CN101797377A - Fibrin sealant and preparation method thereof - Google Patents
Fibrin sealant and preparation method thereof Download PDFInfo
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- CN101797377A CN101797377A CN200910077444A CN200910077444A CN101797377A CN 101797377 A CN101797377 A CN 101797377A CN 200910077444 A CN200910077444 A CN 200910077444A CN 200910077444 A CN200910077444 A CN 200910077444A CN 101797377 A CN101797377 A CN 101797377A
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Abstract
The invention provides a fibrin sealant which comprises a fibrin polymer and a thrombin-like enzyme, wherein a ratio of the thrombin-like enzyme to the fibrin polymer is 0.01 to 100 IU:1 mg. The fibrin sealant can be used on the aspects of hemostasis, healing promotion, defect enclosing, bonding prevention, tendon suture fixation tendon injury, molding repair of bony defect, regeneration of skin tissue, carrier slow release and the like.
Description
Technical field
The present invention relates to a kind ofly can to stop blooding, can do culture medium, can be used for pharmaceutical carrier and alternative skin, the Fibrin Glue of tissues such as skeleton, cartilage, nerve and preparation method thereof, relate more specifically to a kind of fibrin sealant and preparation method thereof.
Background technology
Fibrin sealant (Fibrin Sealant, be called for short FS), its mechanism of action is the final stage that simulate blood is solidified, under the effect of thrombin and calcium ion, make Fibrinogen be converted into fibrin monomer, further polymerization changes the fibrin net into, and under the effect of the XIII factor, form crosslinked stable fibrin net, thereby bring into play its hemostasia effect.In recent decades, the purposes of FS no longer is confined to hemostasis, promotes healing, sealing is damaged, prevent this four big function of adhesion, but by means of the cohesive of FS, can be used for sewing up in the tendon that flesh is strong damages, by means of the plasticity of FS, can be used for the moulding reparation of bony defect.FS is combined analgesia, antitumaous effect time that can the significant prolongation medicine with some analgesic, chemotherapeutics etc. as slow-released carrier.
The fibrin sealant of Sheng Chaning mainly contains two kinds of reagent now, and reagent I mainly contains fibrin, the Stuart factor III of high concentration; Reagent II mainly contains thrombin and calcium chloride.After reagent I and reagent II dissolving mixing, thrombin energy hydrolysis of fibrin is former, makes it to be converted into fibrin monomer.Monomer is because negative charge reduces, and spontaneous polymerization becomes unsettled soluble fibrin polymers.Simultaneously, at Ca
2+Under the condition that exists, thrombin can activity factor X III.The interchain residue covalent bond of factor X III energy some peptide of catalysis forms stable insoluble fibrin polymer at last.After polymer forms, by following mechanism performance hemostasis, sealing, adhesive effect: 1, polymer is woven into nettedly, can catch erythrocyte and platelet, forms blood clot, the performance anastalsis; 2, after the insoluble fibrin polymer forms, its mechanical strength significantly strengthens, but adhesion organization and seal damagedly simultaneously can stimulate capillary endothelial cell and fibroblastic growth, and be that support forms granulation tissue, thereby promote wound healing with the fibrin net; 3, the membranaceous gel that forms at wound surface can prevent that lysosomal enzyme leaks to the extracellular in the slough cell, avoids surrounding tissue further impaired, and is hemorrhage because of reducing wound surface simultaneously, alleviates the machine degree, can prevent the tissue adhesion.
Commercially produced product occurred in European countries the beginning of the eighties.Formally recorded in 1998 and enter the Europe pharmacopeia.Manufacturers adopt multiple virus inactivating method as processing such as heating, UV irradiation, S/D deactivations, to improve its security of products by strict plasma screening.Austrian ImmunoAG company in 1998 make Fibrin Glue---Tisseel/Tissucol is first xanthan gum production that is gone on the market by drugs approved by FDA.The fibrin sealant product that has gone on the market abroad at present also has Beriplast P; Hemaseel, Biocol, Quix2il etc.Also there are nearly ten families in the manufacturer of external producd fibers albumin glue, and the man unit of domestic existing number begins to develop and declare production.This novel external hemorrhage of Fibrin Glue has caused people's extensive concern, and applying in surgical field also is subjected to paying close attention to widely.
The fibrin sealant product basically face with before to avoid Fibrinogen and thrombin or thrombin activation thing premature reaction, make Fibrinogen under the effect of thrombin and calcium ion, be converted into fibrin facing the time spent, again under the effect of the X III factor, become stable fibrin net, thereby bring into play its hemostasia effect.Before for example Tisseel/Tissucol uses, extract two kinds of lysates with two syringes respectively, dissolve corresponding freeze-dried component, respectively solution after two kinds of dissolvings is extracted out with two syringes again, and two syringes are assembled into the duplex syringe by a threeway; During use, two kinds of solution while mixed in equal amounts are sprayed on the wound surface, play the effect of hemostasis wound closure.A kind of fibrin film referring to publication number CN1911440A patent mixes Fibrinogen and thrombin during use immediately, forms the fibrin film of the network-like densification of one deck.This class instantaneity gel at first will thaw or dissolve, and mixture is mixing as far as possible, applies then on hemorrhage wound.This process time GPRS suitable accurately, before fibrin polymerization, easily washed away by blood, and if polymerization is just used Fibrin Glue after finishing, then be difficult for being bonded on the wound.This uses inconvenience clinically, and cost is higher.
At present, fibrin sealant all is to obtain under the effect of thrombin, thrombin is mainly derived from mammals such as cattle, pig, but the mammal thrombin is a kind of strong antigen, can cause various immunoreation at human body after using thrombin, and have for example risk propagated such as foot and mouth disease, bovine spongiform encephalopathy of disease that people and mammal suffer from altogether.Though can solve subproblem, utilize the probability of human thrombin viral infection still to exist, and the source is few, the cost height with human thrombin.Utilize people's thrombin itself to prepare Fibrin Glue, complicated operation, the time, longer feasibility was poor.
Summary of the invention
Problem at the prior art existence, the invention provides a kind of fibrin sealant, it can be used for stopping blooding, promote healing, seals aspects such as damaged, as to prevent to sew up in adhesion, the tendon the strong damage of flesh, bony defect moulding reparation, skin tissue regeneration, slow-released carrier.Fibrin sealant of the present invention is for comprising the gel of fibrin polymer and batroxobin at least, and wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.Described fibrin polymer is to be formed in that the effect of batroxobin is following by fibrin (or Fibrinogen), the shaping that aggregates into for fibrin monomer, can adherent and degradable gel.
The present invention is significantly different with most of fibrin sealants.Fibrin sealant of the present invention is fully to act on Fibrinogen by batroxobin earlier, make Fibrinogen be converted into fibrin, form fibrin polymer, then fibrin sealant of the present invention is prepared into various dosage forms, for example fibrin film, fibrillarin powder, fibrin sponge etc.The fibrin sealant of this form can be avoided some shortcomings of the instantaneity Fibrin Glue of prior art, and for example complicated operation is wayward; The easy degeneration of Fibrinogen is difficult for preserving; The easy inactivation of thrombin activity etc.
The inventor is through number of research projects, determined the proper proportion scope of batroxobin and fibrin polymer in the fibrin sealant of the present invention.That is, the proportion of batroxobin and fibrin polymer should be 0.01～100IU: 1mg.Be lower than 0.01IU: during the ratio of 1mg, can't form colloidal state, and be higher than 100IU: during 1mg, cost is too high, causes waste.Preferred 0.1～10IU: 1mg, the colloid elasticity and the bonding force that form in this ratio are good, also are easy to prepare various dosage forms.
In fibrin sealant of the present invention, before mixing formed gel, Fibrinogen (or fibrin) and batroxobin can be respectively the solution form, and wherein, fibrin or fibrinogen concentration are greater than 1mg/ml.When being lower than 1mg/ml, the colloid water content is excessive, not easy-formation.Preferred 10～100mg/ml, good in colloidal elasticity of this scope and bonding force, be easy to prepare various dosage forms.Batroxobin concentration is greater than 0.1IU/ml, and when being lower than 0.1IU/ml, gelation time is long, and the colloid water content is excessive, easy-formation not, and preferred 20～1000IU/ml, in this scope, gelation time is suitable, and colloidal elasticity and bonding force are good, are easy to prepare various dosage forms.
The present invention also provides a kind of gel, and it is to be mixed by fibrin or Fibrinogen and batroxobin to form, i.e. fibre-bearing protein polymer and batroxobin, and wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.This gel can be used for preparing protein blocking agent, pharmaceutical carrier, culture medium, artificial person soma etc.
The present invention also provides a kind of pharmaceutical carrier, it comprises by fibrin or Fibrinogen and batroxobin and mixes the gel that forms, be fibre-bearing protein polymer and batroxobin, wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.
The present invention also provides a kind of culture medium, it comprises by fibrin or Fibrinogen and batroxobin and mixes the gel that forms, be fibre-bearing protein polymer and batroxobin, wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.
Fibrinogen with change a kind of fibrin into after batroxobin contacts, so Fibrinogen and fibrin all can be applicable to the present invention.
Fibrin refers to any type of fibrin, includes but not limited to fibrin I, des BB fibrin and fibrin II.Fibrin I is meant the fibrin monomer that dissociates Fibrinogen fibrinopeptide A produces.Des BB fibrin is meant the dissociate fibrin monomer of fibrinopeptide B of Fibrinogen.Fibrin II is meant the dissociate fibrin monomer of fibrinopeptide A and B of Fibrinogen.Fibrin can derive from animal bloods such as human blood or Sanguis Bovis seu Bubali, Sanguis sus domestica, horse blood.
Batroxobin is a kind of active enzyme of thrombin-like that has, be mainly derived from snake venom, also can derive from the enzyme of other animal with similar batroxobin function, the snake venom of preferred Crotalinae (Viperidae) and Viperidae (Crotalidae) Serpentis, the batroxobin content height of this two subfamilies snake venom.For example, batroxobin is in the Brazilian spearhead Agkistrodon halys in source; Defibrase derives from the Changbai Mountain agkistrodon halys ussuriensis; China's Effect of Agkistrodon acutus Enzyme derives from Agkistrodon acutus Serpentis poison; The gabonase enzyme source is in Gabon's viper etc. of whistling.Batroxobin can obtain by any way, comprises natural extract, gene recombinaton, cell engineering, protein engineering, tissue culture etc.
Batroxobin (Thrombin-like Enzyme) is to thrombin similar function one albuminoid enzyme to be arranged.Batroxobin has the arginine ester enzymatic activity, can directly act on Fibrinogen, fibrinogen molecule is made up of two pairs of α-chains, beta chain and γ-chains, per 3 peptide chains (α, β, γ) are twisted into strand, form two streak peptide chains, have disulfide bond that fibrinogen molecule is stablized at N-terminal.The N-of α and β peptide chain end has one section 16 and 14 amino acid whose little peptides respectively, be called fibrinopeptide A (fibrinopeptideA, FPA) and B (FPB).Cracking goes out fibrinopeptide A on the Fibrinogen Aa chain for only making of having when batroxobin is hydrolyzed, and a then cracking that has goes out fibrinopeptide B, or cracking goes out fibrin A and B peptide, but the speed difference.Hydrolysis discharges fibrinopeptide, makes fibrin monomer head and the tail polymerization and solidifies.Cause blood plasma or Fibrinogen coagulation external as coagulant, but batroxobin does not activate the Stuart factor III in vivo, it is crosslinked that the fibrin clot that is generated by the batroxobin hydrolysis does not produce side chain, to the wording depth sensitivity of fibrinolysin, be easy to be removed by natural reticuloendothelial system or normal fibrinolytic effect.
Nearly all batroxobin is glycoprotein, but different with thrombin be that its oligonucleotide chain is to be connected with N, rather than serine.More single-minded than thrombin to fibrinogenic effect, its activity is not suppressed by inhibitor such as Antithrombin III heparin, hirudin, soybean trypsins.Most of batroxobins are to thermally-stabilised, even also stable to heat when low concentration.The grumeleuse that batroxobin forms can not cause contraction.
Except that fibrin polymer and batroxobin, also can contain Ca in the fibrin sealant of the present invention
2+, ion concentration is at 1～200mmol; Albumen in the Stuart factor III that from blood plasma, obtains, Fn Fiberonectin and other blood plasma; Composition with antibacterial or bactericidal action: for example gentamycin, amikacin etc.; The material that Profilin is decomposed, for example aprotinin; Promote the cell growth factor of cell growth etc.
A marked feature of the present invention is different with most of fibrin sealants, and this fibrin sealant is fully to act on Fibrinogen by batroxobin earlier, makes Fibrinogen be converted into fibrin, forms the noncrosslinked fibrin polymer.Then fibrin sealant of the present invention is prepared into various dosage forms, for example fibrin film, fibrillarin powder, fibrin sponge etc.The fibrin sealant of this form can be avoided some shortcomings of instantaneity Fibrin Glue, and for example complicated operation is wayward; The easy degeneration of Fibrinogen is difficult for preserving; The easy inactivation of thrombin activity etc.
Fibrinous existing way of the present invention comprises two kinds: one, solid form, be meant that mainly Fibrinogen is converted into fibrin under the effect of batroxobin, and behind the formation gel, the dry product that utilizes any dewatered mode to obtain.Two, liquid form can obtain liquid fibrin by the present or following technology solubilising noncrosslinked fibrin glue that may occur.The mode of implementing now solubilising mainly contains utilizes pH value less than 5 acidic buffer (for example: acetic acid, aspartic acid, glutamic acid and salt thereof etc.) or carbamide.
At present, it is bottom that a kind of commercially available fibrin sealant " Tachocomb " adopts the lyophilized collagen sponge, with complicated production technology components such as Fibrinogen, aprotinin, thrombin is sticked on the collagen sponge molding then.Though more convenient in the use, owing to used organic solvent in the production process, there is the potential safety hazard of producing, and reduced the activity of each component, increased production cost, prolonged the production cycle.
Compare with " Tachocomb ", fibrin sealant of the present invention, production technology is simple, utilizes batroxobin to make Fibrinogen be converted into fibrin, forms uncrosslinked gel, then also can the various dosage forms of dry preparation.
" Tachocomb " uses thrombin of beef, and thrombin of beef is a kind of strong antigen, can cause various immunoreation at human body behind the use thrombin of beef.Thrombin of beef causes the antibody that cross-immune reaction produced, and may cause thrombosis because of its normal inhibitory action that weakens antithrombin AT-III.In the market, thrombin of beef all is mixed with the cattle labile factor, cross-immune reaction can take place after entering human body, and the immune complex that is produced is eliminated through blood circulation, can cause the shortage of human blood coagulation V thus, and may cause the severe haemorrhage tendency.And batroxobin (mainly coming from the Serpentis class) because Serpentis and human race relation far do not carry the human virus that can infect, has reduced the chance of viral infection.Structurally there are bigger difference in batroxobin and people and mammiferous thrombin, so can not cause cross-immune reaction in vivo.Avoid the impaired and symptom that causes bleeding of human body intravascular coagulation function.In addition, batroxobin because formation is the fibrin of non-crosslinked, causes the fibrin clotted texture loose in vivo, is easily removed by fibrinolytic system, can not cause thromboembolism.
" Tachocomb " is to do to contain loading matter with collagen fiber, applies the Fibrin Glue composition above, comprises human fibrinogen, thrombin of beef, aprotinin bovine and makes the riboflavin that powder is indicated color.Make Fibrinogen under the effect of thrombin, form the fibrin net by being pressed on the wound 3-5 minute during use.
The present invention establishedly contains the noncrosslinking fibrin polymer of batroxobin or adds batroxobin in addition, oozing out on the contact surface of blood with wound, Fibrinogen under the effect of the thrombin in batroxobin and human blood in the blood is converted into fibrin monomer, fibrin monomer in noncrosslinked fibrin and the blood is crosslinked rapidly under the effect of the X III factor again, form fine and close crosslinked fibrin net, Fibrin Glue is bonded on the wound.This process was finished in tens seconds.Animal experiment shows that the noncrosslinking Fibrin Glue of the present invention all is better than Tachocomb on bleeding stopping period and oozing of blood amount.
In addition, the present invention forms gel earlier, so can directly make required shape and size, for example film like, cone, cylinder etc.This is extremely easy to use clinically.Because batroxobin is more single-minded than thrombin to fibrinogenic effect, its activity is not suppressed by inhibitor such as Antithrombin III, heparin, hirudin, soybean trypsins, so batroxobin is subjected to interferential factor few when bringing into play Blood clotting.Batroxobin is to thermally-stabilised, even when low concentration heat is also stablized, so product of the present invention is more easily preserved.The grumeleuse that batroxobin forms can not cause contraction, so utilize product of the present invention, wound healing is good, and cicatrix is little.
The preparation method of fibrin sealant of the present invention can may further comprise the steps: Fibrinogen (or fibrin) and batroxobin are mixed in proportion, form gel then.
The present invention can prepare various preparations, and for example fibrin film adds mould with fibrinogen solution, adds the abundant mixing of batroxobin then, and after question response was completed into gel, fibrin film was made in lyophilization; Fibrin sponge adds mould with fibrinogen solution, adds the abundant mixing of batroxobin then, and question response is completed into the gel post-foaming, and fibrin sponge is made in lyophilization; Hemostasis is pasted, and Fibrin Glue can be applied in hemostasis and stick preparation Fibrin Glue hemostasis subsides, and can add antibacterial or antibacterial in the Fibrin Glue; Suppository, fibrinogen solution is added the stype mould, add the abundant mixing of batroxobin then, after question response is completed into gel, lyophilization promptly can stop sperm to enter the uterus when being used for sticking stifled cervix uteri mouth, because the fibrin degradation in vivo time is 3-6 week, can unite the preparation long acting contraceptive with contraceptive; Powder with fibrinogen solution and the abundant mixing of batroxobin, after question response is completed into gel, is ground into fine powder after the lyophilization, further can be made into various peroral dosage forms, chewable tablet for example, capsule, electuary effervescent tablet etc.; Fibrin Glue of the present invention also can combine with binder and make the fibrin tourniquet bandage.
The present invention can prepare liquid dosage form, because noncrosslinking fibrin of the present invention can exist with liquid form, so be prepared into aseptic liquid, can be directly used in the sealing wound.
The present invention can be degraded and absorbed in vivo owing to fibrin; can be used as pharmaceutical carrier uses; for example with Fibrin Glue and proton pump inhibitor; for example omeprazole, pantoprazole, rabeprazole etc. or H2-receptor antagonist cimetidine, ranitidine, nizatidine etc. or mucosa protective agent teprenone or antacid hydrotalcite combine, and treat peptic ulcer.With Fibrin Glue and analgesic, for example ketoprofen, nimesulide etc. are used for long-acting analgesic in conjunction with the preparation slow releasing preparation.
The present invention also can be used for cell culture, is that carrier adds the required inorganic ions of each seed amino acid, cell growth factor, cell growth etc. with noncrosslinking Fibrin Glue.Culture medium of the present invention is more suitable for mammiferous cell growth, for example epithelial cell, epidermis cell, fibroblast etc.
Fibrin Glue of the present invention has the favorable tissue compatibility, the transmission of energy mediated cell signal and the effect between the cell, can promote adhesion and the propagation and the tissue regeneration of cell by the release of some cytokines, have certain mechanical strength and plasticity, form new tissues such as corresponding skin, skeleton, cartilage, nerve on the Fibrin Glue of the present invention so epidermis cell, periosteum cell, chondrocyte etc. can be compound in.
In order to understand essence of the present invention better,, describe in detail but do not limit the present invention below by description to better embodiment of the present invention.
Description of drawings
The ion exchange chromatography figure of batroxobin preparation among Fig. 1: the embodiment 1;
Fig. 2: the affinity chromatograph chromatogram of batroxobin preparation among the embodiment.
The specific embodiment
The used test material of the present invention if no special instructions, is commercially available purchase product.
The preparation of Fibrinogen and batroxobin
Embodiment 1
Fibrinogenic preparation of A and buffer thereof: the pig whole plasm is cooled to below 10 ℃ in 37 ℃ of thawings, constantly being stirred to final concentration with the slow adding of percent by volume 50% ethanol of pre-cooling-20 ℃ is 10% ethanol, make 12 hours precipitation process, 5000 change 30 minutes centrifugal.Again precipitation is dissolved in neutral Tri/ citrate-lysine buffer, about 1.5% (percentage by weight) of final concentration of protein.Make 10～12 hours the ethanol precipitation second time 10% then at 4 ℃ of reuse pre-cooling 50% ethanol.5000 change 30 minutes centrifugal, dissolution precipitation becomes 40mg/ml, aseptic filtration then, every bottle of 5ml lyophilizing of packing.Fibrinogenolysis liquid is for containing 0.02M PH7.4 sodium citrate buffer solution.
Preparation of category-B thrombin and buffer thereof:
Method 1: take by weighing long eyebrow agkistrodon halyx pallas venom in vain (culturing factory) 10g,, with 4000rpm/min centrifugal 30 minutes, get supernatant with 0.02mol/LpH7.2Tris-HCl buffer extraction 4 hours available from Liaoning Agkistrodon halys of radically reforming.To precipitate with method and extract once again, and merge supernatant, and add ammonium sulfate to 30% saturation, the centrifugal precipitation of going adds ammonium sulfate again to saturation 75%, the centrifugal supernatant that goes.Resolution of precipitate in 400ml pH7.2 phosphate buffer, is added cold acetone to 40%, and the centrifugal precipitation of abandoning adds cold acetone to 65% in supernatant, and the centrifugal supernatant of abandoning is deposited in 40 ℃ of vacuum drying ovens and dries.Promptly get batroxobin dry powder.Batroxobin dissolving buffer is a 0.02M PH7.4 buffer.
Method 2: take by weighing long eyebrow agkistrodon halyx pallas venom in vain (culturing factory) 10g,, with 4000rpm/min centrifugal 30 minutes, get supernatant with 0.02mol/LpH7.2Tris-HCl buffer extraction 4 hours available from Liaoning Agkistrodon halys of radically reforming.To precipitate with method and extract again once, merge supernatant, adjust pH to 7.2, get extracting solution.With on the extracting solution in the good DEAE-Sepharose chromatographic column of balance, earlier do not adsorb impurity with 0.02mol/L pH7.2Tris-HCl buffer solution elution, the buffer solution that reuse contains 0～0.5mol/L NaCl carries out gradient elution, collects the active peak of batroxobin, sees Fig. 1.Get active component and collect liquid A.
Above-mentioned active component is collected liquid A go up, do not adsorb impurity with containing 0.5mol/L NaCl0.05mol/L Tris-HCl pH7.0 buffer solution elution earlier in the good affinity column of balance; Collect active component with containing 0.1mol/L eluant 1.5mol/L NaCl 0.05mol/L Tris-HCl pH7.0 buffer solution elution then, obtain active component and collect liquid B, see Fig. 2.
Get active component and collect liquid B, with molecular cut off 10, the bag filter of 000D is concentrated into about 20ml Polyethylene Glycol, is concentrated solution C.Detecting its thrombin activity is 10000 ius/ml, filtration sterilization, and lyophilizing gets lyophilized powder.Batroxobin dissolving buffer is a 0.02M PH7.4 buffer.
The preparation of fibrin sealant of the present invention
Embodiment 2
Become 50mg/ml standby the fibrinogenolysis of preparation, the preparation batroxobin is made every milliliter 100 iu.Get 2 milliliters of fibrinogen solutions, inject mould, add and contain 1% the abundant mixing of glycerols thrombin 60 ius, it is reacted completely, become colloidal, lyophilization.
Embodiment 3
Fibrinogen and batroxobin solution are added the mixed of 10 iu batroxobins by Fibrinogen 100mg, fully react postlyophilization, be ground into fine powder, incapsulate.
Embodiment 4
Fibrinogen and batroxobin solution are added the mixed of 1000 iu batroxobins by Fibrinogen 100mg, fully react postlyophilization, be ground into fine powder, incapsulate.
Embodiment 5
Fibrinogen and batroxobin solution are added the mixed of 10000 iu batroxobins by Fibrinogen 100mg, fully react postlyophilization, be ground into fine powder, incapsulate.
Embodiment 6
Fibrinogen and batroxobin solution are added the mixed of 5000 iu batroxobins by Fibrinogen 100mg, fully react postlyophilization, be ground into fine powder, incapsulate.
Embodiment 7
Get 10 liters of blood plasma and add the 40000IU batroxobins, fully mixing react 30 minutes, 4000 rev/mins centrifugal 25 minutes, abandon supernatant, cleans 2 times, adding 400ml 0.2M carbamide dissolves.Solution is added in the bag filter, is acetic acid-sodium-acetate buffer dialysis of 5.0 to the 0.1M pH value.Solution after the dialysis is shone with cobalt 60.By every bottle of 1ml dress as in the cillin bottle promptly.
Embodiment 8
In one of embodiment 2-7, add one or more of following composition of appropriate amount selectively:
1.Ca
2+, its ion concentration is at 1～200mmol;
2. the albumen in the Stuart factor III that from blood plasma, obtains, Fn Fiberonectin and other blood plasma;
3. the composition that has antibacterial or bactericidal action, for example gentamycin, amikacin etc.;
4. Fibrinogen is stablized enhancer, for example cationic protein, polycationic compounds, poly-D-lysine, basic protein, methylene blue, streptomycin etc.;
5. the material that decomposes of Profilin, for example aprotinin;
6. promote the material of tissue growth, for example fibroblast growth factor, growth hormone etc.;
7. the antitumor drug that merges during as tumor embolism, for example cisplatin etc.
The effect experiment of fibrin sealant of the present invention
Embodiment 9
45 of animal health Wistar rats (180～230g), male and female half and half.Be divided into 3 groups of blank groups at random, matched group is Tachocomb (commercially available), and experimental group is the batroxobin fibrin film.
Femoral artery otch model rats by intraperitoneal injection pentobarbital sodium 40mg/kg faces upward after the anesthesia and is fixed on the operating-table.The pars inguinalis unhairing is peeled off femoral artery, clamps proximal part with hemostatic clamp, tremulous pulse is laterally cut off the otch that accounts for diameter 1/3.Wipe the blood of ejection immediately, put the fibrin glued membrane that comprises batroxobin (contain the 40mg Fibrinogen by 1 milliliter and add the batroxobins preparation of 40 units) or Tachocomb in wound, open hemostatic clamp, cover gauze immediately, perhaps directly cover gauze, press 1min with the counterweight that 150g is heavy.Remove counterweight, continue to observe 30min; Or do not carry out any processing and observe 30min.Blood loss in record bleeding stopping period and the 30min.3min is no longer hemorrhage to be " hemostasis " to remove behind the counterweight.The results are shown in Table 2.
Table 2
| Group | Number of animals (only) | Bleeding stopping period (minute) | Amount of bleeding (ml) |
| Blank group | ??15 | ??6.8±1.2 | |
| Matched group | ??15 | ??＜5 | ??2.5±0.8 |
| Experimental group | ??15 | ??＜2 | ??1.2±0.6 |
More than the description of better embodiment of the present invention is not limited the present invention, those skilled in the art can make various changes or distortion according to the present invention, only otherwise break away from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (15)
1. a fibrin sealant comprises fibrin polymer and batroxobin, and wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.
2. the described fibrin sealant of claim 1 is characterized in that, the ratio of described batroxobin and fibrin polymer is 0.1～10IU: 1mg.
3. the described fibrin sealant of claim 1 is characterized in that, described batroxobin is venin-derived batroxobin.
4. the described fibrin sealant of claim 3 is characterized in that, described snake venom is the snake venom of Crotalinae and/or Viperidae Serpentis.
5. the described fibrin sealant of claim 1 is characterized in that, described fibrin polymer is formed in that the effect of batroxobin is following by Fibrinogen or fibrin.
6. the described fibrin sealant of claim 5 is characterized in that, before the formation fibrin polymer, described Fibrinogen or fibrin are the solution form, and concentration is greater than 1mg/ml.
7. the described fibrin sealant of claim 6 is characterized in that, described Fibrinogen or fibrinous concentration are 10～100mg/ml.
8. the described fibrin sealant of claim 5 is characterized in that, before the formation fibrin polymer, described batroxobin is the solution form, and concentration is greater than 0.1IU/ml.
9. the described fibrin sealant of claim 8 is characterized in that, the concentration 20～1000IU/ml of described batroxobin.
10. the described fibrin sealant of one of claim 1～9 is characterized in that, described fibrin sealant also comprises one or more following components:
1) Ca
2+, ion concentration is at 1～200mmol;
2) the Hageman factor I and/or the Fn Fiberonectin that from blood plasma, obtain;
3) has the composition of antibacterial or bactericidal action;
4) material of Profilin decomposition;
5) promote the cell growth factor that cell is grown.
11. the described fibrin sealant of one of claim 1～9 is characterized in that, the dosage form of described fibrin sealant is gel, glued membrane, powder, hemostasis subsides, suppository or fibrin sponge.
12. the preparation method of the described fibrin sealant of claim 1 is characterized in that, may further comprise the steps:
Formation comprises the gel of fibrin polymer and batroxobin.
13. the preparation method of the described fibrin sealant of claim 12 is characterized in that, further may further comprise the steps:
Described gel made be used for the hemostatic preparation.
14. a pharmaceutical carrier comprises fibrin polymer and batroxobin, wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.
14, a kind of culture medium comprises fibrin polymer and batroxobin, and wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.
15. a gel comprises fibrin polymer and batroxobin, wherein, described batroxobin is 0.01～100IU: 1mg with the ratio of fibrin polymer.
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| Application Number | Title | Priority Date | Filing Date |
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| CN2009100774444A Active CN101797377B (en) | 2009-02-11 | 2009-02-11 | Fibrin sealant and preparation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105007841A (en) * | 2012-12-31 | 2015-10-28 | 乔治·D.·福卢什 | Lyophilized fibrin sealant for high volume hemorrhage |
| CN106389393A (en) * | 2016-11-23 | 2017-02-15 | 中国人民解放军第三军医大学第附属医院 | Biological protein glue-triamcinolone acetonide slow release agent as well as preparation method and application of slow release agent |
| WO2017172854A1 (en) * | 2016-03-29 | 2017-10-05 | George Pins | Compositions and methods for wound healing |
| CN111447944A (en) * | 2017-07-13 | 2020-07-24 | 血栓治疗公司 | Compositions and methods for wound closure |
| CN111888376A (en) * | 2020-06-24 | 2020-11-06 | 四川大学华西医院 | Cisplatin-loaded fibrin glue composite system combined treatment system |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA953212A (en) * | 1970-06-01 | 1974-08-20 | Peter G. Sesin | Process for the isolation of thrombin-like material from pit viber venom |
| CN1102569A (en) * | 1993-11-09 | 1995-05-17 | 刘高东 | Method for prodn. of absorptive biochemical haemostatic with function of bonding tissue |
-
2009
- 2009-02-11 CN CN2009100774444A patent/CN101797377B/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105007841A (en) * | 2012-12-31 | 2015-10-28 | 乔治·D.·福卢什 | Lyophilized fibrin sealant for high volume hemorrhage |
| WO2017172854A1 (en) * | 2016-03-29 | 2017-10-05 | George Pins | Compositions and methods for wound healing |
| US10322206B2 (en) | 2016-03-29 | 2019-06-18 | Worcester Polytechnic Institute | Compositions and methods for wound healing |
| CN106389393A (en) * | 2016-11-23 | 2017-02-15 | 中国人民解放军第三军医大学第附属医院 | Biological protein glue-triamcinolone acetonide slow release agent as well as preparation method and application of slow release agent |
| CN111447944A (en) * | 2017-07-13 | 2020-07-24 | 血栓治疗公司 | Compositions and methods for wound closure |
| CN111888376A (en) * | 2020-06-24 | 2020-11-06 | 四川大学华西医院 | Cisplatin-loaded fibrin glue composite system combined treatment system |
| CN111888376B (en) * | 2020-06-24 | 2022-06-24 | 四川大学华西医院 | Cisplatin-loaded fibrin glue composite system combined treatment system |
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| Publication number | Publication date |
|---|---|
| CN101797377B (en) | 2012-01-25 |
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Address after: 100176 No. 8 prosperous street, Beijing economic and Technological Development Zone, Beijing Applicant after: Beijing Science Sun Pharmaceutical Co., Ltd. Address before: 100176 No. 8 prosperous street, Beijing economic and Technological Development Zone, Beijing Applicant before: Beijing Saisheng Pharmaceutical Co., Ltd. |
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