyophilizing, be prepared into the brain protein hydrolysate for injection that labelled amount is total nitrogen 30mg; Average loading amount is about 0.66 gram.Assay and undue toxicity's experiment for sodium chloride.
Undue toxicity's experimental technique: with reference to 2010 editions second appendix 98(XI C of Chinese Pharmacopoeia) experimental technique.Particularly, on approbation mice health is qualified, and body weight 17-20 gram raises 7 days before test, normally raises.Inspection technique: injection volume: 0.5ml/, injection speed: 4-5s fast injection 0.5ml sample, complete from the injection of mouse tail passages through which vital energy circulates.All mice in 48 hours, is observed after administration.Every a collection of 5 mices, if there is death, then get 10 and test.
Take brain protein hydrolysate for injection prepared by embodiment 1-5 Cerebrolysin Vial stock solution, with normal saline, be mixed with the sample solution of 6.0mg/ml, for above-mentioned undue toxicity's detection.
Sodium chloride content and the undue toxicity of table 3 embodiment 1-5 test comparison
Note: "-" represents, need not carry out the 2nd undue toxicity's experiment.
Embodiment 7: quality evaluation (chromatography)
This product is by the standard YBH33522005 of State Food and Drug Administration and two checks of < < Chinese Pharmacopoeia > > version in 2010, and result is up to specification.
Particularly, the brain protein hydrolysate for injection of embodiment 1-5 is faint yellow loose block; Biuret reaction; The HPLC discriminating chromatogram of need testing solution and the chromatogram of contrast solution are basically identical; PH value is 7.0～7.4; Protein checks that solution is for clarification; Moisture does not surpass 3.0%; Clarity and the color of solution are up to specification; Other material is up to specification; Bacterial endotoxin is up to specification; Content uniformity is 0.66 gram of left and right; Particulate matter meets fixing; Visible foreign matters is up to specification; Sterility test is up to specification; Free amino acid is 150～180mg/ bottle.
Embodiment 8: the comparison that freeze-dry process improves
Take embodiment 1 as reference, adopt identical technique, carried out the interpolation comparative experiments of freeze-dry process mannitol adjuvant, result is as follows.
From result, at identical experiment condition, the sodium chloride content in Cerebrolysin Vial reduced, be conducive to the raising of freeze-dry process quality, particularly, can reduce largely the consumption of adjuvant, both reduce the risk of toxic and side effects, also produced economic worth.
Sodium chloride content and the undue toxicity of table 4 embodiment 1-5 test comparison
Claims (9)
1. a Cerebrolysin Vial, is to take animal brain as raw material, through the step of pepsin hydrolysis and trypsin hydrolyzing, makes; It is characterized in that, in preparation process, add remove the electrodialysis process step of sodium chloride or before pepsin hydrolysis with the acid for adjusting pH that mixes of sulphuric acid and hydrochloric acid;
In described Cerebrolysin Vial, by quality ratio, the percentage composition of sodium chloride is less than 1.0 times of percentage composition of total nitrogen;
Described animal brain is selected from one or more of Medulla sus domestica, Medulla Bovis seu Bubali or Medulla caprae seuovis;
Described pepsin hydrolysis and the step of trypsin hydrolyzing are first to carry out carrying out trypsin hydrolyzing after pepsin hydrolysis again; Or first carry out carrying out again pepsin hydrolysis after trypsin hydrolyzing;
And, in described preparation process, do not adopt the step of chromatographic process separation.
2. Cerebrolysin Vial as claimed in claim 1, is characterized in that, in described Cerebrolysin Vial, by quality ratio, the percentage composition of sodium chloride is less than 0.8 times of percentage composition of total nitrogen.
3. Cerebrolysin Vial as claimed in claim 1, is characterized in that, in described Cerebrolysin Vial, by quality ratio, the percentage composition of sodium chloride is less than 0.6 times of percentage composition of total nitrogen.
4. Cerebrolysin Vial as claimed in claim 1, is characterized in that, in described Cerebrolysin Vial, by quality ratio, the percentage composition of sodium chloride is less than 0.4 times of percentage composition of total nitrogen.
5. the Cerebrolysin Vial as described in claim 1-4 any one, is characterized in that, described pepsin hydrolysis, and regulate pH value is 1.2-3.0.
6. the Cerebrolysin Vial as described in claim 1-4 any one, is characterized in that, described trypsin hydrolyzing, and regulate pH value is 6.5-9.
7. a preparation for Cerebrolysin Vial, is characterized in that, the Cerebrolysin Vial of usining described in claim 1-6 any one claim is as unique active component, and described preparation is selected from peroral dosage form, injection.
8. a method of preparing the Cerebrolysin Vial described in claim 1-6 any one claim, is to take animal brain as raw material, through the step preparation of pepsin hydrolysis and trypsin hydrolyzing; It is characterized in that, in preparation process, add remove the electrodialysis process step of sodium chloride or before pepsin hydrolysis with the acid for adjusting pH that mixes of sulphuric acid and hydrochloric acid;
Described animal brain is selected from one or more of Medulla sus domestica, Medulla Bovis seu Bubali or Medulla caprae seuovis;
Described pepsin hydrolysis and the step of trypsin hydrolyzing are first to carry out carrying out trypsin hydrolyzing after pepsin hydrolysis again; Or first carry out carrying out again pepsin hydrolysis after trypsin hydrolyzing;
And, in described preparation process, do not adopt the step of chromatographic process separation.
9. method as claimed in claim 8, is characterized in that, has adopted electrodialytic step; Concrete technology step is:
(1) animal brain adds water, and defibrination is heated to 90-100 ℃, insulation 10-20 minute;
(2) cooling, adjusts pH 1.2-3.0 with hydrochloric acid, adds pepsin reaction, obtains pepsin enzyme hydrolyzate;
(3) homoiothermic, sodium hydroxide is adjusted pH 7.5-8, adds trypsin reaction;
(4) filter ultrafiltration, fill, sterilizing;
Wherein, in described preparation process, after step (2) and in step (3) before, also comprise the electrodialytic processing step of step (5), step (5) is specific as follows; The pepsin hydrolysis liquid of step (2) is added in the cathode pool of electrodialytic cell, between cathode pool and anode pool, with anion exchange membrane, isolate; In anode pool, add the sodium chloride solution of equivalent 0.01-1.0mo1/L, put into electrode, regulation voltage steady statue, electrodialysis is carried out in energising continuously, controls the temperature of electrodialytic cell, until cathode pool liquid pH value reaches suitable value; Take out pepsin enzyme hydrolyzate, carry out step (3) and (4).
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| CN103656607B (en) * | 2013-12-17 | 2015-05-20 | 弘美制药（中国）有限公司 | Cerebroprotein hydrolysate in piracetam and cerebroprotein hydrolysate tablets and preparation method of cerebroprotein hydrolysate |
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| CN100998861A (en) * | 2006-01-09 | 2007-07-18 | 陈卓涛 | Cerebroprotein hydrolysate sodium chloride injection |
| CN100998860A (en) * | 2006-01-09 | 2007-07-18 | 王德奎 | Troxerutin cerebroprotein hydrolysate sodium chloride injection liquid |
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