EP2938275A1 - Colle de fibrine sous forme lyophilisée pour hémorragie de grande abondance - Google Patents

Colle de fibrine sous forme lyophilisée pour hémorragie de grande abondance

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Publication number
EP2938275A1
EP2938275A1 EP13868100.2A EP13868100A EP2938275A1 EP 2938275 A1 EP2938275 A1 EP 2938275A1 EP 13868100 A EP13868100 A EP 13868100A EP 2938275 A1 EP2938275 A1 EP 2938275A1
Authority
EP
European Patent Office
Prior art keywords
fibrin
composition
clotblock
bleeding
lyophilized
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP13868100.2A
Other languages
German (de)
English (en)
Inventor
George D. Falus
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
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Filing date
Publication date
Priority claimed from US13/731,126 external-priority patent/US8741845B1/en
Application filed by Individual filed Critical Individual
Publication of EP2938275A1 publication Critical patent/EP2938275A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32—Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001—Use of materials characterised by their function or physical properties
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/106—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028—Polypeptides; Proteins; Degradation products thereof
    • A61L26/0042—Fibrin; Fibrinogen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding
    • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F04—POSITIVE - DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS FOR LIQUIDS OR ELASTIC FLUIDS
    • F04C—ROTARY-PISTON, OR OSCILLATING-PISTON, POSITIVE-DISPLACEMENT MACHINES FOR LIQUIDS; ROTARY-PISTON, OR OSCILLATING-PISTON, POSITIVE-DISPLACEMENT PUMPS
    • F04C2270/00—Control; Monitoring or safety arrangements
    • F04C2270/04—Force
    • F04C2270/042—Force radial
    • F04C2270/0421—Controlled or regulated

Definitions

  • the present invention is related to a method to produce a two component or bi-layer system consisting of a sterile biocompatible lyophilized desAB fibrin polymer or fibrin II polymer composition from concentrated desAB fibrin monomer or fibrin II monomer in acid solution, and lyophilized thrombin for application as an adhesive sealant component and hemostatic agent.
  • the preparation (invention) trademarked ClotBlock is presented in various solid shapes and thickness that may be used to stop bleeding or seal tissue in vivo with and without compression. It is particularly related to need of affixing a fibrin Clot without a biodegradable support or a support that can be detached after application over a bleeding wound in order to seal tissue and control vascular, epidermal, bone or internal hemorrhage.
  • the invention may include a biodegradable support made of hyaluronic acid or other biodegradable polymer,
  • the synthetic adhesives are used for the tight sealing of vessels or of lungs and for "gluing" the edges of skin incisions. These glues are eliminated, in general after the scaring of the wound, by biodegradation, absorption or by simple detachment in the form of scabs.
  • Various technologies have been developed for the formulation of tissue adhesives. Some of them are of synthetic origin, such as the glues based on cyanoacrylates (2-butyl cyanoacrylate, 2-octyl cyanoacrylate), or synthetic polymers, and others contain biological materials such as collagen or fibrin which in addition have hemostatic properties.
  • sealants As a result of their hemostatic and adhesive properties, sealants, and particularly fibrin sealants have been extensively used in most surgical specialties for over two decades to reduce blood loss and post-operative bleeding because of the ability to adhere to human tissue as they polymerize (1 , 2, 3). These compounds are used to seal or reinforce the sealing of wounds that have been sutured or stapled; they can also be used with pressure over an injured area. Fibrin sealants are biological adhesives that mimic the final step of the coagulation cascade. (4) The main components of the sealant are fibrinogen, plasma proteins and factor XIII on the one hand and thrombin, and calcium chloride on the other. The components are often extracted from human plasma or produced by recombinant techniques. Mixing fibrinogen and thrombin creates a polymer barrier (fibrin) that simulates the last stages of the natural coagulation cascade to form a structured fibrin clot similar to a physiological clot.
  • tissue adhesives and sealants have to be employed in combination with compression methods, sutures and/or staples, and adhesive patches so as to reduce the tissue-bonding strength required for acceptable performance.
  • tissue adhesives and sealants have to be employed in combination with compression methods, sutures and/or staples, and adhesive patches so as to reduce the tissue-bonding strength required for acceptable performance.
  • the use of strong compression, sutures and/or staples is undesirable, inappropriate or impossible, (e.g. in bone, interventional radiology).
  • the adhesive matrix In order to form a physical barrier that resists the flow of blood, the adhesive matrix must form in a matter of seconds a strong fibrin interface, bond with tissues in the midst of flowing blood and remain at the lacerated site to form a clot.
  • the ability to adhere to human tissue of each of the product presentations in a form of a square 7 a round patch a sphere , a cylinder, or a cone is related to the composition and method of production of fibrin and its interaction (combination) with thrombin to stimulate the coagulatory cascade.
  • the essential aspect of the technology is the ability to bypass the cleavage process of fibrinogen to produce a fibrin monomer and its subsequent polymerized, lyophilized and capable to absorb blood to form a fibrin clot.
  • the agent starts the Clot formation process from an already stabilized I fibrin polymer that absorb the blood into a lyophilized crosslinked polymer containing the necessary components to stimulate the coagulatory cascade (thrombin) and form a physical barrier that turns into a functional fibrin clot within two minutes of application. (6)
  • the lyophilization process in subsequent layers over a biodegradable removable support facilitates its application, and allows for long-term storage, transportation and readiness.
  • the monomer can be mixed with a volume of about 1 % to about 5% of glycerol to achieve a specific viscoelastic profile that is adapted to the type of application.
  • the absorption of blood by the cake turns the lyophilized fibrin into a gel, which forms the fibrin clot at sites of injury (7).
  • the fibrin gel that seals the wound is formed as a result of the absorption of blood by the lyophilized bilayer material, which maintains covalent bonds while changing from solid to gel state.
  • the clot is mechanically stable, well integrated into the wound and more resistant to lysis by plasmin compared with a non- cross-linked clot [8] or other fibrin sealants.
  • the inclusion of calcium independent transglutaminase facilitates the transglutaminase-mediated cross-linking of the aC-domains polymers in fibrin promoting integrin clustering and thereby increasing cell adhesion and spreading, which stimulates fibrin to bind avb3-, avb5-and a5b1 -integrins on endothelial cells [9].
  • the oligomerization also promotes integrin-dependent cell signaling via focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), which results in an increased cell adhesion and cell migration [10].
  • FAK focal adhesion kinase
  • ERK extracellular signal-regulated kinase
  • Lyophilized Fibrin II is obtained from fibrin II monomer polymerization: US patent application 12/487,057 (allowed), which describes a method of preparing a fibrin monomer.
  • the ClotBlock sealant composition uses a lyophilized fibrin polymer obtained from neutralization of fibrin monomer.
  • composition of parts and method of production of the fibrin II described in this patent application as well as the process of neutralization and crosslinking of the polymer are critical to the performance of the proposed technology which depends on the characteristics of fibrin itself (thickness of the fibers, the number of branch points, the porosity, and the permeability and other polymerization characteristics define clotting factors.
  • the Clot produced by ClotBlock creates opaque matrices of thick fibers, and therefore tube formation proceeds at a faster rate than in transparent matrices.
  • concentration of thrombin to produce a fibrin monomer and thus the release-rate of FPA also has an important impact on the polymerization process.
  • the fibrin polymer can be produced and lyophilized in various sizes, thickness and forms in order to adapt to the type of application (Fig 2A, 2B, 2C). It can be configured in small spheres or cylinders % inch diameter to be introduced through a laparoscopic port or a vessel in cases on interventional radiology; it can also be molded in round or square flat solid blocks of various sizes in 1 ⁇ 4; 1 ⁇ 2 a 1 " thickness for use in spleen laceration, or organ resection, or placed over an adhesive bandage to cover deep skin cuts. The lyophilized form can also be soaked in water and used as a sealing paste or gel.
  • the present invention lies within the domain of biological adhesives and tissue sealants, which are biodegradable and nontoxic, intended for therapeutic use, for example, as an adjunct to hemostasis in laparotomy or laparoscopic surgery, or as primary treatment in orthopedic surgery, trauma (spleen laceration), interventional radiology and large-bed wounds.
  • the present invention relates to biocompatible adhesive fibrin polymer, which is bio-reabsorbable and nontoxic, for surgical or therapeutic use. It also relates to a bilayer application containing bioactive substances, which can be released in a given site to stimulate coagulation.
  • the invention relates to a process for producing such an adhesive polymer.
  • ClotBlock is an excellent hemostatic agent candidate control moderate to severe bleeding. Its different presentations maximize the hemostatic effect in various types of surgical and trauma applications.
  • the agent is durable, easy to store, poses minimal risk, requires little training to use, and is highly effective against moderate to severe bleeding.
  • Fig. 2 Possible shapes: A Spheres and cylinders; B Patch or Block; C Bandage
  • Fig. 4 Polymerization, cross-linking and stabilization of fibrin in the presence of ACTIVA and Activa + Factor XIII.
  • Fig. 6 Control of spleen laceration bleeding as primary treatment without packing or sutures. By application of CloBlockT, hemostasis was achieved within 5 minutes of application.
  • Fig.7 Control of intraoperative bleeding as primary treatment in liver injury grade IV by application of ClotBlock. Hemostasis was achieved within 5 minutes . Hemostatic
  • ClotBlock in gel form as an adjunct to hemostasis to control intraoperative bleeding in partial hepatectomy. Hemostasis was achieved within 5 minutes of application.
  • Fig. 8. Microscopic examination under UV light comparing the trace in fluorescence trapped in interstitial spaces in kidney and liver at 2 weeks (A) with 5 weeks (B) after application. .
  • Fig 9. Detection of antibodies that might be produced in swine against Thrombin, using a sandwich ELISA (enzyme linked immunosorbent assay).
  • Fig. 10 Detection of antibodies that might be produced in swine against Thrombin, using a sandwich ELISA (enzyme linked immunosorbent assay).
  • Fig 1 1 Human fibroblast exposed to ClotBlock preparations, there was a total absence of damage or toxicity to the cells, and absence of any bacterial or fungal contamination; the cells appeared slightly larger than in control untreated cultures.
  • the agent is a novel fibrin sealant (pure fibrin II made by neutralization of fibrin II monomer) supplemented by thrombin, and designed to promote hemostasis in cases of severe bleeding, and to stop hemorrhage with minimal
  • the present fibrin sealant can be used 1 ) as an adjunct or as primary treatment for severe bleeding; or 2) shaped for delivery through laparoscopic port or used as a compression in organ resection, or 3) placed on support for use in cases of skin laceration; or 4) shaped to be delivered through catheters in cases of interventional radiology; or 5) shape to seal intramedullary bleeding arising from orthopedic surgery or trauma.
  • Each of the presentations consists of a lyophilized bilayer (fig. 1) comprising: 1 ) a fibrin II polymer produced by neutralization of fibrin monomer in acetic acid solution (pH 3.4) with HEPES buffer (pH 8.3)j and crosslinked, by activated Factor XIII and calcium independent tranglutaminase ; 2) a layer of thrombin at a concentration of 20 NIH units/ml dissolved in HEPES water solution in a proportion of 1 ml for every 4 ml of fibrin; and 3) the option of adding a third layer PLGA fibronectin embedded microspheres between the fibrin and the thrombin layers.
  • the lyophilized bilayer is applied over lacerated bleeding tissue, which absorbs the blood to form a sticky, gummy gel barrier and subsequently a fibrin clot as blood is absorbed by the fibrin. 1 ) The agent seals the wound within 2 minutes, and 2) binds together the lacerated tissue.
  • ClotBlock has been developed in several formulations, which vary in shape, elasticity, and clotting strength as needed.
  • ClotBlock is produced in two layers. It consists of a lyophilized fibrin polymer topped by a layer of lyophilized thrombin.
  • the first layer contains crosslinked fibrin polymer produced by neutralization of fibrin monomer in acetic acid solution mixed with a buffer solution composed of 150 mM NaCI, 50 mM HEPES, 3 mM CaCI 2 , 0.12 g/mL Activa (calcium independent transglutaminase enzyme) and 21 Lowey Units of Factor XIII per ml of Neutralization buffer, pH 8.5.according to method described in U.S. Patent Application No. 12/487,057 and incorporated by reference herein. These two solutions are mixed in a ratio of 1 :1 inside a sterile mold. . To this Composition 1 % to 5% of glycerol can be added, depending on desired flexibility of the block
  • This mold is sealed inside a sterile TYVEK® (Registered trademark of E.I. DuPont Co., Wilmington (DE) bag and incubated at 37°C for four hours.
  • the second layer contains a solution of thrombin in a proportion of 1 :4 to fibrin, which is dissolved in HEPES buffer at the concentration of 20 units/mL.
  • Step 1 Each component is sterilized by filtration through a 0.22 micron Millipore filter. Each layer is poured into a silicon mold of the desired shape (round, oval or square) to produce a "cake" of approximately 3 ⁇ 4 to 1" thick, which can be supported by a removable or biodegradable polymer of mesh such as polyglactin mesh, or sD,L-lactide polymer synthetic mesh, polylactic acid (PLA)/poly(glycolide-co-lactide) copolymer (PLGA) membrane or polyglycolic acid (PGA) mesh; or by a self-adhesive bandage for use in cases of cutaneous lacerations.
  • a removable or biodegradable polymer of mesh such as polyglactin mesh, or sD,L-lactide polymer synthetic mesh, polylactic acid (PLA)/poly(glycolide-co-lactide) copolymer (PLGA) membrane or polyglycolic acid (PGA) mesh; or by a
  • Step 2 Clotblock is then lyophilized at a condenser temperature of -40 C to -50 C, shelf temperature of 21 ° C, during 18-72 hours at a pressure of 200-400 millitor.
  • Step 3 Each piece of ClotBlock is packaged in plastic bags hermetically sealed to prevent moisture loss and maintain sterility.
  • Step 4 ClotBlock is applied with moderate compression directly over the wound for 1 to 2 minutes. Within 3 minutes a fibrin clot is formed over the wound.
  • ClotBlock can be shaped in small cylinders 14" diameter and 1 " to 2" long, which can be delivered through a laparoscopic port into an intracavitary wound, or through a catheter to seal a bleeding vessel.
  • ClotBlock can be dissolved in water at a proportion of 4:2 to form a liquid gel that can be applied with a single syringe on a laceration or wound.
  • ClotBlock can placed over a self-adhesive or non-self adhesive, which is a bi-layer system consisting of flexible fabric adhesive bandage and a sterile biocompatible lyophilized desAB fibrin polymer or fibrin II polymer composition from concentrated desAB fibrin monomer or fibrin II monomer in acid solution, that may be used to stop bleeding or seal cutaneous tissue.
  • This delivery method is particularly related to need of arresting bleeding from large or deep skin cuts by affixing a fibrin Clot compressed by a bandage.
  • CLOTBLOCK sealant forms a fibrin clot stronger and faster than other sealants.
  • the adhesive is expected to adhere to lacerated tissue and bind the opposing tissues together with a strength that is significantly higher than that observed for fibrin sealants.
  • the figure shows the formation of strong gamma dimmers during fibrin cross-linking with calcium independent transglutaminase enzyme and factor XIII at 1 minute. At this time gamma dimmers are not yet present in the fibrinogen sample.
  • Protocol Evaluation of ClotBlock for the control of intraoperative bleeding as primary treatment in partial nephrectomy.
  • the five minute time to hemostasis is defined by the Blood Products Committee of the Food and Drug Administration as the maximum time to demonstrate efficacy in achieving hemostasis.
  • CloBlock can stop profuse bleeding within 5 minutes of application in cases traumatic spleen laceration.
  • the five minute time to hemostasis is defined by the Blood Products Committee of the Food and Drug Administration as the maximum time to demonstrate efficacy in achieving hemostasis.
  • a grade 4 injury is defined as a 7 cm long full-thickness parenchyma laceration (created sharply by an 1 1 blade scalpel).
  • a spot in the middle of the liver was selected to produce the liver injury with a scalpel.
  • the position was calculated by approximation to the suprahepatic vessels and some branches of the portal vein.
  • the spot was marked with a marker.
  • either a 40CC block of ClotBlock of the patch type treate with Alexa fluorescent dye with a PLGA membrane as support or a 3x3 inch piece of GelFoam was compressed was against the wound for 2 minutes.
  • Fluid resuscitation with Lactated Ringer's was begun immediately after injury. LR was infused as necessary to re-establish a MAP within at least 80% of the pre-injury MAP if possible. Resuscitation was continued for the entire observation period. At the end of the 60 minute study, each animal's MAP and the total resuscitation volume infused were recorded.
  • Primary endpoints Proportion of successes achieving hemostasis within 5 minutes following injury.
  • the pre-specified primary endpoint is the time to hemostasis, defined as the time interval from application to termination of bleeding or oozing from the parenchyma. If recurrent bleeding from the sheath site occurred following initial hemostasis, the timing and duration of additional non-compressible application required to reestablish complete hemostasis was also recorded.
  • ClotBlock significantly decreases the bleeding time and blood loss, and significantly improves the adhesion between lacerated and damaged tissue.
  • ClotBlock placed over a self-adhesive bandage for the control of severe cutaneous bleeding as primary treatment.
  • ClotBlock significantly decreases the bleeding time and blood loss, and significantly improves the adhesion between lacerated and damaged tissue. 4. Safety Studies
  • two controls (group 3) were followed and euthanized after 2 weeks, and two animals were euthanized after 4 weeks. Necropsy was performed and tissue samples from main organs were obtained.
  • Acute Toxicity was assessed by macroscopic evaluation at necropsy and by histological studies. Irritation of tissues and tissue vessels to which the agents ere in contact was assessed looking for evidence of acute and/or chronic inflammation as signs or irritation in the histology. Thrombosis, fistula, and abscess formation was assessed for all organs
  • Hyaline droplets intracytoplasmic, proximal tubular epithelium, minimal to mild, multifocal
  • ClotBlock To examine the fate of ClotFoam in vivo, a batch of ClotBlock was prepared using fluorescein-tagged human fibrinogen as tracer. This preparation of ClotBlock was applied to the six animals of Group 1 in the liver grade IV wound procedure (4.3), which were
  • ClotBlock was determined by either the total absence of fluorescent traces in the samples, or by the level of fluorescense observed at 2 weeks and 4 weeks.
  • Serum samples were collected from experimental animals subjected to liver grade IV injury (4.3), pre- and post-treatment on Day 0, Day 7, and Day 21 days post- surgery and stored frozen at -20°C until analysis.
  • Antibodies generated to the components that are used in the formulation of ClotBlock were tested by enzyme-linked immunosorbent assay (ELISA).
  • a sandwich ELISA enzyme linked immunosorbent assay
  • the bottom surfaces of 96-well microtiter plates were coated overnight at 4°C with Fibrin (10 mg/ml in PBS, pH 7.0, Sigma-Aldrich) or thrombin (n 10 mg/ml in PBS, pH 7.0, Sigma-Aldrich). All wells were washed 5 times with PBS. Samples of swine serum were applied at 1 :20 final dilution in PBS, incubated for 1 hr at room temperature and washed 5 times with PBS.
  • Enzyme horseradish peroxidase
  • pig IgG Sigma-Aldrich
  • Substrate was prepared by dissolving one capsule of substrate (2,2'-Azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) di-ammonium salt, 10 mg/capsule, Sigma-Aldrich) in 100 ml of phosphate-citrate buffer, pH 5.0, and adding H2O2 (0.25 ml of 3% solution). Following incubation for 10 min at room temperature, optical density at 405 nm was determined using a BioTek EX800 microplate reader.
  • Each targeted component was diluted 1 :100 in phosphate-buffered saline (PBS), pH 7.4, coated onto microtiter plate wells, and incubated overnight at 4°C.
  • PBS phosphate-buffered saline
  • the wells were blocked with 0.25% (wt/vol) nonfat dry milk/0.2% Tween 20 in PBS (blocking buffer) and then incubated with 50 ⁇ _ of a 1 :10 dilution of animal serum in blocking buffer for 1 hour at
  • the normal range was determined with 5 normal animal sera. An elevated antibody level is defined as greater than two standard deviations above the normal mean. Each plate included wells incubated with all reagents except for the diluted serum, which provided the background absorbance that was subtracted from all results. Antibodies to purified
  • thrombin were determined as described for antibodies to prothrombin, except that purified thrombin (5 ⁇ g/mL) was used to coat the microtiter plate wells. Inhibitory anti-factor V antibodies binding to the factor V C2 domain are associated with hemorrhagic
  • Antibodies to human factor V were identified by coating microtiter plate wells with the murine monoclonal antihuman factor V antibody 6A5 (50 ⁇ _ of 2.5 pg/mL overnight at 4°C). The wells were washed and blocked, after which they will were incubated with 50 ⁇ _ of 5 ⁇ g/mL human factor V. The wells were incubated with a 1 :10 dilution of animal plasma for 1 hour at 37°C. Bound IgG was detected as described above (standard sandwich ELISA).
  • the acidic Fibrin Monomer was sterile filtered in a biological safety cabinet using a Nalg- Nunc 500 ml. device (Cat # 450-0045, nitrocellulose membrane, 0.45 m filter).
  • the general experimental protocol included preparation of sample solutions which were then plated on Potato dextrose agar (PDA, Sigma-Aldrich, Cat#P2182) and Tryptic soy agar (TSA, Sigma-Aldrich, Cat# T4536) gels in Petri dishes for growth. The PDA and TSA gels were incubated and observed at the indicated periods of time for colony growth (mold and/or bacteria).
  • PDA Potato dextrose agar
  • TSA Tryptic soy agar
  • the sample was incubated for 30 min at 37 °C and evaluated for colony growth using the naked eye at the time periods indicated in the Results and Discussion section.
  • the samples were run in duplicate or triplicate with multiple samples indicated with a 1 , 2 and 3 designation in data tables.
  • the scale used for evaluation is as follows:
  • Table 3 shows the results of studies of microorganism growth analysis on PDA and TSA of the sterile components of FIBRIN_ClotFoam.
  • PDA Potato Dextrose Agar
  • the growth data indicate that sterile components yielded no significant growth even after 1 1 days. Furthermore, the following techniques could be used for sterilization.
  • ClottBlock preparations were prepared an d tested under sterile conditions. These preparations were tested for biocompatibility with human fibroblasts (HF) and human epithelial cells (A549 cell line, ATCC).
  • HF human fibroblasts
  • ATCC human epithelial cells
  • HFs Normal human fibroblasts
  • Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and maintained at 37°C in a humidified 5% CO2 atmosphere (CO2 incubator).
  • Human epithelial cell line A549 was maintained in Minimal Essential Medium supplemented with 10% fetal bovine serum and 2 mM glutamine.
  • ClotFoam preparations were mixed and immediately delivered into individual dishes. The cultures were returned to the CO2 incubator and examined daily for a total of five days. ClotFoam material and medium was removed from all cultures, and adherent cells were stained with crystal violet (0.1 % in 2 % ethanol).
  • ClotBlock is biocompatible, and does not inhibit, but rather stimulate, the growth and differentiation of cells; which is an important attribute in wound healing agents.

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  • Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Materials Engineering (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Surgery (AREA)
  • Materials For Medical Uses (AREA)

Abstract

La présente invention concerne un agent hémostatique de fibrine sous forme lyophilisée, ClotBlock, qui est conçu pour être utilisé en tant que traitement principal ou d'appoint en cas d'hémorragie modérée à grave. Il peut être appliqué directement sur la plaie au cours d'une procédure de laparotomie ou sous forme de colle non invasive. Sa technologie de réticulation génère une colle de fibrine adhésive résistante et sûre, requise pour une hémostase de grande abondance. Les propriétés de fixation du tourteau ainsi que la formation rapide et la stabilité du caillot de fibrine garantissent la formation d'un caillot de fibrine résistant et stable dans un délai de 1 à 5 minutes selon la taille de la plaie. L'agent est sans danger, biocompatible, biodégradable et peut être conservé à température ambiante pendant un an.
EP13868100.2A 2012-12-31 2013-12-28 Colle de fibrine sous forme lyophilisée pour hémorragie de grande abondance Withdrawn EP2938275A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US13/731,126 US8741845B1 (en) 2009-06-18 2012-12-31 Lyophilized fibrin sealant for high volume hemorrhage
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JP2016507279A (ja) 2016-03-10
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AU2013370223A1 (en) 2015-08-06
CN105007841A (zh) 2015-10-28

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