CN105007841A

CN105007841A - Lyophilized fibrin sealant for high volume hemorrhage

Info

Publication number: CN105007841A
Application number: CN201380074087.0A
Authority: CN
Inventors: 乔治·D.·福卢什
Original assignee: Individual
Current assignee: Individual
Priority date: 2012-12-31
Filing date: 2013-12-28
Publication date: 2015-10-28
Also published as: CA2896866A1; EP2938275A1; WO2014106136A1; JP2016507279A; AU2013370223A1

Abstract

ClotBlock is a lyophilized fibrin hemostatic designed for use an adjunct or primary treatment in moderate to severe hemorrhage. It can be applied directly to the wound in a laparotomy procedure or as non-invasive sealant. Its crosslinking technology generates a strong and safe adhesive fibrin sealant required for high volume hemostasis. The attachment properties of the cake as well as the rapid formation of and stability of the fibrin clot ensures that a strong stable fibrin clot is formed within 1 to 5 minutes depending on the grade of the wound. The agent is safe, biocompatible, biodegradable and can be stored at room temperature for one year.

Description

For the lyophilizing fibrin sealant of bleeding profusely

Related application

The application is the U.S. Patent application the 12/487th submitted on June 18th, 2009, the continuation-in-part application of No. 057 (mandate), U.S. Patent application the 12/487th, No. 057 on November 6th, 2012 receive mandate notice and before 6 days February in 2013 payment authorization expense.The all description wherein set forth, accompanying drawing and instruction are hereby expressly incorporated by reference all clearly, and we require the priority of the instruction wherein made clearly.

Technical field

The present invention relates to a kind of method, with the bi-component be made up of the lyophilizing desAB fibrin polymer of the sterile biocompatible from the concentrated desAB fibrin monomer in acid solution or fibrin II monomer or fibrin II polymer composition and lyophilized thrombin produced or two-layer system, thus for the application as adhesive sealant component and hemorrhage.The ClotBlock indicating preparation (the present invention) trade mark is rendered as various physical form and thickness, and ClotBlock can when using and do not use pressing for stopping blooding or closing tissue in vivo.It refers more particularly to is not having biodegradable support member or is needing when separable support member to paste fibrin clot after being applied in bleeding wounds, with closed tissue with to control angiorrbagia, epidermis hemorrhage, osteorrhagia or inner hemorrhage.The present invention can comprise the biodegradable support member be made up of hyaluronic acid or other biodegradable polymer.

The field of the invention is biological adhesive and tissue sealant, biological adhesive and tissue sealant are biodegradable and nontoxic, be intended to be used for the treatment of purposes, such as, as auxiliary agent for cutting open the belly or the hemostasis of laparoscopic surgery, or as the primary treatment of orthopedics, wound (spleen laceration), interventional radiology and trunk bed wound.

On the one hand, the present invention relates to biocompatible adhesive fibrin polymer, it is that biology can absorb with nontoxic, for surgery or therapeutic use again.It also relates to the dual-layer applications containing bioactive substance, and it can in the release of given site to stimulate blood coagulation.On the other hand, the present invention relates to a kind of technique for the production of this binder polymer.

A large amount of In vivo study shows, ClotBlock is good hemorrhage candidate for control moderate to severe bleeding.Its different displaying maximizes the haemostatic effect at various types of surgical operation and wound application.This reagent is durable, is easy to store, and causes minimum risk, needs a small amount of training to use, and is be efficient for moderate to severe bleeding.

Background technology

The severe haemorrhage of organ removal, wound or large area skin wound is difficult to control sometimes.Recently, based on fibrinous paster through test, IV/V level wound is closed to use cellulose or gelatin support member.But these technology can not be applied to the step of some type, such as interventional radiology, plastic surgery or laparoscopic surgery.In addition, " support member " that these products use stayed in the body before biodegradation, caused the serious inflammatory reaction to foreign body.

Current solution and restriction.Normally, composite adhesives is used for blood vessel or pulmonary vascular deadend and the edge for " bonding " skin incision.Usually formed after cicatrix at wound, by biodegradation, absorption or eliminate these glue by simply departing from the form of incrustation.Develop the preparation of various technology for tissue adhesive.Some of them are synthesis sources, such as based on the glue of cyanoacrylate (2-butylcyanoacrylate, 2-octyl cyanoacrylate) or the polymer of synthesis and containing other tissue adhesives of biomaterial (such as there is collagen protein or the fibrin of haemostatic properties in addition).

Due to their hemostasis and bond properties, sealer, and particularly fibrin sealant in the past twenty years between be widely used in most of surgical specialties, with due to when their polymerization (1,2,3) ability and reducing adhering to tissue time is lost blood and postoperative hemorrhage.These compounds be used for close or reinforce be sewn or stitching wound close; They also can use together with pressing above damage field.Fibrin sealant is the biological adhesive of the final step of simulation coagulation cascade.(4) on the one hand, Fibrinogen, plasma protein and Ⅹ III factors that the key component of sealer is, and be thrombin and calcium chloride on the other hand.Component is usually extracted and is produced from human plasma or by recombinant technique.Composite fibre proteinogen and thrombin produce polymer barrier (fibrin), and polymer barrier simulates the final stage of natural coagulation cascade, to form the structural fibers fibrin clot being similar to physiology grumeleuse.

There is several available commercial product (Floseal, Gelfoam, Evicel, Bioglue, surgicel, tachoseal etc.) (3,5).But these products have significant limitation, widely using when which prevent their severe haemorrhages in operation and in emergency medicine, plastic surgery and interventional radiology and laparoscopic surgery.For all existing hemorrhage that intracavity is hemorrhage be designed to be used as slight hemorrhage to moderate in auxiliary agent and need hard pressing.Be used in exploitation and/or use that MIN to press in hemorrhage tissue adhesive and sealant compositions one of main restriction run into be that they can not when bleeding profusely and the enough strong bonding of organization formation, and stable grumeleuse can not be produced in application in 10 minutes.Therefore, tissue adhesive and sealer must combinationally use with pressing method, stitching and/or staple and adhesive patch, organize bond strength to reduce needed for acceptable performance.But, under having many situations, use strong pressing, stitching and/or staple to be undesirable, inappropriate or impossible (such as, in bone, interventional radiology).

At present optional method: in order to form the physical barriers of opposing blood flow, adhesive stroma in seconds must form firmly fibrin interface, makes it to be bonded together with tissue in the blood of flowing, and is retained in and tears position to form grumeleuse.Each product attaches to spheroid with square, circle, the representation of cylinder or vertebral body is attached to ability in tissue and compositions and the interaction (combination) of the method for producd fibers albumen and it and thrombin to stimulate coagulation cascade relevant.The importance of this technology is, walks around fibrinogenic cracking process, with produce fibrin monomer and it polymerization subsequently, lyophilizing and can absorbing blood to form the ability of fibrin clot.The clot forming process of this reagent is from stable I fiber type protein polymer, I fiber type protein polymer is by blood absorption to containing being necessary that the lyophilizing cross linked polymer of component is to stimulate coagulation cascade (thrombin) and to form physical barriers, and physical barriers was transformed into functional fibre fibrin clot in two minutes of application.(6)

In our method, these results are obtained by following steps: lyophilizing fibrin network a) applying polymerization crosslinking, the lyophilizing fibrin network absorbing blood of polymerization crosslinking is to form very sticky gel-like matrix, and gel-like matrix adheres to tears tissue; And b) bind thrombin layer, thrombin layer contributes to forming firmly fibrin clot fast, and fibrin clot is stablized by the non-calcium dependent T-5398 mixed in the product and by the XIII factor in blood.

The freeze-drying process being positioned at the layer subsequently above biodegradable removable supporter promotes its application, and allows long storage periods, transport and preparation.

In addition, not yet untapped based on fibrinous product solve stop gash or trunk bed skin wound hemorrhage, this causes so serious hemorrhage and usually need suture or stitching.

Compositions.The form of ownership of current technology---by changing the fibrin monomer that pH value makes square, spheroid, cylinder or cone in acid solution combine, be polymerized under the T-5398 (non-calcium dependence) of activation and the existence of the XIII factor (calcium dependence) (calcium relies on and non-calcium relies on) and calcium chloride, pH is neutralized by buffer solution.Once fibrin polymer is formed and crosslinked, the second layer of thrombin and combined and lyophilizing subsequently.(Fig. 1)

Monomer can mix with the glycerol of about 1% to about 5% volume, to realize the specific viscoelasticity profile being suitable for the type applied.Make lyophilized fibronectin fibrillarin become gel by the absorption of block to blood, this gel forms fibrin clot in the site of damage (7).

Under the condition of coagulating agent, the non-calcium dependent T-5398 in blood and the activation XIII factor are by contributing to this process by covalent bond stable fibers fibrin clot.

Determinant attribute.Polymerization/bonding.The fibrin gel of wound closure is formed by the double layer material absorbing blood of lyophilizing, keeps covalent bond when the double layer material of lyophilizing becomes gel state from solid.Grumeleuse is mechanically stable, is incorporated into wound well, and compared with noncrosslinking grumeleuse [8] or other fibrin sealants, can the dissolving of more resistance to fibrinolysin.Comprise non-calcium dependent T-5398 and be conducive to the crosslinked of aC-territory polymer in transglutaminase mediated fibrin, thus promote integrin cluster, and thus increase cell adhesion and diffusion, this stimulates fibrin in conjunction with avb3-, avb5-and a5b1 integrin on endotheliocyte [9].Oligomerization is also via the cellular signal transduction that focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) promote integrin to rely on, and this causes cell adhesion and the cell migration [10] of increase.The existence of calcium ion enhances the process of from inflammatory reaction to coagulation cascade (first stage), and activates the XIII factor.

In vitro and in vivo the adhesion characteristics of living human's tissue and the kinetics of polymerization of recommendation reagent are tested.The ample evidence of the ability of the various displayings that the data obtained provide ClotBlock to stop blooding, and the hemostasis of the intraperitoneal wound brought out in solid organ or soft tissue is realized with minimum pressing.Further, in pig model, the intramedullary bone in knee joint and hip replacement can be stopped hemorrhage.Lyophilized fibronectin fibrillarin II is obtained by fibrin II monomer polymerization: U.S. Patent application 12/487057 (mandate) describes the method preparing fibrin monomer.ClotBlock sealant compositions uses the fibrin polymer of the lyophilizing obtained from the neutralization of fibrin monomer.The compositions of part and the neutralization of production method and polymer of the fibrin II that present patent application describes are extremely important with the crosslinked performance of process to proposed technology, and the performance of technology depends on the characteristic of fibrin itself (fiber thickness, branch point number, porosity and permeability) and limits other polymerization property of thrombin.The grumeleuse that generated by ClotBlock produces the opaque substrate of thick fiber, and with than residuite faster speed carry out pipe formation.The concentration of thrombin is for generation fibrin monomer, and therefore the rate of release of FPA also has important impact to polymerization process.Concentration, diluent and pH value described by ClotBlock structure produce best fibrin structure with the speed accelerated.ClotBlock introduces

Fibrin polymer can be produced and lyophilizing with all size, thickness and form, to be adapted to the type (Fig. 2 A, Fig. 2 B, Fig. 2 C) applied.It can be configured to the spherula of 1/4 inch diameter or cylinder to be introduced into by laparoscopic port or the blood vessel in interventional radiology; It also can be molded as circle or 1/4, the square flat solid block of the various sizes of 1/2a1 thickness, for spleen laceration or organs resection, or be placed on above cohesive bandage to cover deep skin otch.Lyophilized form also may be dipped in water, and is used as to close cream or gel.

Summary of the invention

Accompanying drawing explanation

Fig. 1 shows the distribution of ClotBlock layer.

Fig. 2 shows possible shape: A spheroid and cylinder; B paster or block; C binder.

The comparison of adhesion strength in the tissue that Fig. 3 shows Clotcake and Tissel, Floseal and Evicel.

Fig. 4 shows fibrinous polymerization under ACTIVA and the Activa+XIII factor exists, crosslinked and stable.

Fig. 5 shows the control of the intraoperative hemorrhage as the coagulation in partial nephrectomy, by application ClotBlock, application is shown 5 minutes in (median 3.2 ± 1.4 minutes) and forms solid grumeleuse.

Fig. 6 shows as the hemorrhage control of the rupture of spleen of the coagulation without wrapping or stitching.By application CloBlock, stop blooding realizes in application 5 minutes.

Fig. 7 shows the control of the intraoperative hemorrhage of the coagulation as the hepatic injury of IV level, and by application ClotBlock, stop blooding realizes in application 5 minutes.The haemostatic effect of the ClotBlock of gel form as auxiliary agent hemostasis with the intraoperative hemorrhage controlling hepatietomy a good appetite suddenly appearing in a serious disease, realization in application 5 minutes of stopping blooding.

2 weeks (A) and the track being captured in the fluorescein in the intercellular substance of kidney and liver time 5 weeks (B) after Fig. 8 microscopy shown under w light compares application.

Fig. 9 shows the antibody using sandwich ELISA (enzyme-linked immunosorbent assay) to detect issuable antithrombase in pig.

Human fibroblasts culture is exposed to ClotBlock preparation

A, HF-are untreated

B, HF+ClotBlock, 5 days

Figure 10 shows the antibody using sandwich ELISA (enzyme-linked immunosorbent assay) to detect issuable antithrombase in pig.

Figure 11 shows the human fibroblasts being exposed to ClotBlock preparation, does not damage completely or toxicity cell, and without any antibacterial or fungal contamination; Cell seems slightly larger than untreated control cultures.

A ,-untreated, 5 days

B, fibroblast+ClotBlock, 5 days

Figure 12 shows the HEP's culture (A549) being exposed to ClotBlock preparation, does not damage completely or toxicity cell, and without any antibacterial or fungal contamination; Cell seems slightly larger than untreated control cultures.

C, A549 cell-untreated, 5 days

D, A549 cell+ClotBlock, 5 days

Detailed description of the invention

We have developed a kind of hemorrhage, and can be shaped with support member in a variety of manners, trade mark is Clotblock.This reagent is novel fibrin sealant (pure fibrin II, by in and fibrin II monomer make), this fibrin sealant is supplemented by thrombin, and be designed to promote hemostasis when severe haemorrhage, and stop when minimum pressing by the wound of organs resection, wound and/or solid organ wound, soft tissue, skeleton and trunk bed cause hemorrhage.This sealer promote blood coagulation, and provide hemostasis and damaged tissues surface between cohesive.This fibrin sealant can use 1) as the auxiliary agent of severe haemorrhage or as coagulation; Or 2) be configured as by laparoscopic port transmission or in organs resection, be used as pressing, or 3) be placed on the situation for skin lacerations on support member, or 4) be configured as when intervention radiation by conduit transmission; Or 5) be configured as the intramedullary hemorrhage closed and caused by plastic surgery or wound.

The each thing that presents be made up of lyophilizing bilayer (Fig. 1) comprises: 1) fibrin II polymer, is neutralized and be cross-linked by the non-calcium dependent T-5398 of XIII Summing Factor of activation to produce by the fibrin monomer in acetic acid solution (pH3.4) and HEPES buffer (pH 8.3); 2) with the ratio of every 4ml fibrin 1ml, the concentration be dissolved in HEPES aqueous solution is the layer of the thrombin of 20NIH unit/ml; And 3) select to add the microsphere embedding third layer PLGA fibronectin between fibrin and thrombin layer.

Lyophilizing dual-layer applications in the bleeding tissue of tearing, the double-deck absorbing blood of lyophilizing with form viscosity, colloidal gel barrier and blood is absorbed by fibrin subsequently time form fibrin clot.1) this reagent was at 2 minutes inner sealing wounds, with 2) make together with the tissue bond of tearing.

ClotBlock has developed several dosage form, changes as required on shape, viscoelasticity and clot strength.

Compositions and application

ClotBlock makes two layers.It is made up of the lyophilized thrombin layer at lyophilizing fibrin polymer and top.Ground floor contains crosslinked fibrin polymer, according to the U.S. Patent application be hereby expressly incorporated by reference the 12/487th, the method described in No. 057, crosslinked fibrin polymer is produced by the neutralization of the fibrin monomer in the acetic acid solution mixed with buffer solution, and buffer solution comprises 150mM NaCl, 50mM HEPES, 3mM CaCl ₂, 0.12g/mLActiva (non-calcium dependent T-5398) and every milliliter of neutralization buffer PH8.5 the XIII factor of 21Lowey unit.This two kinds of solution are mixed with the ratio of 1:1 in aseptic mould.Depend on the flexibility of the expectation of block, the glycerol of 1% to 5% can be added in the composition.

This mould is sealed in aseptic (registered trade mark of E.I E.I.Du Pont Company, Wilmington (DE)) bag, and 4 hours are hatched at 37 DEG C.

The second layer comprises thrombin and fibrinous ratio is the solution of 1:4, and thrombin is dissolved in HEPES buffer with the concentration of 20 units/mL.

Step 1: each component filters sterilizing by the microfilter of 0.22 micron.Every one deck pours the silicon mould of intended shape (circular, oval or square) into, to produce about 3/4 to 1 " thick " caking "; it can by removing or biodegradable polymer support of netting; net is poly lactose net such as; or sD, L-lactide polymer polymer fabric, polylactic acid (PLA)/PLGA copolymer (PLGA) film or polyglycolic acid (PGA) net; Or supported by the self-adhering fascia of the situation for skin lacerations.

Step 2: then lyophilizing Clotblock at the condenser temperature of-40 DEG C to-50 DEG C, the shelf temperature of 21 DEG C, continues 18-72 hour under the pressure of 200-400 millitorr.

Step 3: be packaged in plastic bag, to prevent water loss and to keep aseptic to every sheet ClotBlock gas-tight seal.

Step 4: directly apply to apply ClotBlock appropriateness presses 1 minute to 2 minutes on wound.On wound, fibrin clot is formed in 3 minutes.

Optional transmission method

ClotBlock can be shaped as 1/2 " diameter and 1 " to 2 " long small column, this can be passed to the wound of intracavity by laparoscopic port, or carrys out closed hemorrhage blood vessel by conduit.

ClotBlock can be dissolved in the water with the ratio of 4:2, and to form liquid gel, liquid gel can be applied on laceration or wound with single syringe.

ClotBlock can be positioned over above self-adhesion or non-Autoadhesive, ClotBlock is two-layer system, this two-layer system forms by flexible fabric adhering bandage with from the lyophilizing desAB fibrin polymer of the sterile biocompatible of the concentrated desAB fibrin monomer in acid solution or fibrin II monomer or fibrin II polymer composition, and ClotBlock may be used for hemostasis or closed skin histology.This transmission method particularly relates to be needed to stop the hemorrhage of large or dark skin incision by binder pressing adhering fibre fibrin clot.

Example

Haemostatic properties is tested in zooscopy, and its display CLOTBLOCK sealer forms fibrin clot more by force and faster than other sealers.Expect that binding agent adheres to tear tissue and utilize a kind of strength by relative tissue bond together, this strength is significantly higher than viewed fibrin sealant.

Following laboratory test zoopery is implemented alternately (for the rat of III level and IV liver wound, rabbit and pig model).

1. the isolated experiment of benchmark preparation: adhere to and coagulant property

We carry out with isometric transducer the measurement (adhering to and clot strength in tissue) that bonds and stretch in Sprague-Dawley liver tissues of rats.

Stretch and measure: two the maximum leaves being separated liver.The keeper of isometric transducer will be fixed on after a leaf is attached to.Another leaf is positioned over the par of the gauze in container, and container can progressively raise and reduce, and contacts with the liver sheet in the keeper of sensor to produce.All 1cm is produced in these two pork liver plates ²damaged area.The preparation of the test being used for tissue adherence is placed between two panels.Specimen is placed on the contact position of the baseline pressure of 0 gram.At different time points (exposing and 1 minute that contacts, 5 minutes and 10 minutes), record the pressure needing to separate completely them.We compare the interior adhesion of tissue of ClotBlock, Tissel, Floseal and Evice.Fig. 5 shows the result adhered in tissue.The adhesion that ClotBlock produces after 10 minutes is stronger than Evicel by 150%, and than the contrast strong by 800% (Fig. 3) in adhesive model in tissue.

2. the molecular chemistry of fibrin polymerization

We conducted the efficiency (Fig. 4) that molecular chemistry analysis is polymerized (pH neutralizes) to be compared fibrin monomer with the T-5398 that non-Ca relies on by the activation XIII factor and stablizes (being cross-linked).

2.1. ACTIVA is determined on the stable impact of fibrin

Identifiable, FXIII is at Ca ²⁺there is the crosslinked of lower catalysis fibre protein polymer, to produce insoluble fibrin grumeleuse.But the non-calcium existed in reactant mixture relies on whether T-5398 can the crosslinked of catalysis fibre albumen be not certifiable.Also the cooperative effect that there is non-calcium dependent T-5398 and the activation XIII factor can not be confirmed whether.In order to follow these reactions, fibrin stands non-calcium dependent transglutamin-ase 9 ferment treatment, is first as concentration dependent reaction, and reacts as time dependence afterwards.

Monitoring concentration dependent and time dependence (1 minute, 5 minutes, 10 minutes) reaction, the amount of the acid fiber protein solution of 2mg/mL and variable concentrations (1.0-20.0U/ml) (there is 2mM CaCl at 60mM Tris buffer (pH8.4) ₂) in Activa rapid mixing, with realize neutralization.Make fibrin visual by antifibrin original antibody (1:50).Com-parison and analysis is a) by the fibrin of non-calcium dependent T-5398 when 1 minute, 5 minutes and 10 minutes (Fig. 4) and fibrinogenic crosslinked and crosslinked by the fibrin during concentration of non-calcium dependent T-5398 at 20U/ml, 19U/ml and 1U/ml.

Fibrin shown in figure 1 minute time under non-calcium dependent T-5398 and the XIII factor exist crosslinked period forms strong gamma dimmer.Now gamma dimmer is not also present in fibrinogenic sample.

3. the experiment in animal model

We have carried out the research of intracavity intraoperative hemorrhage with the formation of assessment by the fibrin clot of blood absorption in pig (pig) model, and determine the ability of Bleeding control.

Goal in research: stop moderate to the Standard surgical of severe haemorrhage to put into practice during comparing ClotBlock and laparoscopic partial nephrectomy; And determine whether stop blooding is safe, and intraoperative hemorrhage (minimizing loss of blood in art) can be controlled.

3.1. scheme: assessment ClotBlock is as the control of the intraoperative hemorrhage of the coagulation in partial nephrectomy.

Use six Yorkshire crossbreeding sows, 2.5 months ages, body weight 37 ± 2kg.The program is ratified with use committee through the animal care mechanism of TMCI.Animal is by 25% of open laparotomy ventrotomy excision kidney.After bringing out damage, 2.5 of the 60CC ClotBlock bilayer of the compositions #1F containing 1% glycerol " the circular grumeleuse of diameter is pressed against excision in fact.

Result: after bleeding profusely 1 minute in interlobular arteries, in 5 minutes of application, * (median 3.2 ± 1.4 minutes) forms solid grumeleuse, all realizes stopping blooding (Fig. 5) in all six animals.

* within five minutes, bleeding stopping period is defined as the maximum time that the effect realizing hemostasis is shown by the blood products committee of Food and Drug Administration.

3.2 assessment ClotBlock are as without wrapping or the hemorrhage control of the rupture of spleen of coagulation sewed up.

The object of this research determines that whether CloBlock can stop massive hemorrhage in application in 5 minutes in traumatic spleen laceration.

Method: use eight Yorkshire crossbreeding sows, 2.5 months ages, body weight 37 ± 2kg.The program is through animal care mechanism and use committee's approval.

Animal stands 1 inch cut (sharply being caused by 11 blade scalpels) at the horizontal mid portion of spleen.After bringing out damage, the compositions #1F containing 1% glycerol 60CC ClotBlock 2.5 " the circular grumeleuse of diameter is compressed against in laceration and continues 2 minutes.All animals all realize in 5 minutes stopping blooding (Fig. 6) in application.

Result: all animals (n=6) all realize hemostasis in application 5 minutes *, and median is 3.2 ± 1.4 minutes.

* five minutes bleeding stopping period are defined as by the blood products committee of Food and Drug Administration the maximum time that the effect realizing hemostasis is shown.

3.3. the control of ClotBlock as the intraoperative hemorrhage of the coagulation in the hepatic injury of IV level is assessed.

Ten four-head Yorkshire crossbreeding sows, at 2.5 months ages, body weight 37 ± 2kg, is divided into 3 groups at random.The animal of the 1st group (n=6) stands 4 grades of hepatic injury and with 40CC ClotBlock process by open cutting open the belly.The animal via of the 2nd group (n=6) is subject to similar process and processes with GelFoam (Pfizer); Be subject to similar process with the animal via of the 3rd group (n=4), stopped by sew up wound hemorrhage, and use saline solution process further.

In two kinds of hemostatic treatment groups, apply ClotBlock or Gelfoam and 2 minutes are pressed to wound.(Fig. 7) for the object of this model, 4 grades of damages are defined as the long complete abundant matter laceration (sharply being caused by 11 blade scalpels) of 7cm.These damages are all consistent with the Society of Surgery of U.S. wound organ injury ratio system.

Select the point in the centre of liver, produce hepatic injury with scalpel.This position calculation is close to blood vessel on liver and more pylic branches.With labelling, this point is marked.After bringing out damage, be no matter the 40CCClotBlock grumeleuse of the patch-type processed with Alexa fluorescent dye (it is using PLGA film as support member), or the sheet of 3 × 3 of GelFoam inches is all by pressing 2 minutes to wound.

At once lactated Ringer (LR) is used to start liquid resuscitation after injury.Infusion LR is to re-establish the MAP at least 80% of the MAP before injured possibly as required.Recovery continues the whole observation period.At the end of the research of 60 minutes, record the MAP of every animal and total recovery volume of infusion.

After completing this process, make all animals (Gemfoam) euthanasia of the 2nd group.When research completes two weeks, make the half (Clotblock of the animal in the 1st group, the half of the n=3) and 3rd group (contrast saline, n=2) euthanasia, and after completion 4 weeks time make second half (the 1st group of n=3 and the 3rd group n=2) euthanasia.Implement postmortem and obtain histological sample from some organs.

Outcome measurement

Primary Endpoint: damage in latter 5 minutes the ratio successfully realizing stopping blooding.Preassigned Primary Endpoint is bleeding stopping period, and bleeding stopping period is defined as and stops interval that is hemorrhage or that ooze out from essence from being applied to.If from vagina vasorum site Recurrent Bleeding after initial hemostasis, also record recovers time and the persistent period of the application of the additional non-pushed completely needed for hemostasis.

Secondary endpoints: maintain the total blood loss needed for median blood pressure in 10% of baseline and the resuscitation fluid scale of construction.

Result: the terminal that the animal (damage of IV level) in the 1st group and the 2nd group has been shown in table 1.The measurement result of the IV level hepatic injury processed with ClotBlock (the 1st group) and GelFoam (the 2nd group).All values is all reported as meansigma methods ± SEM

Conclusion: realize hemostasis with all animals of ClotBlock process in application 5 minutes.ClotBlock significantly reduces bleeding time and blood loss, and significantly improves the bonding of tearing between damaged tissue.

3.4 assessments are positioned over above self-adhering fascia and control the hemorrhage ClotBlock of serious skin for as coagulation

Ten Yorkshire crossbreeding sows, 2.5 months ages, body weight 37 ± 2kg.Random packet.The animal via of the 1st group (n=5) is by 3 of groin injury place " long deep skin laceration, and with the ClotBlock process be positioned over above self-adhering fascia 10 minutes.The animal via of the 2nd group (n=5) by similar process, and does not process.

Outcome measurement

Primary Endpoint: damage in latter 10 minutes the ratio successfully realizing stopping blooding.Secondary endpoints: total blood loss.

Result: the terminal that the animal in the 1st group and the 2nd group has been shown in table 2.All values is all reported as meansigma methods ± SEM

Table 2. group	Total blood loss (ml)	Stop blooding when 10 minutes
			1(n＝5)	20±13	5
2(n＝5)	210±112	0

Conclusion: realize stopping blooding in 10 minutes in application with all animals of the ClotBlock process of form of bandage.ClotBlock significantly reduces bleeding time and blood loss, and significantly improves the bonding of tearing between damaged tissue.

4. safety research

In 6 pigs of the 1st group (n=6) wherein 3 by opening abdomen and stand 4 grades of hepatic injury and with 40CC ClotBlock process, implementing euthanasia when 2 weeks after operation completes, and made its excess-three euthanasia 4 weeks time.Similarly, two contrasts (the 3rd group) then, and implement euthanasia after 2 weeks, make two animal euthanasias after 4 weeks.Implement postmortem and obtain tissue sample from major organs.

The safety of reagent is evaluated by the formation of the assessment of toxicity, physiological side effect, biocompatibility, delayed hematoma and/or edema and immunology risk.Physiology and pathological observation comprise: mortality rate/sickness rate; Body weight, food consumption, organ weight.

4.1 acute toxicityassessed by macroscopic evaluation during postmortem and Histological research.By the evidence of finding acute and/or chronic inflammatory disease as signal or histology stimulate evaluate before the tissue of this reagent contacted and vascular tissue stimulate.Evaluate thrombosis, fistula and abscess to all organs to be formed.

4.2 the evaluation of delayed hematoma: under peplos or essence hematoma formed risk.After application 21 days time naked eyes and histological observation formed to delayed hematoma and edema.Little hematoma forms the touched lump that is defined as visible or≤diameter 4cm and does not accompany relevant lapsing to.

4.3. pathology and histology

We analyze lung, kidney,liver,spleen all treated Histological injury after 2 weeks and 4 weeks of operation, and the sample compared with ClotBlock process and the inflammation data contrasting (using normal saline solution process), inflammation comprises apoptosis and leukocyte infiltration.Also Histological evaluation's inflammation and edema are formed.

Once put to death animal, collect organ, to be fixed in 10% formalin and to be embedded in paraffin mass.Histological section's hematoxylin and eosin dyeing and 100X and 400X in optics and microscope check.These microscope slides are assessed by the veterinary pathologist of committee's certification, as shown in table 3.

result:

the summary of Morphologic Diagnosis:

Heart:

Without significantly finding

Lung:

1. pulmonary atelectasis, mild to moderate, multifocal

2. edema, is low to moderate slight, acute most, fills the air

3. hemoglobin crystal, minimum, focus, accompanies slight neutrophilic granulocyte inflammation

Kidney:

1. granular cast, in pipe, minimum, multifocal

2. transparent water droplet, in ooecium slurry, proximal renal tubular epithelial cells, is low to moderate slight, multifocal most

Stomach wall:

Fibre bundle, focus, with central chamber, slightly to moderate granuloma, neutrophilic granulocyte, hair shaft lymphoplasmacytic inflammation, and intermediate edema

Summarize pathology in table 4 to find.

conclusion: contrast (stitchings) and non-stitching process animal in injury of abdominal wall site all containing slight chronic inflammatory disease and fibrosis.The animal only processed in injury of abdominal wall site, the adjacent peritoneal surface place of hepatic injury site and crystal seed has the sign of the granulomatous inflammation relevant to foreign body (ClotBlock).Although the animal of process does not show significantly hemorrhage histological evidence at arbitrary clinical site, the contrast sewed up to present under serious peritonitis and peplos hepatitis and with sign that is downright bad and Fibrotic hematoma.Thromboembolism sign is not had in any other organ.Control animal has extramedullary hemopoiesis sign in spleen, is interpreted as the hemorrhage response to previous procedure damage.The change of pointing out remarkable hemorrhagic anemia is not seen in arbitrary animal.

All pigs (process and matched group) produce adhesion when injured with when treating or sew up.Therefore adhesion is not caused by ClotBlock, although may be promoting factor as the ClotBlock of other fibrin sealants.

The pharmacokinetic profile of the reagent 4.4. studied by biodegradation.Determine in vivo by the biodegradable elimination of proteolytic enzyme.

method: in order to the final result of the ClotFoam in examination volume, use fluorescently-labeled human fibrinogen to prepare a collection of ClotBlock as tracer.Six animals of 1st group of this ClotBlock formulation application in IV level liver wound progress (4.3), and 2 weeks (n=3) and 4 weeks (n=3) make animal euthanasia after application.Once put to death animal, collect organ, be fixed on 10% formalin and be embedded in paraffin mass.Histological section checks with 100X and 400X in fluorescence microscope.The elimination of ClotBlock is by there is not fluorescence trace in sample completely or being determined by the fluorescence level observed when 2 weeks and 4 weeks.

Result: eliminate Clotblock (Fig. 8) in 4 weeks in all organs in application.

4.5. immunne response is assessed

The potential antibody response of assessment to ClotBlock.

Method: collect blood serum sample from the laboratory animal standing IV level hepatic injury (4.3), be collected in before postoperative process with process after 0 day, 7 days and 21 days, and refrigerated storage at-20 DEG C until analyze.The antibody that the component of the preparation for ClotBlock is generated is detected by elisa (ELISA).

In order to detect the antibody of the issuable component for ClotBlock in pig, construct sandwich ELISA (enzyme-linked immunosorbent assay).The bottom surface of 96 hole microtitration plates at 4 DEG C with fibrin (in PBS 10mg/ml, pH value 7.0, Sigma-Aldrich company) or thrombin (in PBS in n10mg/ml PBS, pH value 7.0, Sigma-Aldrich company) bag spent the night.With PBS to the porose washing of institute 5 times.The sample PBS of porcine blood serum, with the final dilution factor application of 1:20, is at room temperature hatched 1 hour, and is washed 5 times with PBS.It is porose that the rabbit anti-pig IgG antibody (Sigma-Aldrich company) that enzyme (horseradish peroxidase) connects is applied to institute with PBS with 1:20 dilution, at room temperature hatches 1 hour, and wash 5 times with PBS.By by substrate (2,2'-azine group-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts, 10mg/ capsule, Sigma-Aldrich company) a capsule dissolves in 100ml phosphate citrate buffer (pH 5.0) and add H ₂o ₂(3% solution of 0.25ml) prepares substrate.Then, at room temperature hatch 10 minutes, use the EX800 microplate reader of BioTek to determine the optical density of 405nm.

Each target components phosphate buffered saline (PBS) (PBS (pH 7.4) with 1:100 dilution, be coated in micro titer plate well, and at 4 DEG C overnight incubation.This some holes 0.25% (wt/vol) defatted milk powder/0.2%Tween 20 in PBS (Block buffer) closes, and then at 37 DEG C, hatches 1 hour with the animal serum 50 μ L of the 1:10 dilution in Block buffer.In conjunction with IgG and IgM detect as the standard ELISA system of secondary antibodies.

Normal range is determined with 5 parts of Normal animal serums.The antibody horizontal raised is defined as two standard deviations being greater than more than normal average value.Each plate comprises the hole of hatching with all reagent except dilute serum, and this hole provides the background absorption deducted from all results.The antibody of the thrombin of purification by the determination of antibody being described as thrombinogen, except the thrombin (5 μ g/mL) of purification for bag by microwell plate.Inhibition anticoagulin (anti-factor) V antibodies is in factor Ⅴ C2 domain, and this is relevant to hemorrhage clinical presentation.Human blood coagulation (human factor) V antibody is by wrapping by micro titer plate well identification with Mus monoclonal anti labile factor antibody 6A5 (50 μ L 2.5 μ g/mL, 4 DEG C whole night).Washing and closed each hole, afterwards, will hatch each hole with 50 μ L 5 μ g/mL human blood coagulation V.At 37 DEG C, each hole is hatched 1 hour with the animal blood slurry of 1:10 dilution.Detect the IgG (standard sandwich ELISA) of combination as described above.

It is highly important that and will consider, then propose be test in rat and pig for human body component across species model produce enzyme system.Even if animal produces the antibody for any ClotBlock component, this still positive result can not can be extrapolated to the mankind.Homology experimental system is not had at preclinical phase.

Statistical analysis

By the comparison of quantitative variable between 6 random groups in use Kruskall-Wallis test execution two treatment weapons.Mann-Whitney U test evaluates the difference between each random group by being used for.Statistical analysis adopts Stata, and 9.0 versions (Stata, institute stands, TX) carry out, and P<0.05 thinks statistically-significant difference.

Result and conclusion: when testing for fibrin or thrombin, do not observe the significant difference (Fig. 9 and 10) of OD reading from the pig of contrast and ClotBlock process at the serum of the 0th day, the 7th day or collection in the 21st day.Our conclusion is that experiment pig does not produce detectable anti-ClotBlock antibody.

5. sterilizing

The aseptic preparation of clotcake is studied.

Use Nalg-Nunc company 500mL device (catalogue #450-0045, nitrocellulose filter, the filter of 0.45m) aseptic filtration acid fiber protein monomer in Biohazard Safety Equipment.

Increment study: general experimental program comprises prepares sample solution, potato dextrose agar (PDA subsequently in culture dish, Sigma-Aldrich company, catalog number (Cat.No.) P2182) and Tryptic Soy Agar (TSA, Sigma-Aldrich company, catalog number (Cat.No.) T4536) gel is coated with sample solution for growth.Hatch in cycle instruction time of colony growth (mould and/or antibacterial) and observe PDA and TSA gel.

Sample hatches 30 minutes at 37 DEG C, and the time cycle assessment colony growth with the naked eye indicated by results and discussions part.This sample grows, with the multiple sample of 1,2 and 3 sign in tables of data with binary fission or tripartition form.For assessment of scoring as follows:

table 2. colony counting key

Symbol	Counting
		-	There is no visible growth
+	1-199 visible colonies
		++	200-399 visible colonies
+++	> 400 visible colonies

Table 3 illustrates the result of the research that the growth of microorganism on PDA and TSA of the sterile components of FIBRIN_ClotFoam is analyzed.

The aseptic C of * is used for the zooscopy in SUNY, at 4 DEG C, store one week

Increase data to show, even if sterile components does not produce significant growth after 11 days yet.In addition, following technology may be used for sterilizing.

6. biocompatibility

Two ClottBlock preparations are aseptically prepared and test.The biocompatibility of these preparations is tested with human fibroblasts (HF) and HEP (A549 cell line, ATCC).

Normal human fibroblast (HF) is bought by business and to obtain and the Eagle culture medium (being supplemented with 10% hyclone) improved with Dulbecco in 60mm tissue culture plate builds culture, and at the 5%CO of humidity ₂atmosphere (CO ₂incubator) in remain on 37 DEG C.HEP system A549 remains in the minimal essential medium being supplemented with 10% hyclone and 2mM glutamine.When fibroblast and epithelial culture reach sub-fusion, contrast and the mixing of sodium benzoate ClotFoam preparation, and be delivered to independent ware device immediately.Culture is turned back to CO ₂incubator and daily check totally five days.ClotFoam material and medium remove from all cultures, and attached cell crystal violet (0.1%, in 2% ethanol) dyes.

Result: main detection does not damage completely or toxicity cell, and without any antibacterial or fungal contamination.

Be exposed in the human fibroblasts culture of ClotBlock preparation in Figure 11 (a, b), cell seems than the untreated culture of contrast and disperses a little more greatly or more.And in Figure 12 in (a, b) in HEP be also.

Conclusion: ClotBlock is biocompatible, and does not suppress, but stimulates, the growth and differ entiation of cell; This is the important attribute of Wound-healing agent.

Claims

1., for pressing minimum the fibrin sealant compositions depressing Bleeding control, described compositions comprises:

A) layer of the desAB fibrin II polymer of lyophilizing, and

B) layer of the thrombin of lyophilizing.

2. compositions according to claim 1, wherein, prepares described fibrin polymer by the reaction of fibrin II monomer and neutralization buffer, adds described neutralization buffer with the volume ratio of 1:1.

3. compositions according to claim 2, also comprises the glycerol of about 1% to about 5% added based on volume-volume.

4. compositions according to claim 2, wherein, described neutralization buffer comprises:

150mM NaCl，

50mM HEPES，

3mM CaCl ₂，

0.12g/mL Activa (non-calcium dependent T-5398); And

The XIII factor of the 21Lowey unit in every mL neutralization buffer.

5. neutralization buffer according to claim 4, wherein, the pH of described neutralization buffer is 8.5.

6. compositions according to claim 2, wherein, uses the non-calcium dependent T-5398 be mixed in described neutralization buffer to carry out crosslinked described fibrin polymer.

7. compositions according to claim 1, wherein, the feature of described compositions is for being biocompatible.

8. compositions according to claim 1, when described compositions being applied to bleeding wounds and organizing, described compositions absorbing blood thus formed gel, described gel for forming stable fibrin clot on wound.

9. compositions according to claim 8, wherein, in pressing or when not pressing, realized hemostasis in one minute that described compositions is applied to wound tissue.

10. an equipment for Bleeding control, wherein, compositions according to claim 1 can be placed on bonding support member or use when not having support member.

11. equipment according to claim 10, wherein, described bonding support member is biodegradable and removes.

12. equipment according to claim 10, wherein, the biodegradable adhesive bandage of described bonding support member right and wrong.

13. compositionss according to claim, wherein, described fibrin polymer is lyophilizing in the mould with various shape and size.