CN104623626A - Stable compound coenzyme preparation as well as preparation method and applications thereof - Google Patents
Stable compound coenzyme preparation as well as preparation method and applications thereof Download PDFInfo
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Abstract
The invention provides a stable compound coenzyme preparation and a preparation method thereof. The main compositions and proportions of the stable compound coenzyme preparation are as follows: 90-120 U of coenzyme A (CoA), 0.1-0.3 mg/ml coenzyme I (NAD), 1-5 mg/ml glutathione (GSH), 1-2.5 mg/ml adenosine triphosphate (ATP), 1.8-12 mu g/ml flavin mononucleotide (FMN), 3-13 mu g/ml flavin adenine dinucleotide (FAD), 0.1-0.4 mg/ml adenosine diphosphate (ADP), 0.2-0.4 mg/ml adenosine monophosphate (AMP), 1.2-15 mu g/ml of adenosine methionine (SAM), 1-5 mg/ml calcium gluconate, 0.5-1.5 mg/ml cysteine hydrochloride, and 0.6-5 mg/ml mannitol. The preparation method overcomes the defects that high-speed centrifugalization is adopted, freeze-drying conditions are not clear and the product stability is poor in the existing method, and the stable compound coenzyme preparation is widely applied to the practices of preparing drugs for treating cardia-cerebrovascular diseases and digestive system diseases.
Description
The application is that the application number submitted on July 17th, 2013 is 201310298764.9, denomination of invention is the divisional application that " a kind of stable Coenzyme Complex preparation, its preparation method and application " is applied for.
Technical field
The invention belongs to field of pharmaceutical preparations, the core of invention there are provided a kind of stable Coenzyme Complex medicine preparation, its preparation method and application.
Background technology
Coenzyme is important and indispensable cofactor in body material's metabolism.Coenzyme composition is because being rich in various active composition (coenzyme A, nadide, GSH, ATP etc.) and mutual Synergistic function thereof, and to the dynamic equilibrium etc. of the metabolism of body, protein synthesis, tissue and physiological function, there is important facilitation, be clinical treatment acute, chronic hepatitis, idiopathic thrombocytopenic purpura, coronary atherosclerosis, renal failure and other diseases important ancillary drug (more than Qin Bangyong will person of outstanding talent etc., 2009; Shen Zhong Ming Zhujun is firm, and 200710037422.6; Rebecca A.Faulkner, Andrew D.Nguyen etc., 2013).
Yeast is unicellular eukaryote, containing various enzymes (Paul N.Black, Nils J.Faergeman, etc., 2000 that the biological substance metabolism such as sugar, fat, protein, nucleic acid is required in its cell; Jasmina Marjanovic, Dominika Chalupska, etc., 2010; Ren Lei, Ren Qiao, 200810063121.5), the Biocoen injection developed the external seventies is exactly (Qin Liu, Rodrigo M.P.Siloto, etc., 2011 based on yeast; Zheng Xuechang, 200410058086.x).
At present, the Patents had no in domestic and foreign literature about stable Coenzyme Complex medicine preparation compositions is reported.Chinese patent retrieves two sections of Patents documents.Wherein " a kind of preparation method of Coenzyme Complex and compound coenzyme medicine and clinical purposes " thereof; application number: 2004158086.X; describe a kind of method manufacturing Coenzyme Complex, and carried out protecting (Zheng Xuechang, 200410058086.x) to the method and produced product.Its independent claims are " a kind of manufacture methods ", and other claim is dependent claims." a kind of preparation method of compound coenzyme medicine ", application number: 201010503237.3, describes the another kind of manufacture method of Coenzyme Complex, and has carried out protecting (Liao Yuanqiu, 201010503237.3) as independent claim to the method.The all not mentioned manufacture product of these two patents, as the stability data required by medicine, has no way of investigating its stability; Meanwhile, above-mentioned document does not relate to galenic pharmacy data, does not consider prescription composition from preparation research angle.
But the stability of current micromolecule polypeptide class and enzyme drug is the significant challenge of pharmacy industry formulation science man now.The multiple stress causing enzyme and the reversible and irreversible change of micromolecule polypeptide class is there is in environment.These difficulties propose the requirement to the reality stablizing these sensitive micromolecule polypeptides and enzyme.In Coenzyme Complex constituent, all there is structural unstability in the many kinds of substances such as CoA, SAM, ADP, AMP, ATP, GSH, under different temperature, acid-base value, oxidoreduction factor or blocking factor, have different stabilities.The committed step of formulation development, needs select and add certain stabilizing agent, excipient etc., and carries out research to these components and proportioning and grope, obtain stable clinical pharmaceutical formulation (Wang Song, 2010; William Ai Erdeqi, Neil Cooley etc., 200680012072.1).So preparation composition is the key of preparation, and the stability of preparation is an indispensable Testing index, is the basic assurance of clinical application quality.Meanwhile, freeze-dry process is also the key technology that can not be ignored, and has not yet to see the relevant report about freeze-dry process condition in Coenzyme Complex preparation.But, for this, the micromolecule polypeptide class medicine again with certain surface activity more responsive for ratio of specific heat of Coenzyme Complex, temperature and time suitable in freeze-drying process is found to be not a nothing the matter, lyophilisation condition is controlled bad, drug appearance not only can be caused bad, the more important thing is and can cause product atrophy after lyophilizing, namely cause commercially producing middle percent defective very high, and official written reply quality is unstable, is difficult to preparation and preserves for a long time.
In addition, with regard to the method that above-mentioned authorized two patents itself relate to, certain drawback is there is: first in production application process, high speed centrifugation (20000-35000CPM and 10000-15000G) is used in two kinds of methods, centrifugation time longer (30-60 minute), such condition is very high to instrument and equipment prescription, and power consumption is greatly, be difficult to aborning realize, severe contamination is caused to environment; Secondly, directly ultrafiltration is carried out to centrifugal liquid in " a kind of preparation method of compound coenzyme medicine " patent, in actual production process, easily cause the blocking of ultrafiltration post, greatly reduce the rate of filtration, even make filtration stop, significant wastage is caused to filler; Again, the according to said method Coenzyme Complex raw material that goes out of small-scale production, its stability is more difficult control also, poor reproducibility between batch; Finally, in two patents, freeze-dry process condition is indefinite, is conventionally difficult to again repeat to obtain stable freeze-drying prods.
It can thus be appreciated that, invent a kind of simple and easy to do method and substitute complex steps there is important innovative value and real world applications prospect.
Summary of the invention
My company has carried out a large amount of literature search analyses and experimental work in Coenzyme Complex development, and provide a kind of stable Coenzyme Complex preparation and preparation method thereof first, the method is different from existing preparation method, more simple, repeatable strong.The proportioning of all the components in said preparation and preferred content range are studied simultaneously, provide a kind of mix proportion scheme of optimization.Especially, surprisingly find that in crude drug, add a certain proportion of calcium gluconate, cysteine hydrochloride and mannitol three kinds of compositions by a certain percentage can play fabulous stablizing effect under study for action.
In whole development process, we compared for different buffer systems, as weak acid and salt buffer system (HAc-NaAc), weak base and salt buffer system (NH thereof
3h
2o-NH
4cl), the acid salt of Multiple Weak Acid and the secondary salt buffer system (NaH of correspondence thereof
2pO
4-Na
2hPO
4).Wherein acetic acid is as buffer agent, easily makes stock solution pH unstable, differs greatly before and after lyophilizing; NH
3.H
2o--NH
4cl buffer agent, has certain zest; Phosphate buffer is in the solution containing phosphorus, and range of accommodation is wide, wayward.We also compared for different antioxidants effect in the formulation, as glutathion and vitamin C etc., result show with glutathion and vitamin C effect undesirable; We also provide a comparison conventional excipient, as sucrose, calcium phosphate two hydrate etc., serve as excipient with sucrose, and can cause the transient rising of blood glucose when injecting, we think that this is very unfavorable to diabetes patient; And with calcium phosphate as excipient, in testing process, finding that in solution, phosphate anion has a small amount of increase, pH value has fluctuation.Finally, we surprisingly find, add calcium gluconate, cysteine hydrochloride and mannitol three kinds of compositions in Coenzyme Complex preparation, and these three kinds of compositions mix by a certain percentage can play fabulous stablizing effect, and this effect is confirmed by great many of experiments.Remarkable as the effect of pharmacy buffer agent using calcium gluconate, pH value of solution is stablized, add without harmful ion, more surprisingly, after adding calcium gluconate, serve a kind of effect of nutritional supplement, zoopery shows, after injection adds the Coenzyme Complex solution of calcium gluconate, the capillary permeability of mice reduces, myocardial contraction is strengthened, the adjustment that blood ion balances to a certain extent; We are using cysteine hydrochloride as antioxidant, find that its antioxidation is extremely strong, and are easily rapidly absorbed, and also can play the effect of certain supplementary simultaneously; We select mannitol as excipient, contrasted the physicochemical property of it and other excipient before, find that mannitol outstanding behaviours is no hygroscopicity, fast drying, chemical stability is good, so, the product appearance characteristic that we draw after figuration is good, and three kinds of compositions mix by a certain percentage, greatly strengthen the stability of Coenzyme Complex, extend the shelf life, clinical effectiveness is given prominence to.
The present invention adopts the slow-speed of revolution centrifugal (4390G, 20000-35000CPM and 10000-15000G far below introducing in existing patent), successful, energy savings first in process of production; The present invention simultaneously discloses the freeze-dry process of Coenzyme Complex freeze-dried powder first, this technique is by groping for a long time, repeatedly, experiment draws repeatedly, effect is good, controllability is strong, and reference value is large, greatly reduces the figuration existed in product freeze-drying process bad, Moisture high UCL, the problems such as poor stability.1. the preparation provided in the present invention is realized by following scheme:
1) freezing fresh yeast bacterium is taken out, add pure water stirring and evenly mixing; Suspension is heated to 90 ~ 97 DEG C, is cooled to 20 DEG C immediately and makes thalline breaking cellular wall.2810G-5120G after abundant breaking cellular wall, collects and slightly puts on clear liquid for centrifugal 20 minutes by 4 ~ 10 DEG C.Breaking cellular wall by temperature difference is adopted in this step, the slow-speed of revolution is centrifugal, overcome in current existing method the weak point adopting high rotating speed (20000-35000CPM and 10000-15000G) centrifugal, reduce the requirement to machine, thus saved cost, be more suitable for large-scale production, add repeatability between criticizing.
2) get the active carbon dress post of corresponding crude extract supernatant volume, balance cylinder by pure water rinsing.Crude extract supernatant adjust pH to 5 ~ 5.5 upper prop, uses cholamine elution, collects eluent;
3) by eluent evaporation and concentration, concentrated solution is adjusted to pH value 7.0, with upper anion-exchange column after 2 ~ 5 times of pure water dilutions, with 20mM pH8.0 phosphate buffer eluting, collects eluate sample.This step overcomes the shortcoming of direct ultra-filtration in existing method, and because the eluent molecules amount obtained in previous step is 5000-8000 dalton, so in practical operation, the blocking easily causing ultrafiltration to live, reduces the rate of filtration greatly, even makes filtration stop;
4) be 5000 daltonian ultrafilter ultrafiltration by elution samples molecular cut off, access filtered solution with clean container;
5) in step 4) filtered solution in, thin up, after dilution, every milliliter contains coenzyme A (CoA) 90-120U, nadide (NAD) 0.1-0.3mg/ml, glutathion (GSH) 1-5mg/ml, adenosine triphosphate (ATP) 1-2.5mg/ml, flavin mononucleotide (FMN) (FMN) 1.8-12 μ g/ml, flavin adenine dinucleotide (FAD) (FAD) 3-13 μ g/ml, adenosine diphosphate (ADP) (ADP) 0.1-0.4mg/ml, adenosine monophosphate (AMP) 0.2-0.4mg/ml, ademetionine (SAM) 1.2-15 μ g/ml;
6) in step 5) diluent in add following material, and make its final concentration be: calcium gluconate 1.5-4mg/ml, cysteine hydrochloride 0.8-1.2mg/ml, mannitol 0.6-5mg/ml are mixed with middle product; The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, collects filter liquor;
7) by step 6) filter liquor of collecting is through degerming, fill;
8) after fill terminates, conventional pre-freeze is carried out to product, after reaching requirement, carry out primary drying, redrying, tamponade.Especially, freeze-dry process is as follows:
During pre-freeze, drain temperature is set as-46--50 DEG C, and drain reaches less than-46 DEG C and keeps starting to fall cold-trap after 2-4 hour; Condenser temperature starts evacuation lower than when-45 DEG C, primary drying, and drain temperature is set as-20 DEG C, and then drain temperature is with 4-7 DEG C/h of intensification, and vacuum is set as 0.17 ± 0.01mbar, and vacuum degree control scope is≤0.20mbar or≤5.10V; Redrying, drain temperature keeps 5-8 hour after being set as 39-41 DEG C to temperature, and vacuum is set as 0.09 ± 0.02mbar, and vacuum degree control scope is≤0.14mbar or≤4.80V; Lyophilizing rear pressing cover, censorship obtains the stable Coenzyme Complex lyophilized formulations product of pharmaceutical properties.
2., for observing the stability of above-mentioned Coenzyme Complex preparation, test as follows:
Study on the stability index under each condition comprises: appearance character, pH value, each constituent content etc.
1) influence factor's test:
(1) high temperature
Test sample is placed in study on the stability incubator, at 37 DEG C (because the usual storage temperature of enzyme drug is less than 20 DEG C, so 37 DEG C are calculated high temperature for such medicine) place 10 days under condition, sample in the 5th day and the 10th day, detect above-mentioned related index.
(2) high humidity
Test sample is placed in study on the stability incubator, in 25 DEG C, place 10 days under RH75% ± 5% condition, in above-mentioned indexs of correlation such as the 5th day and sampling detection moisture absorption weightening finishes in the 10th day.
(3) strong illumination
Test sample is put in lighting box or other suitable illumination container, places 10 days under illumination 4500Lx ± 500Lx condition, detects above-mentioned index of correlation the 5th day and sampling in the 10th day.
2) accelerated test:
Sample thief, in 25 DEG C, places six months in relative humidity 75 ± 5% drying baker.The above-mentioned index of correlation of detection is respectively sampled in the 1st, 2,3 and 6 months at duration of test.
3) long-time stability:
Sample thief, less than 20 DEG C lucifuges are deposited, and detect above-mentioned index of correlation at duration of test in the 3rd, 6,9,12,18,24 and sampling in 36 months.
3. embodiment 1-3 provides under thalline breaking cellular wall centrifugal rotational speed is respectively 2400G, 4390G and 6320G condition (existing methodical rotating speed 20000-35000CPM and 10000-15000G), the content of each main component of Coenzyme Complex crude drug of preparation, its medium speed is under 2400G (embodiment 1) condition, impurity content is more, and clarity of solution is poor; Rotating speed is that each component content of the Coenzyme Complex raw material obtained under 4390G (embodiment 2) condition is optimum, and this technique is selection process; Rotating speed is under the condition of 6320G (embodiment 3), and Contents of Main Components, compared with embodiment 2, can slightly reduce, and analyzing reason may be that rotating speed strengthens, and causes the slightly large effective ingredient of molecular weight also can lose a part;
Embodiment 4-8 provides based on the Coenzyme Complex stock solution that obtains by the selection process of embodiment 2, and condition is respectively and does not add any adjuvant (embodiment 4), only adds buffer agent calcium gluconate (embodiment 5), adds calcium gluconate and cysteine hydrochloride (embodiment 6), adds calcium gluconate, cysteine hydrochloride and mannitol (embodiment 7), adds cysteine hydrochloride and mannitol (embodiment 8) is mixed with middle product.Middle product molecular cut off is 5000 daltonian ultrafilter ultrafiltration, obtain finished product after degerming, fill, lyophilizing, its stability is investigated, under wherein adding calcium gluconate, cysteine hydrochloride and mannitol (embodiment 7) condition, each constituent content is stablized, satisfactory for result;
Embodiment 9 summarizes the screening contrast test to different buffer, antioxidant and excipient various combination in R&D process, totally 27 groups, experimental result shows again, the combination of calcium gluconate, cysteine hydrochloride and mannitol affects insensitive on high temperature (37 ± 2 DEG C), high humidity (RH75% ± 5%) and strong illumination (illuminance 4500Lx ± 500Lx), and pH value is stablized; Under Acceleration study condition (25 DEG C, relative humidity 75+5%), within six months, each component content is stablized; Under long-time stability experiment condition (18 ± 2 DEG C, relative humidity 60 ± 5%), in 36 months, each component content is stablized;
Embodiment 10 provides the animal pharmacodynamic experiment after the dissolving of Coenzyme Complex lyophilized injectable powder, and result shows that Coenzyme Complex improves CPR rat SOD, Na
+-K
+-ATPase vigor, improves the damage of histiocyte energy metabolism to antioxidant radical, and apoptosis-related genes Fas is expressed and reduces, apoptotic cell obviously reduces, and has protective effect to the rat after CPR, is significantly improved heart beating time-out and CPR prognosis.
4. the mass parameter of Coenzyme Complex medicine preparation and standard as follows:
The present invention is surprised to find that and is confirmed by great many of experiments in Coenzyme Complex, adds calcium gluconate, cysteine hydrochloride and mannitol three kinds of compositions (its preferred content scope is: cysteine hydrochloride 0.5-1.5mg/ml, calcium gluconate 1-5mg/ml, mannitol 0.6-5mg/ml) by a certain percentage, fabulous stablizing effect can be played, be better than other compound modes, substantially prolongs the shelf-life, reduce the requirement to preservation condition, be the preparation compositions having no bibliographical information, embody creativeness and the novelty of invention; The present invention adopts the slow-speed of revolution centrifugal in process of production, improves the operability of large-scale production, decreases the pollution of loss to energy and environment, reduces the requirement to equipment; The invention provides detailed lyophilisation condition, simple and practically to operate, official written reply is reproducible, has very strong practical value, and these features were not addressed in existing document; The invention still further relates to concrete freeze-dry process, specific parameters of freeze-drying process successfully prepares lyophilizing to meet one of necessary requirement taking enzyme preparation.
Detailed description of the invention
Further illustrate the present invention by the following examples, but not as limitation of the present invention.
Embodiment 1:
1) freezing fresh yeast bacterium is taken out, add pure water stirring and evenly mixing; Suspension is heated to 90 ~ 97 DEG C, is cooled to 20 DEG C immediately to thalline breaking cellular wall.2400G after abundant breaking cellular wall, collects and slightly puts on clear liquid for centrifugal 20 minutes by 4 ~ 10 DEG C;
2) get the active carbon dress post of corresponding crude extract supernatant volume, balance cylinder by pure water rinsing.Crude extract supernatant adjust pH to 5 ~ 5.5 upper prop, with ethanol ammonia elution;
3) by eluent evaporation and concentration, ion-exchange chromatography is carried out: regulate concentrated solution pH value 7.0, with upper anion-exchange column after 2 ~ 5 times of pure water dilutions, by 20mM pH8.0 phosphate buffer elution samples;
4) by elution samples molecular weight 5000 Dalton ultrafilter ultrafiltration, access filtered solution with clean container, this filtrate is Coenzyme Complex stock solution.
Each constituent content testing result is as follows:
Interpretation of result: solution impurity content is high, and clarity is low, only have Coenzyme Complex A to reach reference standard, other component contents are all below standard.
Embodiment 2:
1) freezing fresh yeast bacterium is taken out, add pure water stirring and evenly mixing; Suspension is heated to 90 ~ 97 DEG C, is cooled to 20 DEG C immediately to thalline breaking cellular wall.4390G after abundant breaking cellular wall, collects and slightly puts on clear liquid for centrifugal 20 minutes by 4 ~ 10 DEG C;
2) get the active carbon dress post of corresponding crude extract supernatant volume, balance cylinder by pure water rinsing.Crude extract supernatant adjust pH to 5 ~ 5.5 upper prop, uses cholamine elution;
3) by eluent evaporation and concentration, ion-exchange chromatography is carried out: regulate concentrated solution pH value 7.0, with upper anion-exchange column after 2 ~ 5 times of pure water dilutions, by 20mM pH8.0 phosphate buffer elution samples;
4) by elution samples molecular weight 5000 Dalton ultrafilter ultrafiltration, access filtered solution with clean container, this filtrate is the multiple proenzyme liquid of compound.
Product analysis result is as follows:
Interpretation of result: clarity of solution is high, without visible foreign matters, each constituent content all meets and exceeds reference standard.
Embodiment 3:
1) freezing fresh yeast bacterium is taken out, add pure water stirring and evenly mixing; Suspension is heated to 90 ~ 97 DEG C, cool immediately to 20 DEG C to thalline breaking cellular wall.6320G after abundant breaking cellular wall, collects and slightly puts on clear liquid for centrifugal 20 minutes by 4 ~ 10 DEG C;
2) get the active carbon dress post of corresponding crude extract supernatant volume, balance cylinder by pure water rinsing.Crude extract supernatant adjust pH to 5 ~ 5.5 upper prop, uses cholamine elution;
3) by eluent evaporation and concentration, ion-exchange chromatography is carried out: regulate concentrated solution pH value 7.0, with upper anion-exchange column after 2 ~ 5 times of pure water dilutions, by 20mM pH8.0 phosphate buffer elution samples;
4) by elution samples molecular weight 5000 Dalton ultrafilter ultrafiltration, access filtered solution with clean container, this filtrate is Coenzyme Complex stock solution.
Product component analysis result is as follows:
Interpretation of result: although Contents of Main Components reaches reference standard all, but compared with embodiment two acquired results, there is no obvious superiority, and the content of the coenzyme A making molecular weight larger, nadide, adenosine triphosphate, flavin mononucleotide (FMN) and ademetionine reduces.
In sum, in preparation method, centrifugal rotational speed is that 2810-5120G (4000-6000rpm) can satisfy the demands, and promotes rotating speed and can not play the effect obviously promoted for active constituent content in stock solution.According to the optimum stock solution of embodiment 2 gained, freeze-dry process is groped, further its ratio of adjuvant is furtherd investigate:
Embodiment 4:
Based on the Coenzyme Complex that the technique of embodiment two obtains, do not add any auxiliaries and become middle product.The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, degerming, fill, repeatedly attempts using different lyophilisation condition, to temperature (pre-freezing temperature attempted-70--76 DEG C ,-60--64 DEG C ,-50--58 DEG C ,-46--50 DEG C ,-40--47 DEG C; Drain second time baking temperature attempted 37-40 DEG C, 39-41 DEG C, 41-44 DEG C, 45-48 DEG C; ), retention time (1-3 hour pre-freeze retention time, 2-4,4-6 hour, second time dry retention time 2-5 hour, 5-8 hour), the condition such as heating gradient and vacuum control scope grope in detail, finally show that following freeze-dry process effect is better :-46--50 DEG C of drain temperature pre-freeze, reach less than-46 DEG C to keep starting to fall cold-trap after 2-4 hour, evacuation after cold-trap,-20 DEG C of drain primary dryings, drain temperature is with 4-7 DEG C/h of intensification, vacuum is set as 0.17 ± 0.01mbar, and vacuum degree control scope is≤0.20mbar or≤5.10V.39-41 DEG C of drain redrying keeps 5-8 hour, and vacuum is set as 0.09 ± 0.02mbar, and vacuum degree control scope is≤0.14mbar or≤4.80V.Finished product is obtained after lyophilizing.
Carry out study on the stability to product, analysis result is as follows:
1) influence factor's test:
(1) high temperature
Test sample is put in sealing clean container, places 10 days under 37 ± 2 DEG C of conditions, in the 5th day and sampling in the 10th day, detects related index.Testing result sees the following form:
(2) high humidity
Test sample is put in constant humidity hermetic container, in 25 ± 2 DEG C, place 10 days under RH75% ± 5% condition, the 5th day and sampling in the 10th day, detection related index.Testing result sees the following form:
(3) strong illumination
Test sample is put in lighting box or other suitable illumination container, places 10 days under illumination 4500Lx ± 500Lx condition, detects the 5th day and sampling in the 10th day.Testing result is as following table:
2) accelerated test:
Sample thief, in 37 ± 2 DEG C, places six months in relative humidity 75 ± 5% drying baker.Respectively sample once in the 1st, 2,3 and 6 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
3) long-time stability:
Sample thief is lower than 18 ± 2 DEG C of placements, place under the condition of relative humidity 60 ± 5%, respectively sample once in the 3rd, 6,9,12,18,24 and 36 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
Embodiment 5:
Based on the Coenzyme Complex that the technique of embodiment two obtains, every ml soln adds calcium gluconate 3mg/ml, is mixed with middle product.The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, degerming, fill, carries out lyophilizing by embodiment 4 method, obtains finished product after lyophilizing.
Carry out stability test to product, analysis result is as follows:
1) influence factor's test:
(1) high temperature
Test sample is put in sealing clean container, places 10 days under 37 ± 2 DEG C of conditions, in the 5th day and sampling in the 10th day, detects related index.Testing result sees the following form:
(2) high humidity
Test sample is put in constant humidity hermetic container, in 25 ± 2 DEG C, place 10 days under RH75% ± 5% condition, at related indexes such as the 5th day and sampling detection moisture absorption weightening finishes in the 10th day.Testing result sees the following form:
(3) strong illumination
Test sample is put in lighting box or other suitable illumination container, places 10 days under illumination 4500Lx ± 500Lx condition, detects the 5th day and sampling in the 10th day.Testing result is as following table:
2) accelerated test:
Sample thief, in 37 ± 2 DEG C, places six months in relative humidity 75 ± 5% drying baker.Respectively sample once in the 1st, 2,3 and 6 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
3) long-time stability:
Sample thief, in 18 ± 2 DEG C, is placed under the condition of relative humidity 60 ± 5%, and respectively sample once in the 3rd, 6,9,12,18,24 and 36 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
Embodiment 6:
Based on the Coenzyme Complex that the technique of embodiment two obtains, every ml soln adds calcium gluconate 3mg/ml, adds stabilizing agent hydrochloric acid Cys2 .0mg and is mixed with middle product.The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, degerming, fill, carries out lyophilizing by embodiment 4 method, obtains finished product after lyophilizing.
Carry out stability test to product, analysis result is as follows:
1) influence factor's test:
(1) high temperature
Test sample is put in sealing clean container, places 10 days under 37 ± 2 DEG C of conditions, in the 5th day and sampling in the 10th day, detects related index.Testing result sees the following form:
(2) high humidity
Test sample is put in constant humidity hermetic container, in 25 ± 2 DEG C, place 10 days under RH75% ± 5% condition, at related indexes such as the 5th day and sampling detection moisture absorption weightening finishes in the 10th day.Testing result sees the following form:
(3) strong illumination
Test sample is put in lighting box or other suitable illumination container, places 10 days under illumination 4500Lx ± 500Lx condition, detects the 5th day and sampling in the 10th day.Testing result is as following table:
2) accelerated test:
Sample thief, in 37 ± 2 DEG C, is placed six months in relative humidity 75 ± 5% drying baker.Respectively sample once in the 1st, 2,3 and 6 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
3) long-time stability:
Sample thief is at 18 ± 2 DEG C, place under the condition of relative humidity 60 ± 5%, respectively sample once in the 3rd, 6,9,12,18,24 and 36 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:.
Embodiment 7:
Based on the Coenzyme Complex that the technique of embodiment two obtains, every ml soln adds calcium gluconate 3mg/ml, adds stabilizing agent cysteine hydrochloride 1.0mg, 3mg/ml mannitol and is mixed with middle product.The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, degerming, fill, carries out lyophilizing by embodiment 4 method, obtains finished product after lyophilizing.
Carry out stability test to product, analysis result is as follows:
1) influence factor's test:
(1) high temperature
Test sample is put in sealing clean container, places 10 days under 37 ± 2 DEG C of conditions, in the 5th day and sampling in the 10th day, detects related index.Testing result sees the following form:
(2) high humidity
Test sample is put in constant humidity hermetic container, in 25 ± 2 DEG C, place 10 days under RH75% ± 5% condition, at related indexes such as the 5th day and sampling detection moisture absorption weightening finishes in the 10th day.Testing result sees the following form:
(3) strong illumination
Test sample is put in lighting box or other suitable illumination container, places 10 days under illumination 4500Lx ± 500Lx condition, detects the 5th day and sampling in the 10th day.Testing result is as following table:
2) accelerated test:
Sample thief, in 37 ± 2 DEG C, is placed six months in relative humidity 75 ± 5% drying baker.Respectively sample once in the 1st, 2,3 and 6 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
3) long-time stability:
Sampling sample is in 18 ± 2 DEG C, place under the condition of relative humidity 60 ± 5%, respectively sample once in the 3rd, 6,9,12,18,24 and 36 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
Embodiment 8:
Based on the Coenzyme Complex that the technique of embodiment two obtains, every ml soln adds stabilizing agent cysteine hydrochloride 1.0mg, 3% mannitol is mixed with middle product.The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, degerming, fill, carries out lyophilizing by the method for embodiment 4, obtains finished product after lyophilizing.
Carry out stability test to product, analysis result is as follows:
1) influence factor's test:
(1) high temperature
Test sample is put in sealing clean container, places 10 days under 37 ± 2 DEG C of conditions, in the 5th day and sampling in the 10th day, detects related index.Testing result sees the following form:
(2) high humidity
Test sample is put in constant humidity hermetic container, in 25 ± 2 DEG C, place 10 days under RH75 ± 5% condition, at related indexes such as the 5th day and sampling detection moisture absorption weightening finishes in the 10th day.Testing result sees the following form:
(3) strong illumination
Test sample is put in lighting box or other suitable illumination container, places 10 days under illuminance 4500Lx ± 500Lx condition, detects the 5th day and sampling in the 10th day.Testing result is as following table:
2) accelerated test:
Sample thief, in 37 ± 2 DEG C, is placed six months in relative humidity 75 ± 5% drying baker.Respectively sample once in the 1st, 2,3 and 6 months at duration of test, strictly detect its character, pH value, color and its related substances it, measurement result sees the following form:
3) long-time stability:
Sample thief, in 18 ± 2 DEG C, is placed under the condition of relative humidity 60 ± 5%, and respectively sample once in the 3rd, 6,9,12,18,24 and 36 months at duration of test, strictly detect its character, pH value, color and its related substances, measurement result sees the following form:
Embodiment 9:
For different buffer systems, antioxidant and excipient composition, we have done 27 organizing, stability contrast tests, and compound mode is as follows:
Experimental result shows: in 27 kinds of combinations, Portugal-salt-Gan (calcium gluconate, cysteine hydrochloride and mannitol) combination affects insensitive on high temperature (37 ± 2 DEG C), high humidity (RH75% ± 5%) and strong illumination (illuminance 4500Lx ± 500Lx), and pH value is stablized; Under Acceleration study condition (25 ± 2 DEG C, relative humidity 75+5%), within six months, each component content is stablized; Under long-time stability experiment condition (18 ± 2 DEG C, relative humidity 60 ± 5%), in 36 months, each component content is stablized.At this, stability data does not repeat one by one.
Embodiment 10:
With Coenzyme Complex prepared by the scheme of embodiment 7, to the protective effect of cardiac resuccitation (CPR) rat thyroid afterwards, test as follows:
Use suffocates (succinylcholine) merges 0.5M ice potassium chloride and stops jumping liquid to rat cardiac arrest, starts cardiac resuccitation after 5 minutes.Experiment points three groups: 1) control rats without suffocating, sudden cardiac arrest and cardiac resuccitation; 2) conventional recovery group rat (after cardiac resuccitation 24 hours); 3) Coenzyme Complex experimental group: conventional medicine recovery gives Coenzyme Complex (each props up/10Kg) normal saline dilution pneumoretroperitoneum simultaneously and injects, and recovers latter 12 hours again to Isodose.Often organize eight rats.
(1) change of experimental rat serum thyroid hormones T3, T4 thyrotropin is as following table:
| Grouping | T3(ng/ml) | T4(Hg/dl) | TSH(μIU/ml) |
| Matched group | 0.62±0.04 | 5.39±0.42 | 1.73±0.5 |
| Conventional recovery group | 0.22±0.05 | 1.47±0.35 | 1.45±0.35 |
| Coenzyme Complex group | 0.31±0.4 | 2.51±0.54 | 1.60±0.26 |
Results of statistical analysis shows, there is significant difference (P < 0.05) result between Coenzyme Complex group and matched group and conventional group to show: CPR can cause serum T 3, T4, TSH concentration obviously declines, but conventional recovery group is starkly lower than Coenzyme Complex group, two groups of T3, T4 have marked difference.
(2) malonaldehyde (MDA) content, superoxide dismutase (SOD) vigor, Na in parathyroid tissue
+-K
+-ATPase vigor
as following table:
Coenzyme Complex group is all less than 0.05 with matched group and conventional group than P respectively.
Result shows: in Coenzyme Complex group parathyroid tissue, MDA content is apparently higher than matched group, lower than conventional recovery group; SOD vigor and Na
+-K
+-ATPase vigor, all lower than matched group, is significantly higher than conventional recovery group.
(3) experimental mouse TdT-mediated-dUTP nick end labeling (Tunel labelling) changes as shown in the table:
| Grouping | n | Positive cell number (individual) | Average gray value (%) |
| Matched group | 8 | 4.99±1.36 | 72.03±6.71 |
| Conventional recovery group | 8 | 36.72±3.66 | 38.88±3.46 |
| Coenzyme Complex group | 8 | 27.23±4.00 | 56.24±4.03 |
Coenzyme Complex group is all less than 0.05 with matched group and conventional group than P respectively.
Result shows: Coenzyme Complex group positive cell number and average gray value are all between matched group and conventional recovery group, and the difference between conventional recovery group and Coenzyme Complex group has statistical significance.
(4) research of experimental rat Fas differential expression is as shown in the table:
Interpretation of result: Coenzyme Complex group Fas gene expression positive cell number and average gray compare with conventional recovery group that there were significant differences.
In sum, this embodiment shows:
1) Coenzyme Complex improves CPR rat SOD, Na
+-K
+-ATPase vigor, improves the damage of histiocyte energy metabolism to antioxidant radical, and in exhibit tissue the content of lipid peroxidation catabolite MDA lower than conventional recovery group;
2) Coenzyme Complex makes apoptosis-related genes Fas express reduction, and apoptotic cell obviously reduces, and has protective effect to the rat after CPR;
3) Coenzyme Complex is significantly improved heart beating time-out and CPR prognosis.
Claims (5)
1. a stable Coenzyme Complex lyophilized formulations, its main constituent comprises: coenzyme A (CoA), nadide (NAD), glutathion (GSH), adenosine triphosphate (ATP), flavin mononucleotide (FMN) (FMN), flavin adenine dinucleotide (FAD) (FAD), adenosine diphosphate (ADP) (ADP), adenosine monophosphate (AMP), ademetionine (SAM), calcium gluconate and cysteine hydrochloride, mannitol.
2. Coenzyme Complex preparation as claimed in claim 1, after water dissolution, when coenzyme A (CoA) concentration is 90-120 unit/ml, preferably other constituent content proportionings are nadide (NAD) 0.1-0.3mg/ml, glutathion (GSH) 1-5mg/ml, adenosine triphosphate (ATP) 1-2.5mg/ml, flavin mononucleotide (FMN) (FMN) 1.8-12 μ g/ml, flavin adenine dinucleotide (FAD) (FAD) 3-13 μ g/ml, adenosine diphosphate (ADP) (ADP) 0.1-0.4mg/ml, adenosine monophosphate (AMP) 0.2-0.4mg/ml, ademetionine (SAM) 1.2-15 μ g/ml, calcium gluconate 1-5mg/ml, cysteine hydrochloride 0.5-1.5mg/ml, mannitol 0.6-5mg/ml.
3. a preparation method for Coenzyme Complex preparation stable as described in claim 1,2, mainly comprises the following steps:
1) get freezing fresh yeast bacterium, add pure water stirring and evenly mixing; Temperature differential method is utilized to carry out thalline breaking cellular wall (high temperature is 90-97 DEG C, and chilling temperature is less than 20 DEG C); After abundant breaking cellular wall, 2810-5120G is centrifugal, 4-10 DEG C, centrifugal 20 minutes, collects and slightly puts on clear liquid;
2) get and fill post with the isopyknic active carbon of crude extract supernatant, balance cylinder by pure water rinsing; Crude extract supernatant adjust pH, to 5-5.5 upper prop, with ethanol ammonia eluting, collects liquid eluting;
3) by step 2) eluent evaporation and concentration, concentrated solution is adjusted to pH value 7.0, with anion-exchange column upper after the dilution of 2-5 times of pure water, collects elution samples with 20mM pH8.0 phosphate buffer eluting;
4) by step 3) elution samples molecular cut off is 5000 Dalton ultrafilter ultrafiltration, accesses filtered solution with clean container;
5) in step 4) in gained filtered solution, thin up, after dilution, every milliliter contains coenzyme A (CoA) 90-120U, nadide (NAD) 0.1-0.3mg/ml, glutathion (GSH) 1-5mg/ml, adenosine triphosphate (ATP) 1-2.5mg/ml, flavin mononucleotide (FMN) (FMN) 1.8-12 μ g/ml, flavin adenine dinucleotide (FAD) (FAD) 3-13 μ g/ml, adenosine diphosphate (ADP) (ADP) 0.1-0.4mg/ml, adenosine monophosphate (AMP) 0.2-0.4mg/ml, ademetionine (SAM) 1.2-15 μ g/ml;
6) in step 5) diluent in add following material, and make its final concentration be: calcium gluconate 1-5mg/ml, cysteine hydrochloride 0.5-1.5mg/ml, mannitol 0.6-5mg/ml are mixed with middle product; The daltonian ultrafilter ultrafiltration of middle product molecular weight 5000, collects filter liquor;
7) by step 6) filter liquor of collecting is through degerming, fill;
8) after fill terminates, conventional pre-freeze is carried out to product, after reaching requirement, carry out primary drying, redrying, tamponade, obtain the lyophilized formulations product that pharmaceutical properties is stable.
4. preparation method as claimed in claim 3, its freeze-drying process feature and parameter as follows:
1) during pre-freeze, drain temperature is set as-46--50 DEG C, and drain reaches less than-46 DEG C and keeps starting to fall cold-trap after 2-4 hour; Condenser temperature starts evacuation lower than when-45 DEG C, primary drying, and drain temperature is set as-20 DEG C, and then drain temperature is with 4-7 DEG C/h of intensification, and vacuum is set as 0.17 ± 0.01mbar, and vacuum degree control scope is≤0.20mbar or≤5.10V; Redrying, drain temperature keeps 5-8 hour after being set as 39-41 DEG C to temperature, and vacuum is set as 0.09 ± 0.02mbar, and vacuum degree control scope is≤0.14mbar or≤4.80V;
2) lyophilizing rear pressing cover, censorship obtains the stable Coenzyme Complex lyophilized formulations product of pharmaceutical properties.
5. the application of preparation and preparation method thereof in the practice of the medicine for the preparation for the treatment of cardiovascular and cerebrovascular disease and digestive system disease as described in claim 1-4.
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