CN105007841A - Lyophilized fibrin sealant for high volume hemorrhage - Google Patents

Lyophilized fibrin sealant for high volume hemorrhage Download PDF

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CN105007841A
CN105007841A CN 201380074087 CN201380074087A CN105007841A CN 105007841 A CN105007841 A CN 105007841A CN 201380074087 CN201380074087 CN 201380074087 CN 201380074087 A CN201380074087 A CN 201380074087A CN 105007841 A CN105007841 A CN 105007841A
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fibrin
sealant
wound
clot
safe
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CN 201380074087
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Chinese (zh)
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乔治·D.·福卢什
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乔治·D.·福卢什
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L26/00Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061Use of materials characterised by their function or physical properties
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L15/00Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/22Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing macromolecular materials
    • A61L15/32Proteins, polypeptides; Degradation products or derivatives thereof, e.g. albumin, collagen, fibrin, gelatin
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L15/00Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
    • A61L15/16Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
    • A61L15/42Use of materials characterised by their function or physical properties
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L24/00Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/001Use of materials characterised by their function or physical properties
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L24/00Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10Polypeptides; Proteins
    • A61L24/106Fibrin; Fibrinogen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L26/00Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0028Polypeptides; Proteins; Degradation products thereof
    • A61L26/0042Fibrin; Fibrinogen
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61LMETHODS 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
    • A61L2400/00Materials characterised by their function or physical properties
    • A61L2400/04Materials for stopping bleeding
    • FMECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
    • F04POSITIVE DISPLACEMENT MACHINES FOR LIQUIDS; PUMPS FOR LIQUIDS OR ELASTIC FLUIDS
    • F04CROTARY-PISTON, OR OSCILLATING-PISTON, POSITIVE-DISPLACEMENT MACHINES FOR LIQUIDS; ROTARY-PISTON, OR OSCILLATING-PISTON, POSITIVE-DISPLACEMENT PUMPS
    • F04C2270/00Control; Monitoring or safety arrangements
    • F04C2270/04Force
    • F04C2270/042Force radial
    • F04C2270/0421Controlled or regulated

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

用于大量出血的冻干纤维蛋白封闭剂 Lyophilized fibrin sealant for excessive bleeding

[0001] 相关申请 [0001] RELATED APPLICATIONS

[0002] 本申请是2009年6月18日提交的美国专利申请第12/487, 057号(授权)的部分继续申请案,美国专利申请第12/487, 057号已经在2012年11月6日收到授权的通知并且在2013年2月6日之前支付授权费用。 [0002] This application is US Patent Application June 18, 2009, filed on 12/487, 057 (authorized) a continuation in part application, US Patent Application No. 12/487, 057 have been in November 6, 2012 the date of receipt of the notification and authorization to pay licensing fees before February 6, 2013. 其中阐述的所有说明书、附图和教导都明确地结合于此作为参考,并且我们要求其中明确地作出的教导的优先权。 Wherein all the specification, drawings and teachings set forth herein are expressly incorporated by reference, and claims priority of our teachings where expressly made.

技术领域 FIELD

[0003] 本发明涉及一种方法,以产生的由来自酸溶液中的浓缩desAB纤维蛋白单体或纤维蛋白II单体的无菌生物相容的冻干desAB纤维蛋白聚合物或纤维蛋白II聚合物组合物以及冻干凝血酶组成的双组分或双层系统,从而用于作为粘合封闭剂组分和止血剂的应用。 [0003] The present invention relates to a method to produce a sterile biologically compatible desAB concentrated fibrin monomer or fibrin II monomer derived from an acid solution is lyophilized desAB fibrin or fibrin II polymer polymerized compositions, and two-component systems or double lyophilized thrombin, thereby blocking agent for use as an adhesive component and a hemostatic agent. 标有制备(本发明)商标的ClotBlock呈现为各种实体形状和厚度,ClotBlock可以在使用和不使用按压的情况下用于止血或在体内封闭组织。 Preparation of labeled (the present invention) is presented ClotBlock trademark shapes and thicknesses of various entities, ClotBlock may be used in the case of hemostasis and without pressing or sealing tissue in vivo. 它尤其涉及到在没有可生物降解的支撑件或在应用于出血伤口上之后可分离的支撑件的情况下需要粘贴纤维蛋白凝块, 以封闭组织和控制血管出血、表皮出血,骨出血或内部出血。 In particular, it relates to the inside in the absence of the biodegradable support member or support members applied to a bleeding wound after the separable fibrin clot to be pasted to seal tissue and control vascular bleeding, bleeding skin, or bleeding bone bleeding. 本发明可以包括由透明质酸或其它可生物降解的聚合物制成的可生物降解的支撑件。 The present invention may include a support member made of a biodegradable hyaluronic acid or other biodegradable polymer.

背景技术 Background technique

[0004] 器官切除、创伤或大面积皮肤创伤的严重出血有时难以控制。 [0004] organ removal, trauma, or large areas of skin trauma severe bleeding and sometimes difficult to control. 近来,基于纤维蛋白的贴片已经经过测试,以使用纤维素或明胶支撑件封闭IV/V级创伤。 Recently, fibrin-based patch has been tested, using a support member or gelatin closed IV / V level trauma. 然而,这些技术不能应用于某些类型的步骤,诸如介入放射学、整形外科或腹腔镜手术。 However, these techniques do not apply to certain types of steps, such as interventional radiology, plastic surgery or laparoscopic surgery. 此外,这些产品所使用的"支撑件"在生物降解之前留在体内,造成对异物的严重的炎症反应。 In addition, "support" used in these products remain in the body prior to biodegradation, causing severe inflammatory response to foreign substances.

[0005] 当前的解决方案和限制。 [0005] Current solutions and restrictions. 通常地,合成粘合剂用于血管或肺血管的紧密封闭以及用于"粘合"皮肤切口的边缘。 Generally, the synthetic adhesive used for vascular or pulmonary vessels tightly closed and the skin incision edge "bonding." 通常在伤口形成瘢痕之后,通过生物降解、吸收或通过以结痂的形式简单脱离而消除这些胶。 Typically after scar formation of a wound, by biodegradation, by absorption or crusting eliminated from the simple form of these gums. 已经开发各种技术用于组织粘合剂的制备。 Various techniques have been developed for the preparation of tissue adhesives. 其中一些是合成来源的,诸如基于氰基丙烯酸酯(2-丁基氰基丙烯酸酯,2-辛基氰基丙烯酸酯)的胶、 或合成的聚合物以及含有生物材料(诸如另外具有止血特性的胶原蛋白或纤维蛋白)的其他组织粘合剂。 Some of which are synthetic origin, such as based on cyanoacrylate (2-butyl cyanoacrylate, 2-octyl cyanoacrylate) glue, or synthetic polymers and containing biological material (such as having hemostatic properties additional collagen or fibrin) other tissue adhesive.

[0006] 由于它们的止血和粘合性能,封闭剂,和特别是纤维蛋白封闭剂在过去的二十年间已经广泛用于大多数外科专业,以由于当它们聚合(1,2, 3)时粘附到人体组织的能力而减少失血和术后出血。 [0006] due to their hemostatic and adhesive properties, blocking agents, and in particular the fibrin sealant over the past two decades has been widely used in most surgical specialties, when due to their polymerization (1, 2, 3) ability to stick to human tissue and reduce blood loss and postoperative bleeding. 这些化合物用于封闭或加固已经被缝合或钉合的伤口的封闭;它们也可以与在损伤区域上方按压一起使用。 These compounds have been used for closing or closing stitched or stapled wound reinforcement; they may also be used together with the above pressing damaged region. 纤维蛋白封闭剂是模拟凝血级联的最后步骤的生物粘合剂。 The fibrin sealant is a biological adhesive the final step of the coagulation cascade simulation. (4) 一方面,封闭剂的主要组分是的纤维蛋白原、血浆蛋白和XIII因子,以及另一方面是凝血酶和氯化钙。 (4) The main component on the one hand, the blocking agent is fibrinogen, Factor XIII and plasma proteins, thrombin and calcium chloride, and on the other hand. 组分通常提取自人血浆或通过重组技术产生。 Component is typically extracted from human plasma or produced by recombinant techniques. 混合纤维蛋白原和凝血酶产生聚合物屏障(纤维蛋白),聚合物屏障模拟天然凝血级联的最后阶段,以形成类似于生理凝块的结构化纤维蛋白凝块。 Mixing fibrinogen and thrombin generation barrier polymer (fiber protein), a polymer barrier simulate the final stages of the natural coagulation cascade, to form a structure similar to that of physiological fibrin clot clot.

[0007] 存在几个可用的商业产品(Floseal、Gelfoam、Evicel、Bioglue、surgicel、 tachoseal等)(3,5)。 [0007] There are several commercially available products (Floseal, Gelfoam, Evicel, Bioglue, surgicel, tachoseal, etc.) (3,5). 然而,这些产品具有显著的局限性,这阻碍了它们在手术中的严重出血的情况下和急诊医学、整形外科和介入放射学以及腹腔镜手术中的广泛使用。 However, these products have significant limitations, which hinders their case severe bleeding in surgery and emergency medicine, orthopedics and interventional radiology as well as the widespread use of laparoscopic surgery. 用于腔内出血的所有现有的止血剂被设计为用作轻度至中度出血中的助剂并且需要硬按压。 All existing hemostatic agent for the bleeding chamber is designed to be used in mild to moderate bleeding auxiliaries and requires hard pressed. 在开发和/或使用用于最低限度的可按压出血的组织粘合剂和封闭剂组合物中遇到的主要限制之一是它们不能在大量出血时与组织形成足够强的键合,和不能在应用10分钟内产生稳定的凝块。 One of the main limitations encountered in the tissue adhesive and sealant compositions to develop and / or use of a minimal bleeding is depressible they do not form a sufficiently strong bond, and not the tissue in heavy bleeding generating a stable clot within the application 10 min. 因此,组织粘合剂和封闭剂必须与按压方法、缝合和/或缝钉和粘合剂贴片组合使用,以便减少可接受的性能所需的组织键合强度。 Thus, tissue adhesives and sealants must, stitching and / or adhesive patch and staples used in combination with the pressing method, in order to reduce the desired properties of tissue acceptable bond strength. 然而,有许多情况下,使用强按压、缝合和/或缝钉是不希望的、不恰当的或不可能的(例如,在骨、介入放射学中)。 However, there are many cases, using a strong pressing, stitching and / or staple is undesirable, inappropriate or impossible (e.g., bone, Interventional Radiology).

[0008] 目前可选的方法:为了形成抵抗血液流动的物理屏障,粘合剂基质必须在几秒钟内形成牢固的纤维蛋白界面,使之在流动的血液中与组织键合在一起,并保留在撕裂部位以形成凝块。 [0008] The current alternative method: To form a physical barrier against the flow of blood, the adhesive matrix to form a firm fibrin must interface within a few seconds, so that blood flow in the tissue are bonded together, and retained in the tearing to form a clot. 每个产品以正方形、圆形贴附到球体、圆柱体或椎体的表示形式附着到人体组织中的能力与组合物和生产纤维蛋白的方法以及它与凝血酶的相互作用(组合)以刺激凝血级联相关。 Each product in a square, a circle is attached to the representation of a sphere, a cylinder or attached to the vertebrae in the ability of human tissue with the composition and method for producing fibrin and its interaction with thrombin (combined) to stimulate The coagulation cascade related. 该技术的重要方面是,绕过纤维蛋白原的裂解过程,以产生纤维蛋白单体和它的随后的聚合、冻干并能够吸收血液以形成纤维蛋白凝块的能力。 Important aspects of the technology is that bypassing cracking process fibrinogen to fibrin monomer and its subsequent polymerization, and capable of absorbing blood lyophilized ability to form fibrin clots. 该试剂的血块形成过程从已经稳定的I型纤维蛋白聚合物开始,I型纤维蛋白聚合物将血液吸收到含有必要组分的冻干交联聚合物以刺激凝血级联(凝血酶)并且形成物理屏障,物理屏障在应用的两分钟内转变成功能纤维蛋白凝块。 The reagent clot formation begins already stabilized fibrin polymer type I, type I fibrin polymer absorbed into the blood lyophilized crosslinked polymer containing the necessary components to stimulate the coagulation cascade (thrombin) is formed and a physical barrier, a successful transformation can be physical barrier fibrin clot within two minutes of application. (6) (6)

[0009] 在我们的方法中,这些结果通过以下步骤获得:a)应用聚合交联的冻干纤维蛋白网络,聚合交联的冻干纤维蛋白网络吸收血液以形成非常粘的凝胶状基质,凝胶状基质粘附于撕裂组织;和b)结合凝血酶层,凝血酶层有助于快速形成牢固的纤维蛋白凝块,纤维蛋白凝块由掺入在产品中的非钙依赖的转谷氨酰胺酶以及由血中的XIII因子稳定。 [0009] In our approach, the results obtained by the following steps: a lyophilized fibrin network a) cross-linking application, polymeric crosslinked absorbent lyophilized fibrin network of blood to form a highly viscous gel-like matrix, gel matrix adhered to the torn tissue; and b) a layer of bound thrombin, thrombin layer helps to quickly form a solid fibrin clot, fibrin clot by the incorporation of a non-calcium-dependent products in turn glutaminase and stabilized by factor XIII in blood.

[0010] 位于可生物降解的可去除的支持件上方的随后的层的冻干过程促进它的应用,并且允许长期贮存,运输和准备。 [0010] positioned biodegradable removable support member above the subsequent lyophilization process layers facilitate its application, and allow long-term storage, transportation, and preparation.

[0011] 此外,尚未未开发基于纤维蛋白的产品来解决阻止深伤口或大血管床皮肤伤口出血,这导致如此严重的出血而通常需要缝线或缝合。 [0011] In addition, not yet undeveloped fibrin-based products to solve or prevent large blood vessels deep wound bleeding skin wound bed, which leads to such serious bleeding usually require sutures or suture.

[0012] 组合物。 [0012] composition. 目前技术的所有形式一通过改变pH值来使酸溶液中的正方形、球体、圆柱体或圆锥体结合的纤维蛋白单体,在活化的转谷氨酰胺酶(非钙依赖)和XIII因子(钙依赖)(钙依赖和非钙依赖)以及氯化钙的存在下聚合,PH由缓冲溶液中和。 All forms of current technology by changing the pH to a fibrin monomer acid solution square, sphere, cylinder or cone bound, activated transglutaminase (non-calcium-dependent) and Factor XIII (Ca dependent) polymerization (calcium-dependent and calcium-dependent) and the presence of calcium chloride, PH, and a buffer solution. 一旦纤维蛋白聚合物形成并且交联,凝血酶的第二层即被结合并且随后冻干。 Once fibrin and crosslinked polymer formed, the second layer i.e. binding of thrombin and subsequently lyophilized. (图1) (figure 1)

[0013] 单体可以与约1%至约5%体积的甘油混合,以实现适于应用的类型的特定的粘弹性轮廓。 [0013] The monomer mixture may volume glycerol and about 1% to about 5%, to achieve a particular viscoelastic profile adapted to the type of application. 通过块状物对血液的吸收使冻干纤维蛋白变成凝胶,该凝胶在损伤(7)的位点处形成纤维蛋白凝块。 Mass absorption by blood lyophilized fibrin into a gel which is formed fibrin clot at the site of injury (7).

[0014] 在凝结剂的条件下,血液中的非钙依赖的转谷氨酰胺酶和活化XIII因子通过由共价键稳定纤维蛋白凝块而有助于该过程。 [0014] Under the condition of a coagulant, the blood calcium-dependent transglutaminase and activated factor XIII by which stabilized by covalent bonds contributes to the fibrin clot.

[0015] 关键属性。 [0015] The key attribute. 聚合/粘合。 Aggregation / adhesion. 封闭伤口的纤维蛋白凝胶是由冻干的双层材料吸收血液而形成,冻干的双层材料从固体变为凝胶状态时保持共价键。 Wound closure fibrin gel is formed by a double lyophilized blood-absorbable material, holding a covalent bond lyophilized material from a solid to a double gel state. 凝块是机械稳定的,良好地并入到伤口,并且与非交联的凝块[8]或其他纤维蛋白封闭剂相比,能更耐纤溶酶的溶解。 Clot is mechanically stable, well incorporated into the wound, as compared with the non-crosslinked clot of [8] or other fibrin sealant, can be more resistant to dissolution plasmin. 包括非钙依赖的转谷氨酰胺酶有利于转谷氨酰胺酶介导的纤维蛋白中的aC-域聚合物的交联,从而促进整合素集群,并且从而增加细胞粘附和扩散,这刺激纤维蛋白结合内皮细胞 Comprising a non-calcium-dependent transglutaminase facilitate crosslinked polymer domain aC- transglutaminase-mediated fibrin, thereby promoting integrin clustering, and thereby increase proliferation and cell adhesion, which stimulate fibrin binding of endothelial cells

[9]上的avb3-、avb5-和a5bl整合素。 [9] avb3-, avb5- a5bl and integrins on. 寡聚反应还经由黏着斑激酶(FAK)和细胞外信号调节激酶(ERK)促进整合素依赖的细胞信号传导,这导致增加的细胞粘附和细胞迀移[10]。 Oligomerization and also adjusted via extracellular signal focal adhesion kinase (of FAK) kinase (ERK) signaling promotes cell-dependent integrins, which leads to increased cell adhesion and cell Gan shift [10]. 钙离子的存在增强了从炎性反应到凝血级联(第一阶段)的进程,并且激活XIII因子。 Presence of calcium ions enhances the inflammatory reaction from the clotting cascade (first stage) of the process, and activates factor XIII.

[0016] 已经在体外和体内对活体人体组织的粘附特性和推荐试剂的聚合动力学进行了测试。 [0016] have been tested in vitro and in vivo polymerization kinetics of living human tissue adhesion characteristics and reagents recommended. 获得的数据提供ClotBlock止血的各种展示的能力的充分证据,并且以最小的按压实现实体器官或软组织中的诱发的腹腔内伤口的止血。 Sufficient evidence demonstrate the ability to provide various data obtained ClotBlock hemostasis, and hemostasis is achieved with minimum pressing intraperitoneal induced solid organ or soft tissue wounds. 并且,在猪模型中,能停止膝关节和髋关节置换中的髓内骨出血。 And, in a porcine model, you can stop bleeding intramedullary bone knee and hip replacements in. 冻干纤维蛋白II是由纤维蛋白II单体聚合获得:美国专利申请12/487057(授权)描述了制备纤维蛋白单体的方法。 Lyophilized fibrin II is obtained by polymerization of fibrin monomer II: U.S. Patent Application No. 12/487057 (authorization) describes a method of preparing a fibrin monomer. ClotBlock封闭剂组合物使用从纤维蛋白单体的中和作用中获得的冻干的纤维蛋白聚合物。 ClotBlock sealant composition obtained from using lyophilized fibrin monomer and role of fibrin polymers. 本专利申请描述的纤维蛋白II的部分的组合物和生产方法以及聚合物的中和与交联的过程对所提出的技术的性能非常重要,技术的性能取决于纤维蛋白本身的特性(纤维厚度、分支点数目、孔隙率和渗透性)以及限定凝血因子的其它聚合特性。 Compositions and Methods of Production The present patent application describes fibrin II and the process and the crosslinked polymer is important for the performance of the proposed technique, the performance characteristics of the fiber technology depends on protein itself (fiber thickness , the number of branch points, porosity and permeability) and other properties of polymeric defined clotting factors. 通过ClotBlock生成的凝块产生厚纤维的不透明基质, 并且以比透明基质更快的速度进行管形成。 Generating an opaque substrate by thick fibers ClotBlock clot generated, and a transparent matrix faster than the speed of tube formation. 凝血酶的浓度对于产生纤维蛋白单体,并且因此FPA的释放速率也对聚合过程具有重要的影响。 The concentration of thrombin to generate fibrin monomer, and thus the rate of release of FPA also has an important influence on the polymerization process. 为ClotBlock构建的所描述的浓度、稀释液和pH值以加快的速率产生最佳的纤维蛋白结构。 Concentration, dilution and pH as described ClotBlock constructed at an accelerated rate to produce the best fibrin structure. ClotBlock介绍 ClotBlock Introduction

[0017] 纤维蛋白聚合物可以以各种大小、厚度和形式生产和冻干,以适应于应用的类型(图2A、图2B、图2C)。 [0017] The fibrin polymer may be produced in various sizes and lyophilized, form and thickness to accommodate the type of application (FIGS. 2A, 2B, and 2C). 它可以配置成1/4英寸直径的小球体或圆柱体以通过腹腔镜口或在介入放射学中的血管被引入;它也可以模制成圆形或l/4、l/2al厚度的各种尺寸的正方形平坦固体块,用于脾脏裂伤或脏器切除,或放置在粘性绷带上方以覆盖深层皮肤切口。 It may be configured to 1/4 inch diameter spheres or small cylinders through the laparoscopic port or interventional radiology is introduced into a blood vessel; it can also be molded into a circular or l / 4, each of l / 2al thickness sizes square planar solid block, for spleen laceration or organ resection, or adhesive bandage is placed over to cover the deep skin incision. 冻干形式也可以浸泡在水中,并且用作封闭膏或凝胶。 Lyophilized form may also be soaked in water, and used as a paste or gel blocking.

发明内容 SUMMARY

[0018] 本发明的领域在于生物粘合剂和组织封闭剂,生物粘合剂和组织封闭剂是可生物降解和无毒的,旨在用于治疗用途,例如,作为助剂用于剖腹或腹腔镜手术的止血,或作为矫形外科、创伤(脾裂伤)、介入放射学和大血管床伤口的主要治疗。 [0018] Field of the invention to a biological tissue adhesives and sealants, adhesives and biological tissue sealant is a biodegradable and non-toxic, is intended for therapeutic use, e.g., as an additive to or laparotomy laparoscopic surgery to stop bleeding, or as orthopedic, trauma (spleen laceration), interventional radiology and macrovascular major therapeutic wound bed.

[0019] -方面,本发明涉及生物相容性粘合剂纤维蛋白聚合物,其是生物可再吸收和无毒的,用于外科或治疗用途。 [0019] - aspect, the present invention relates to a biocompatible adhesive fibrin polymer, which is bioresorbable and non-toxic, for surgical or therapeutic use. 它也涉及含有生物活性物质的双层应用,它可以在给定的位点释放以刺激凝血。 It also relates to a biologically active substance comprising a double application, which can be released in order to stimulate coagulation at a given site. 另一方面,本发明涉及一种用于生产这种粘合剂聚合物的工艺。 Another aspect, the present invention relates to a method for producing such a binder polymer processes.

[0020] 大量的体内研究表明,ClotBlock对于控制中度至重度出血是很好的止血剂候选。 [0020] a large number of in vivo studies have shown that, ClotBlock control for moderate to severe bleeding hemostatic agent is a good candidate. 它的不同的展示最大化在各种类型的外科手术和创伤应用的止血效果。 It's a different show to maximize the various types of surgery and trauma applications hemostasis. 该试剂耐用,易于储存,造成最小的风险,需少量的培训即可使用,并且是针对中度至重度出血是高效的。 The reagent durable, easy to store, causing minimal risk, require minimal training to use and is for moderate to severe bleeding is efficient.

附图说明 BRIEF DESCRIPTION

[0021] 图1示出了ClotBlock层分布。 [0021] FIG. 1 shows a distribution layer ClotBlock.

[0022] 图2示出了可能的形状:A球体和圆柱体;B贴片或块;C绷带。 [0022] FIG. 2 shows a possible form: A sphere and cylinder; patch or block B; C bandage.

[0023] 图3示出了Clotcake与Tissel、Floseal和Evicel的组织内粘附强度的比较。 [0023] FIG. 3 shows a comparison of the organization and Clotcake Tissel, Floseal Evicel and adhesion strength.

[0024] 图4示出了在ACTIVA和Activa+XIII因子存在下的纤维蛋白的聚合、交联和稳定。 [0024] FIG. 4 shows the polymerization in the presence of fibrin and ACTIVA Activa + XIII factor, and crosslinking stabilization.

[0025] 图5示出了作为肾部分切除术中的一级处理的术中出血的控制,通过应用ClotBlock,示出应用5分钟(中位值3· 2土L 4分钟)内形成固体凝块。 [0025] FIG. 5 shows a control blood loss as a partial nephrectomy of a process by the application ClotBlock, shown applied for 5 minutes (median 3.2 Soil L 4 minutes) the condensate to form a solid Piece.

[0026] 图6示出了作为无包扎或缝合的一级处理的脾破裂出血的控制。 [0026] FIG. 6 shows a control process as a non-wrapping or stapling the rupture of bleeding. 通过应用CloBlock,止血在应用5分钟内实现。 By applying CloBlock, hemostasis achieved within five minutes application.

[0027] 图7示出了作为IV级肝损伤的一级处理的术中出血的控制,通过应用ClotBlock, 止血在应用5分钟内实现。 [0027] FIG. 7 shows a process of controlling blood loss as grade IV liver injury, by applying ClotBlock, hemostasis achieved within 5 minutes in the application. 凝胶形式的ClotBlock的止血效果作为助剂止血以控制肝部分切除中的术中出血,止血在应用5分钟内实现。 ClotBlock hemostatic effect in gel form as hemostatic aids to control bleeding in partial hepatectomy, application in hemostasis achieved within 5 minutes.

[0028] 图8示出了在UV光下的显微镜检查比较应用后2周㈧与5周⑶时捕获在肾脏和肝脏的细胞间隙中的荧光素的轨迹。 [0028] FIG. 8 shows a cell trapped in the kidneys and liver clearance of fluorescein (viii) at 2 weeks and 5 weeks after ⑶ microscopy under UV light Comparison trajectory.

[0029] 图9示出了使用夹心ELISA(酶联免疫吸附测定)检测在猪中可能产生的抗凝血酶的抗体。 [0029] FIG. 9 shows an antibody using a sandwich ELISA (enzyme linked immunosorbent assay) detecting antithrombin in pigs that may arise.

[0030] 人成纤维细胞培养物暴露于ClotBlock制剂 [0030] The human fibroblast cultures were exposed to the formulation ClotBlock

[0031] a、HF-未处理 [0031] a, HF- untreated

[0032] b、HF+ClotBlock,5 天 [0032] b, HF + ClotBlock, 5 days

[0033] 图10示出了使用夹心ELISA(酶联免疫吸附测定)检测在猪中可能产生的抗凝血酶的抗体。 [0033] FIG 10 illustrates antibody using a sandwich ELISA (enzyme linked immunosorbent assay) detecting antithrombin in pigs that may arise.

[0034] 图11示出了暴露于ClotBlock制剂的人成纤维细胞,对细胞完全没有损伤或毒性,并且没有任何细菌或真菌污染;细胞显得比未处理的对照培养物稍大。 [0034] FIG. 11 shows a person exposed to the formulation ClotBlock fibroblasts, no cell damage or toxicity, and without any bacterial or fungal contamination; cells appear slightly larger than the untreated control cultures.

[0035] a、_未处理,5天 [0035] a, _ untreated, 5 days

[0036] b、成纤维细胞+ClotBlock,5 天 [0036] b, fibroblasts + ClotBlock, 5 days

[0037] 图12示出了暴露于ClotBlock制剂的人上皮细胞培养物(A549),对细胞完全没有损伤或毒性,并且没有任何细菌或真菌污染;细胞显得比未处理的对照培养物稍大。 [0037] FIG. 12 shows the effect of exposure to the formulation ClotBlock epithelial cell cultures (of A549), no cell damage or toxicity, and without any bacterial or fungal contamination; cells appear slightly larger than the untreated control cultures.

[0038] c、A549细胞-未处理,5天 [0038] c, A549 cells - untreated, 5 days

[0039] d、A549 细胞+ClotBlock,5 天 [0039] d, A549 cells + ClotBlock, 5 days

具体实施方式 detailed description

[0040] 我们已经开发出一种止血剂,可以以各种形式和支撑件成形,商标为Clotblock。 [0040] We have developed a hemostatic agent, may be shaped in a variety of forms and the support member, under the trademark Clotblock. 该试剂是新型的纤维蛋白封闭剂(纯的纤维蛋白II,由中和纤维蛋白II单体制成),该纤维蛋白封闭剂由凝血酶补充,并且设计成在严重出血的情况下促进止血,以及在最小按压的情况下停止由脏器切除、创伤和/或实体器官伤口、软组织、骨骼和大血管床的创伤造成的出血。 The agent is a novel fibrin sealant (fibrin pure II, fibrin II monomer neutralization is made), the fibrin sealant by the thrombin added, and designed to promote hemostasis in cases of severe bleeding, and stop resection, bleeding wounds and / or solid organ wounds, trauma and soft tissue, bones and large vascular bed caused by the organ with minimal compressions. 该封闭剂促进凝血,并且提供止血以及受损组织的表面之间的粘合性。 The blocking agent to promote clotting and to provide adhesion between the surface and the hemostatic damaged tissue. 本纤维蛋白封闭剂可以使用1)作为严重出血的助剂或作为一级处理;或2)成形为通过腹腔镜端口传送或者在脏器切除中用作按压,或3)放置在支撑件上用于皮肤撕裂的情况,或4)成形为在介入放射的情况下通过导管传送;或5)成形为封闭由整形外科或创伤引起的髓内出血。 This fibrin sealant can be used 1) as a processing aid severe bleeding or one; or 2) formed through a laparoscopic port or transferred as pressed against the organ resection, or 3) placed on a support member with in the case of skin tearing, or 4) is shaped in the case of transmission through a catheter interventional radiology; or 5) is shaped as a closed orthopedic intramedullary caused by trauma or hemorrhage.

[0041] 由冻干双层(图1)组成的每个呈现物包括:1)纤维蛋白II聚合物,通过乙酸溶液(pH3. 4)中的纤维蛋白单体和HEPES缓冲液(pH 8. 3)中和以及通过活化的XIII因子和非钙依赖的转谷氨酰胺酶交联而产生的;2)以每4ml纤维蛋白Iml的比例,溶解于HEPES水溶液中的浓度为20NIH单位/ml的凝血酶的层;和3)在纤维蛋白和凝血酶层之间选择添加嵌入第三层PLGA纤连蛋白的微球体。 [0041] Each presentation from a lyophilized composition bilayer (FIG. 1) is composed comprising: a) fibrin II polymer, fibrin monomer by a solution of acetic acid (pH3 4) and in HEPES buffer (pH 8. 3) neutralization and by activating the transglutaminase, factor XIII and cross-linked non-calcium dependent generated; 2) in a ratio of fibrin Iml 4ml, the concentration of HEPES was dissolved in aqueous solution is 20NIH units / ml thrombin layer; and 3) between the fibrin and thrombin select Add embedded layer a third layer of fibronectin PLGA microspheres.

[0042] 冻干双层应用于撕裂的出血组织,冻干双层吸收血液以形成粘性、胶状凝胶屏障和随后血液由纤维蛋白吸收时形成纤维蛋白凝块。 [0042] The applied bilayer lyophilized torn bleeding tissue, the blood-absorbable bilayer lyophilized to form a viscous, colloidal formed fibrin clot when the gel barrier and then absorbed by the blood fibrin. 1)该试剂在2分钟内封闭伤口,和2)使撕裂的组织结合在一起。 1) The agent in wound closure within 2 minutes, and 2) that the torn tissue together.

[0043] ClotBlock已经开发了几种剂型,根据需要在形状、粘弹性和凝血强度上改变。 [0043] ClotBlock Several formulations have been developed, changes in shape, viscoelasticity and strength required coagulation.

[0044] 组合物及应用 [0044] Compositions and Applications

[0045] ClotBlock制成二层。 [0045] ClotBlock Layer made. 它由冻干纤维蛋白聚合物和顶部的冻干凝血酶层组成。 It consists of layers lyophilized thrombin and lyophilized fibrin polymer top. 第一层含有交联的纤维蛋白聚合物,根据结合于此作为参考的美国专利申请第12/487, 057 号中描述的方法,交联的纤维蛋白聚合物由与缓冲溶液混合的乙酸溶液中的纤维蛋白单体的中和产生,缓冲溶液包括150mM NaCl、50mM HEPES、3mM CaCl2、0. 12g/mLActiva (非钙依赖的转谷氨酰胺酶)以及每毫升中和缓冲液PH8. 5的21L〇wey单位的XIII因子。 A first layer containing a crosslinked fibrin polymer according incorporated herein by reference U.S. Patent Application No. 12/487, 057 described method, the crosslinked fibrin polymer is mixed with a buffer solution of acetic acid fibrin monomers and produce, a buffer solution comprising 21L 150mM NaCl, 50mM HEPES, 3mM CaCl2,0. 12g / mLActiva (non-calcium-dependent transglutaminase) per ml and a neutralizing buffer PH8. 5 in factor XIII 〇wey units. 在无菌模具内以1 :1的比例混合这两种溶液。 Sterile mold in a 1: 1 ratio of the two solutions. 取决于块的期望的柔性,在该组合物中可以添加1% 到5 %的甘油。 Block depends on the desired flexibility in the composition may be 1% to 5% glycerin.

[0046] 这个模具被密封于无菌TYVEK® (E. I杜邦公司的注册商标,威尔明顿(DE)) 袋,并且在37°C下孵育4小时。 [0046] This mold is sealed in a sterile Tyvek® (registered trademark of E. I DuPont, Wilmington (DE)) bags, and incubated at 37 ° C 4 hours.

[0047] 第二层包含凝血酶与纤维蛋白的比例为1:4的溶液,凝血酶以20单位/mL的浓度溶解于HEPES缓冲液中。 Thrombin and fibrin contained in a proportion of [0047] the second layer is a 1: 4 solution of thrombin to a concentration of 20 units / mL dissolved in HEPES buffer.

[0048] 步骤1 :每个组分通过0. 22微米的微孔过滤器过滤来灭菌。 [0048] Step 1: Each component filter sterilized through a Millipore filter 0.22 [mu] m. 每一层倒入期望形状(圆形、椭圆形或方形)的硅模具,以产生大约3/4至1"厚的"结块",它可以由网的可去除或可生物降解的聚合物支撑,网诸如聚乳糖网,或sD,L-丙交酯聚合物合成网、聚乳酸(PLA)/聚(乙交酯-共-丙交酯)共聚物(PLGA)膜或聚乙醇酸(PGA)网;或由用于皮肤撕裂的情况的自粘绷带支撑。 Each layer was poured into a desired shape (round, oval or square) silicon mold to produce about 3/4 to 1 "thick" blocking ", which may be removed from a polymer mesh or biodegradable support, such as polyglactin mesh network, or sD, L- lactide polymer synthetic mesh, polylactic acid (PLA) / poly (glycolide - co - lactide) (PLGA) copolymer film or a polyglycolic acid ( PGA) mesh; or a case where a self-adhesive bandage support skin tearing.

[0049] 步骤2 :然后在_40°C至_50°C的冷凝器温度、21°C的搁架温度下冻干Clotblock, 在200-400毫托的压力下持续18-72小时。 [0049] Step 2: then the condenser temperature _40 ° C to _50 ° C, and lyophilized at shelf temperature Clotblock of 21 ° C, at a pressure of 200-400 milli torr duration 18-72 hours.

[0050] 步骤3 :每片ClotBlock气密密封地包装在塑料袋中,以防止水分流失并且保持无菌。 [0050] Step 3: Each tablet ClotBlock packaged in a hermetically sealed plastic bag to prevent moisture loss and maintain sterility.

[0051] 步骤4 :在伤口上直接施加适度按压1分钟至2分钟的情况下应用ClotBlock。 [0051] Step 4: Application of applying a moderately pressed ClotBlock case where one to two minutes directly on the wound. 在3分钟内在伤口上形成纤维蛋白凝块。 Forming fibrin clot in 3 minutes at the wound.

[0052] 可选的传递方法 [0052] The method of alternative delivery

[0053] ClotBlock可以成形为1/2"的直径和1"到2"长的小圆柱,这可以通过腹腔镜口传递至腔内的伤口,或通过导管来封闭出血的血管。 [0053] ClotBlock can be formed into 1/2 "diameter and 1" to 2 "long small cylinder, which can be passed through to the wound cavity laparoscopic port, or to close the bleeding vessels through a catheter.

[0054] ClotBlock可以以4:2的比例溶解于水中,以形成液体凝胶,液体凝胶可以用单个注射器应用于裂伤或伤口上。 [0054] ClotBlock may be 4: 2 ratio dissolved in water to form a liquid gel, liquid gel may be applied to a single syringe or laceration wound.

[0055] ClotBlock可以放置于自粘或非自粘合剂上方,ClotBlock是双层系统,该双层系统由柔性织物胶粘绷带和来自酸溶液中的浓缩的desAB纤维蛋白单体或纤维蛋白Π 单体的无菌生物相容的冻干desAB纤维蛋白聚合物或纤维蛋白II聚合物组合物组成, ClotBlock可以用于止血或封闭皮肤组织。 [0055] ClotBlock may be placed over the adhesive or self adhesive, ClotBlock a layer system, which layer system of a flexible fabric adhesive bandage and desAB fibrin monomer or fibrin Π from the concentrated acid solution sterile lyophilized biologically compatible monomers desAB fibrin polymer or fibrin II polymer composition, ClotBlock may be closed to stop bleeding or skin tissue. 这种传递方法尤其涉及需要通过绷带按压粘附纤维蛋白凝块来阻止大的或深的皮肤切口的出血。 This delivery method is particularly directed to the pressing need for a bandage adhesion to fibrin clots by preventing large or deep skin incision bleeding.

[0056] 实例 [0056] Examples

[0057] 止血特性已在动物研究中测试,其显示CL0TBL0CK封闭剂比其他封闭剂更强和更快的形成纤维蛋白凝块。 [0057] The hemostatic properties have been tested in animal studies, showing the formation of fibrin and clot faster CL0TBL0CK sealant stronger than the other blocking agents. 期望粘合剂粘附至撕裂组织和利用一种力量将相对的组织结合在一起,这种力量显著高于所观察到的纤维蛋白封闭剂。 Desired adhesive and adhered to the torn tissue using a force opposing the tissue together, this force is significantly higher than the fibrin sealant observed.

[0058] 以下实验室试验用动物实验交互实施(用于III级和IV肝伤口的大鼠、兔和猪模型)。 [0058] The following laboratory test animal experiments embodiment interaction (for rat liver stage III and IV wounds, rabbit and porcine model).

[0059] 1.基准制剂的离体实验:粘附和凝血性能 [0059] 1. In vitro experiments reference formulations: coagulation and adhesion properties

[0060] 我们用等长传感器在Sprague-Dawley大鼠肝组织中进行粘合和拉伸测量(组织内粘附和凝块强度)。 [0060] We isometric sensor and the measurement of tensile adhesion (adhesion and clot strength tissue) in the liver of Sprague-Dawley rats.

[0061] 拉伸测量:分离肝脏的两个最大叶。 [0061] Tensile measurements: the separation of the two largest lobe of the liver. 一个叶附接到之后将固定于等长传感器的保持器。 After a leaf attached to the holder fixed to the other length of the sensor. 另一叶放置于容器内的纱布的平坦部,容器可以逐步升高和降低,以产生与传感器的保持器中的肝片接触。 Another leaf is placed in the container a flat gauze portion, the container can be gradually raised and lowered to make contact with the sensor holder hepatica. 这两个猪肝片中均产生Icm2的损伤面积。 Both tablets are produced liver damage in the area Icm2. 将用于组织粘附的测试的制剂放置在两片之间。 Formulations for tissue adhesion test is placed between the two. 标本放置在〇克的基线压力的接触处。 The specimen is placed at the contact billion grams baseline pressure. 在不同的时间点(暴露和接触的1分钟、5分钟和10分钟),记录下需要完全分开它们的压力。 At different time points (contact exposure 1 minute, 5 minutes and 10 minutes), the records need to be completely separated from the pressure thereof. 我们比较了ClotBlock、 Tissel、Floseal和Evice的组织内粘附。 We compared the adhesion of the organization ClotBlock, Tissel, Floseal and Evice of. 图5示出了组织内粘附的结果。 FIG. 5 shows the result of tissue adhesion. ClotBlock在10分钟后产生的粘附力比Evicel强150%,并且比组织内粘附模型中的对照强800% (图3) 〇 ClotBlock adhesion after 10 minutes produced 150% stronger than Evicel, and stronger than the adhesion of the tissue model control 800% (FIG. 3) square

[0062] 2.纤维蛋白聚合的分子化学 [0062] 2. Chemistry of fibrin polymerization

[0063] 我们进行了分子化学分析以通过活化XIII因子与非Ca依赖的转谷氨酰胺酶比较纤维蛋白单体聚合(pH中和)和稳定(交联)的效率(图4)。 [0063] We performed chemical analysis of molecules in fibrin monomer by comparing the non-activated factor XIII transglutaminase Ca-dependent polymerization (pH neutralization) and stabilization (cross-linking) efficiency (FIG. 4).

[0064] 2. 1.研究确定ACTIVA对纤维蛋白稳定的影响 [0064] 2.1.2 Study to determine the effect on fibrin stabilized ACTIVA

[0065] 可以确认的是,FXIII在Ca2+存在下催化纤维蛋白聚合物的交联,以产生不溶性纤维蛋白凝块。 [0065] it was confirmed that, in the crosslinking of FXIII catalyzed fibrin polymer in the presence of Ca2 + to produce an insoluble fibrin clot. 然而,反应混合物中存在的非钙依赖转谷氨酰胺酶是否能催化纤维蛋白的交联是不可确认的。 However, non-calcium present in the reaction mixture depends whether the transglutaminase catalyzes the crosslinking of fibrin can not be recognized. 也不可确认是否存在非钙依赖的转谷氨酰胺酶和活化XIII因子的协同效应。 We can not confirm the existence of a synergistic effect of transglutaminase and calcium-dependent activation of Factor XIII. 为了遵循这些反应,纤维蛋白经受非钙依赖的转谷氨酰胺酶处理,首先是作为浓度依赖性反应,以及之后作为时间依赖性反应。 In order to follow these reactions, fibrin transglutaminase treatment is subjected to a non-calcium-dependent, concentration-dependent as a first reaction, and thereafter as a time-dependent reaction.

[0066] 监测浓度依赖性和时间依赖性(1分钟、5分钟、10分钟)反应,2mg/mL的酸性纤维蛋白溶液的量与不同浓度(I. 0-20.0 U/ml)的在60mM Tris缓冲液(pH8. 4)(具有2mM CaCl2)中的Activa快速混合,以实现中和。 [0066] Monitoring the concentration-dependent and time (1 minute, 5 minutes, 10 minutes) the reaction, the amount of the acidic solution fibrin 2mg / mL with various concentrations (I. 0-20.0 U / ml) in 60mM Tris buffer (pH8. 4) (having 2mM CaCl2) in Activa rapid mixing, to achieve neutralization. 通过抗纤维蛋白原抗体(I :50)使纤维蛋白可视化。 By anti-fibrinogen antibodies fiber (I: 50) to visualize fibrin. 分析比较a)通过非钙依赖的转谷氨酰胺酶在1分钟、5分钟和10分钟(图4)时的纤维蛋白和纤维蛋白原的交联以及通过非钙依赖的转谷氨酰胺酶在20U/ml、19U/ml和IU/ ml的浓度时的纤维蛋白交联。 Comparative Analysis a) by a non-calcium-dependent transglutaminase cross-linked fibrin and fibrinogen as well as by non-calcium-dependent transglutaminase (FIG. 4) at the time of 1 minute, 5 minutes and 10 minutes 20U / ml, crosslinked fibrin at a concentration of 19U / ml and IU / ml of.

[0067] 图中示出在1分钟时在非钙依赖的转谷氨酰胺酶和XIII因子存在下的纤维蛋白交联期间形成强伽马调光器。 [0067] is shown to form strong gamma dimmer during crosslinked fibrin in the presence of a non-calcium-dependent transglutaminase and Factor XIII in 1 minute. 此时伽玛调光器还不存在于纤维蛋白原的样品中。 At this time, the gamma dimmer not present in a sample fibrinogen.

[0068] 3.在动物模型中的实验 [0068] 3. In experimental animal models

[0069] 我们在猪(猪)模型中进行了腔内术中出血的研究以评估通过血液吸收的纤维蛋白凝块的形成,以及确定控制出血的能力。 [0069] In our porcine (pig) model study to evaluate the cavity bleeding by forming fibrin clot blood absorption, and determining the ability to control bleeding.

[0070] 研究目标:比较ClotBlock与腹腔镜肾部分切除术期间停止中度至严重出血的标准外科实践;并且确定止血是否是安全的,以及能否控制术中出血(最小化术中血液流失)。 [0070] research objectives: stop comparing ClotBlock during laparoscopic partial nephrectomy with standard surgical practice of moderate to severe bleeding; and determine whether it is safe to stop bleeding, and the ability to control bleeding (blood loss minimization surgery) .

[0071] 3. 1.方案:评估ClotBlock作为肾部分切除术中的一级处理的术中出血的控制。 [0071] 3. 1. Program: evaluation control ClotBlock as partial nephrectomy of a process of bleeding.

[0072] 使用六头约克郡杂交母猪,年龄2. 5个月,体重37 ± 2kg。 [0072] Using six hybridization Yorkshire sows, 2.5 months of age, weighing 37 ± 2kg. 该方案经TMCI的动物护理机构和使用委员会批准。 The program approved by TMCI Animal Care and Use Committee institutions. 动物通过开放性剖腹手术切除肾脏的25%。 Animal resection by open laparotomy 25% of the kidney. 在诱发损伤后,含有1 %甘油的组合物#1F的60CC ClotBlock双层的2. 5"直径的圆形凝块压靠在切除的实质上。 After inducing injury, containing 1% glycerol composition # 1F of 60CC ClotBlock bilayer 2.5 "diameter pressed against clot circular cut substantially.

[0073] 结果:小叶间动脉大量出血1分钟后,在应用的5分钟内*(中位数3. 2±1. 4分钟)形成固体凝块,在所有六只动物中均实现止血(图5)。 [0073] Results: After massive bleeding interlobular arteries 1 minute, 5 minutes of the application * (The median 3. 2 ± 1 4 min) to form a solid clot, all six animals in both hemostasis is achieved (FIG. 5).

[0074] *五分钟止血时间由食品和药物管理局的血液制品委员会定义为示出实现止血的功效的最大时间。 [0074] * five minutes to stop bleeding time is defined as the maximum time to achieve hemostasis shows the efficacy of blood products by the Food and Drug Administration committee.

[0075] 3. 2评估ClotBlock作为无包扎或缝合的一级处理的脾破裂出血的控制。 [0075] 3.2 Evaluation ClotBlock rupture of bleeding control without treatment as a wrapping or stapled.

[0076] 本研究的目的是确定CloBlock在创伤性脾裂伤中是否可以在应用5分钟内停止大出血。 [0076] The purpose of this study was to determine whether CloBlock bleeding can be stopped within 5 minutes Application of traumatic laceration of the spleen.

[0077] 方法:使用八头约克郡杂交母猪,年龄2. 5个月,体重37 ± 2kg。 [0077] Method: hybridization using eight Yorkshire sows, 2.5 months of age, weighing 37 ± 2kg. 该方案经动物护理机构和使用委员会批准。 The protocol was approved by the institutional animal care and use committee.

[0078] 动物在脾的横向中间部分经受1英寸切口(由11叶片手术刀锐利地造成)。 [0078] The animals subjected to one inch incision in the transverse direction intermediate portion of the spleen (caused by sharply scalpel blade 11). 在诱发损伤后,在含有1 %甘油的组合物#1F的60CC ClotBlock的2. 5"直径的圆形凝块被压靠在裂伤上持续2分钟。所有动物在应用5分钟内均实现止血(图6)。 After inducing injury, containing 1% glycerol composition # 1F of 60CC ClotBlock 2. 5 "diameter circular clot is pressed against the laceration for 2 minutes in all animals within five minutes it was applied to achieve hemostasis (Figure 6).

[0079] 结果:所有动物(η = 6)均在应用5分钟*内实现止血,中位数为3. 2 ± 1. 4分钟。 [0079] Results: All animals (η = 6) were applied to achieve hemostasis within 5 minutes *, median 3. 2 ± 1. 4 min.

[0080] *五分钟止血时间是由食品和药物管理局的血液制品委员会定义为示出实现止血的功效的最大时间。 [0080] * five minutes to stop bleeding time is defined as the maximum time to achieve hemostasis shows the efficacy of blood products by the Food and Drug Administration committee.

[0081] 3. 3.评估ClotBlock作为IV级肝损伤中的一级处理的术中出血的控制。 [0081] 3. 3. Evaluation of bleeding ClotBlock control stage IV as a liver injury process.

[0082] 十四头约克郡杂交母猪,年龄2. 5个月,体重37±2kg,随机分成3组。 [0082] fourteen hybridization Yorkshire sows, 2.5 months of age, weighing 37 ± 2kg, were randomly divided into 3 groups. 第1组(η =6)的动物通过开放剖腹经受4级肝损伤并且用40CC ClotBlock处理。 Group 1 (η = 6) animals subjected to laparotomy by opening stage 4 liver injury and treated with 40CC ClotBlock. 第2组(η = 6) 的动物经受类似的过程并且用GelFoam (辉瑞)处理;和第3组(η = 4)的动物经受类似的过程,通过缝合伤口停止出血,并且进一步用盐水溶液处理。 Group 2 (η = 6) animals and subjected to a similar process (Pfizer) was treated with Gelfoam; and Group 3 (η = 4) animals subjected to a similar process, by stitching a wound stops bleeding, and further treated with saline solution .

[0083] 在两种止血治疗组中,应用ClotBlock或Gelfoam并且对伤口按压2分钟。 [0083] In both treatment groups hemostasis, application of the wound ClotBlock or Gelfoam and pressing for 2 minutes. (图7)对于这种模型的目的,4级损伤定义为7cm长的全厚实质裂伤(由11叶片手术刀锐利地造成)。 (FIG. 7) for the purposes of this model, injury is defined as grade 4 7cm long full thickness laceration of the substance (11 caused by sharply scalpel blade). 这些损伤都与美国创伤器官损伤比例系统的外科学会一致。 These injuries are consistent with the American Society for Surgery of Trauma proportional system of organ damage.

[0084] 选择肝脏的中间中的点,用手术刀产生肝损伤。 [0084] Select an intermediate point of the liver, resulting in liver damage with a scalpel. 该位置计算为接近肝上血管和门静脉的一些分支。 The position calculation for some branch close to the liver and portal vein of a blood vessel. 用标记对该点作标记。 This point will be marked with the marker. 在诱发损伤后,无论是用Alexa荧光染料(其以PLGA膜作为支撑件)处理的贴片型的40CCClotBlock凝块,还是GelFoam的3 X 3英寸的片均靠对伤口按压2分钟。 After inducing injury, either with Alexa fluorescent dye (which is PLGA film as a support member) of the SMD process 40CCClotBlock clot, or 3 X 3 inches GelFoam sheet are pressed against the wound for 2 minutes.

[0085] 在损伤后即刻用乳酸林格氏(LR)开始液体复苏。 [0085] immediately begin resuscitation with Ringer's lactate (LR) after injury. 根据需要输注LR以可能地重新建立在受伤前的MAP的至少80%内的MAP。 LR infusion may be necessary to re-establish a MAP at least 80% MAP before the injury. 复苏持续整个观察期。 Recovery continued throughout the observation period. 在60分钟的研究结束时,记录每只动物的MP和输注的总复苏体积。 At the end of 60 minutes of research, MP and record the total recovery volume infusion of each animal.

[0086] 在完成该过程后,使第2组的所有动物(Gemfoam)安乐死。 [0086] After completion of the procedure, all the animals of the second group (Gemfoam) euthanasia. 在研究完成两周时,使第1组中的动物的一半(Clotblock,η = 3)和第3组的一半(对照盐水,η = 2)安乐死, 而在完成之后4周时使另一半(第1组η = 3与第3组η = 2)安乐死。 Upon completion of the study two weeks, so that half of the animals in Group 1 (Clotblock, η = 3) and half of the third group (control saline, η = 2) were euthanized, and that the other half at 4 weeks after the completion of ( group 1 η = 3 and group 3 η = 2) were euthanized. 实施尸检和从若干器官获取组织学样品。 Implementation and access to autopsy histology samples from several organs.

[0087] 结果测量 [0087] The result of the measurement

[0088] 主要终点:损伤后5分钟内成功实现止血的比例。 [0088] The primary endpoint: the proportion of successful hemostasis after injury to 5 minutes. 预先指定的主要终点是止血时间,止血时间定义为从应用到终止出血或从实质渗出的时间间隔。 Pre-specified primary endpoint is time to hemostasis, hemostasis is defined as the time from application to terminate bleeding or oozing from the substantial time interval. 如果初始止血后从血管鞘位点复发出血,也记录恢复完全止血所需的附加非按压的应用的时间和持续时间。 If the initial hemostatic sheath recurrent bleeding from the blood vessel site, but also additional non-recording resume complete hemostasis pressing time and duration of application required.

[0089] 次要终点:维持基线的10%内的中位数血压所需的总失血量和复苏液体量。 [0089] Secondary endpoints: maintain the total amount of blood loss and recovery of the liquid within 10% of the median baseline blood pressure required.

[0090] 结果:表1中示出了第1组和第2组中的动物(IV级损伤)的终点。 [0090] Results: Table 1 shows the end of the group 1 and group 2 animals (IV damage level) is. 用ClotBlock (第1组)与GelFoam(第2组)处理的IV级肝损伤的测量结果。 Measurement of liver injury and Gelfoam (Group 2) class IV treated with ClotBlock (Group 1). 所有值均报告为平均值土SEM All values ​​are reported as mean SEM soil

Figure CN105007841AD00101

[0092] 结论:以ClotBlock处理的所有动物在应用5分钟内实现止血。 [0092] Conclusion: All treated animals ClotBlock application in 5 minutes to achieve hemostasis. ClotBlock显著减少出血时间和失血量,并且显著提高撕裂和损伤组织之间的粘合。 ClotBlock significantly reduce the time of bleeding and blood loss, and significantly improve the adhesion between the torn and damaged tissue.

[0093] 3. 4评估放置于自粘绷带上方以用于作为一级处理控制严重皮肤出血的ClotBlock [0093] 3.4 Evaluation placed over the adhesive bandages for the control process as a severe skin ClotBlock bleeding

[0094] 十头约克郡杂交母猪,年龄2. 5个月,体重37 ± 2kg。 [0094] The ten hybridizing Yorkshire sows, 2.5 months of age, weighing 37 ± 2kg. 随机分组。 Randomized. 第1组(η = 5)的动物经受腹股沟受伤处的3"长的深层皮肤裂伤,并且用放置于自粘绷带上方的ClotBlock 处理10分钟。第2组(η = 5)的动物经受相似的过程,并且没有进行处理。 Group (η = 5) animals subjected to the first 3 "long deep laceration of the skin at the groin injury, and placed with adhesive bandages ClotBlock above for 10 minutes. Group 2 (η = 5) animals subjected to similar process and not treated.

[0095] 结果测量 [0095] The result of the measurement

[0096] 主要终点:损伤后10分钟内成功实现止血的比例。 [0096] The primary endpoint: the proportion of successful hemostasis after injury within 10 minutes. 次要终点:总失血量。 Secondary endpoints: total blood loss.

[0097] 结果:表2中示出了第1组和第2组中的动物的终点。 [0097] Results: Table 2 shows the end of the first group and a second group of animals in 2. 所有值均报告为平均值土SEM All values ​​are reported as mean SEM soil

[0098] [0098]

Figure CN105007841AD00102

[0099] 结论:用绷带形式的ClotBlock处理的所有动物在应用10分钟内实现止血。 [0099] Conclusion: All animals treated with the form of a bandage ClotBlock in applications achieve hemostasis within 10 minutes. ClotBlock显著减少出血时间和失血量,并且显著提高撕裂和损伤组织之间的粘合。 ClotBlock significantly reduce the time of bleeding and blood loss, and significantly improve the adhesion between the torn and damaged tissue.

[0100] 4.安全性研究 [0100] 4. Security Studies

[0101] 第1组(η = 6)的6只猪中的其中3只通过开腹经受4级肝损伤并且用40CC ClotBlock处理,在手术完成后2周时实施安乐死,并且在4周时使其余三只安乐死。 [0101] Group 1 (η = 6) 6 3 wherein in pigs subjected to open through stages of liver injury and treated with 4 40CC ClotBlock, euthanized at 2 weeks after completion of surgery, and the at 4 weeks The remaining three were euthanized. 类似地,两个对照(第3组)接着,并且在2周后实施安乐死,在4周后使两只动物安乐死。 Similarly, two control (Group 3) Next, and euthanized 2 weeks after 4 weeks after that the two animals were euthanized. 实施尸检和从主要器官获取组织样品。 Implementation and access to autopsy tissue samples from major organs.

[0102] 通过毒性的评估、生理的副作用、生物相容性、迟发性血肿和/或水肿的形成和免疫学风险来评价试剂的安全性。 [0102] By toxicity evaluation of physiological effects, biocompatibility, delayed hematoma and / or edema formation and immunological risks of assessing the safety of the agent. 生理和病理观察包括:死亡率/发病率;体重、食物消耗、器官重量。 Physiological and pathological observations included: mortality / morbidity; body weight, food consumption and organ weights.

[0103] 4. 1急件毒件由尸检时的宏观评价和组织学研究来评估。 [0103] 4.1 Dispatch toxicity evaluation items are evaluated by macroscopic and histological study at necropsy. 通过寻找急性和/或慢性炎症的证据作为信号或组织学刺激来评价之前接触过此试剂的组织和血管组织的迴邀。 By looking acute and / or chronic inflammation evidence of contact with tissue and vascular tissue invited back to this as a signal reagent prior to stimulation or histological evaluation. 对所有器官评价血栓形成、痿管及脓肿形成。 Evaluation of all organs thrombosis, fistula and abscess formation.

[0104] 4. 2迟发件血肿的评价:包腊下或实质血肿形成的风险。 [0104] 4.2 Evaluation of delayed hematoma member: the packet wax or substantial risk of hematoma formation. 在应用后21天时肉眼和组织学观察到迟发性血肿及水肿形成。 After application 21 days was observed visually and histologically delayed hematoma and edema formation. 小血肿形成定义为可见或<直径4cm的可触及肿块而不伴相关的转归。 Small hematoma formation defined as visible or <4cm diameter without a palpable mass associated with outcome.

[0105] 4. 3.病理学与组织学 [0105] 4. 3. Histology and Pathology

[0106] 我们分析了肺、肾、肝、脾在手术的2周和4周后均经过处理的组织学损伤,并且比较了用ClotBlock处理的样本与对照(用生理盐水溶液处理)的炎症数据,炎症包括凋亡和白细胞浸润。 [0106] We analyzed the lung, kidney, liver and spleen at 2 weeks and 4 weeks were treated histological damage surgery, and compared inflammation data samples and control ClotBlock treatment (solution treated with saline), and inflammation including apoptosis and leukocyte infiltration. 也组织学评价炎症和水肿形成。 Histological evaluation also inflammation and edema formation.

[0107] -旦处死动物,收集器官,固定于10%福尔马林中并且包埋于石蜡块中。 [0107] - Once the animals were sacrificed, organs were collected, fixed in 10% formalin and embedded in paraffin blocks. 组织学切片用苏木精和曙红染色并且在光学和显微镜中的IOOX和400X检查。 Histological sections were stained with hematoxylin and eosin and the optical microscope and checking IOOX and 400X. 这些载玻片通过委员会认证的兽医病理学家进行评估,如表3所示。 These slides were evaluated by a board-certified veterinary pathologist, as shown in Table 3.

[0108] 结果: [0108] Results:

Figure CN105007841AD00111

Figure CN105007841AD00121

[0111] 形杰学诊断的总结: [0111] Jay-shaped diagnosis summary:

[0112] 心脏: [0112] Heart:

[0113] 无显著发现 [0113] found no significant

[0114] 肺: [0114] Lungs:

[0115] 1.肺不张,轻至中度,多灶性 [0115] 1. atelectasis, mild to moderate, multifocal

[0116] 2.水肿,最低至轻度,急性,弥漫 [0116] 2. edema, the minimum to mild, acute, diffuse

[0117] 3.血红蛋白晶体,最小的,病灶,伴轻度中性粒细胞炎症 [0117] 3. hemoglobin crystals, the smallest lesions, with mild neutrophilic inflammation

[0118] 肾脏: [01] Kidney:

[0119] 1.颗粒管型,管内的,最小的,多灶性 [0119] 1. The particle tube, the inner tube, the smallest, multifocal

[0120] 2.透明水滴,卵胞浆内,近端肾小管上皮细胞,最低至轻度,多灶性 [0120] 2. transparent droplets, intracytoplasmic, proximal tubular epithelial cells, low to mild, multifocal

[0121] 腹壁: [0121] abdominal wall:

[0122] 纤维束,病灶,伴有中央腔,轻度至中度肉芽肿,中性粒细胞,毛干淋巴浆细胞炎症,和中度水肿 [0122] fiber bundle, lesions, with a central lumen, mild to moderate granulomatous, neutrophils, lymphoplasmacytic inflammation hair shaft, and moderate edema

[0123] 表4中总结了病理学发现。 [0123] Table 4 summarizes the pathological findings.

[0124] [0124]

Figure CN105007841AD00131

Figure CN105007841AD00141

[0126] 结论:对照(缝合)和非缝合处理的动物在腹壁损伤位点处均含有轻度慢性炎症和纤维化。 [0126] Conclusion: control animals (stapling) and a non-stapling process at the site of injury in the abdominal wall contain mild chronic inflammation and fibrosis. 仅处理的动物在腹壁损伤位点、肝损伤位点和晶种的邻接腹膜表面处具有与异物(ClotBlock)相关的肉芽肿性炎症的迹象。 Only animals treated site of injury in the abdominal wall, liver injury site and the seed with peritoneal signs abutment surface foreign matter (ClotBlock) associated with granulomatous inflammation. 虽然处理的动物没有在任一临床部位表现出显著出血的组织学迹象,但是缝合的对照呈现出严重的腹膜炎和包膜下肝炎以及伴有坏死和纤维化的血肿的迹象。 Although the animal is not treated in any part of a clinical histology showed signs of significant bleeding, but stitched control showing severe peritonitis and subcapsular hepatitis with necrosis and fibrosis, as well as signs of hematoma. 在任何其他器官中没有血栓栓塞迹象。 There is no evidence of thrombus embolism in any other organs. 对照动物在脾中具有髓外造血迹象,解释为对先前手术损伤的出血的响应。 Control animals had evidence of extramedullary hematopoiesis in the spleen, previously interpreted as a response to the surgical trauma bleeding. 在任一动物中未看到提示显著失血性贫血的改变。 No animals in either a change prompted significant blood loss anemia.

[0127] 所有的猪(处理与对照组)当受伤和治疗或缝合时产生粘连。 [0127] All pigs (treatment and control) is generated when the blocking and treatment of injury or stitching. 因此粘连不是由ClotBlock造成的,尽管作为其他纤维蛋白封闭剂的ClotBlock可能是促进因素。 Therefore, adhesion is not caused by ClotBlock, although as ClotBlock other fibrin sealant may be a contributing factor.

[0128] 4. 4.通过生物降解研究的试剂的药物动力学曲线。 [0128] 4.4.3 Study of degradation by biological agents pharmacokinetic profile. 在体内确定通过蛋白水解酶生物降解的消除。 In vivo determination of the proteolytic enzyme eliminated by biodegradation.

[0129] 方法:为了检查体内的ClotFoam的结局,使用荧光标记的人纤维蛋白原作为示踪剂制备了一批ClotBlock。 [0129] Method: To examine the outcome ClotFoam vivo, using fluorescently labeled human fibrinogen was prepared as a tracer ClotBlock number. 该ClotBlock制剂应用于IV级肝伤口过程(4.3)中的第1组的六只动物,并且在应用后2周(η = 3)和4周(η = 3)使动物安乐死。 The ClotBlock formulation to the wound during liver stage IV (4.3) six animals in the first group, and two weeks after application (η = 3) and 4 weeks (η = 3) animals were euthanized. 一旦处死动物,收集器官,固定于10%福尔马林并且包埋于石蜡块中。 Once the animals were sacrificed, organs were collected, fixed in 10% formalin and embedded in paraffin blocks. 组织学切片在荧光显微镜中以IOOX和400Χ检查。 Histological sections in fluorescence microscopy to IOOX and 400Χ inspection. ClotBlock的消除由样品中完全不存在荧光踪迹或通过2周和4周时观察到的荧光水平来确定。 ClotBlock eliminate traces level of fluorescence or fluorescence observed by 2 weeks and 4 weeks complete absence in a sample is determined.

[0130] 结果:在应用4周内在所有器官中消除了Clotblock(图8)。 [0130] Results: Application of 4 weeks to eliminate the Clotblock (FIG. 8) in all organs.

[0131] 4. 5.评估免疫应答 [0131] 4. 5. Evaluation of immune responses

[0132] 评估对ClotBlock的潜在抗体应答。 [0132] evaluation of the antibody response to the potential of ClotBlock.

[0133] 方法:从经受IV级肝损伤(4. 3)的实验动物收集血清样品,收集于术后的处理前与处理后的0天、7天和21天,并且冷冻贮存在-20°c直至分析。 [0133] Methods: Serum samples were collected from the experimental animals subjected to liver damage stage IV (4.3), and collected in the process with day 0, 7 and 21 days after the pre-treatment, and stored frozen at -20 ° c until analysis. 通过酶联免疫吸附试验(ELISA)检测对用于ClotBlock的配制的组分所生成的抗体。 The components for detecting antibody ClotBlock formulated generated by enzyme linked immunosorbent assay (ELISA).

[0134] 为了检测在猪中可能产生的针对ClotBlock的组分的抗体,构建了夹心ELISA (酶联免疫吸附测定)。 [0134] In order for an antibody component ClotBlock in pigs of detecting possible to construct sandwich ELISA (enzyme linked immunosorbent assay). 96孔微量滴定板的底面在4°C下用纤维蛋白(在PBS中10mg/ml,pH值7.0, Sigma-Aldrich 公司)或凝血酶(在PBS 中nlOmg/ml 中PBS,pH 值7.0, Sigma-Aldrich 公司)包被过夜。 The bottom surface 96-well microtiter plate at 4 ° C for fibrin (10mg / ml, pH values ​​in PBS 7.0, Sigma-Aldrich Corporation) or thrombin (nlOmg / ml in PBS and in PBS, pH value 7.0, Sigma -Aldrich company) were coated overnight. 用PBS对所有孔洗涤5次。 For all wells it was washed five times with PBS. 猪血清的样品用PBS以I :20的最终稀释度应用,在室温下孵育1小时,并且用PBS洗涤5次。 I pig serum sample in a PBS: 20 final dilution of applications, incubated for 1 hour at room temperature, and washed five times with PBS. 酶(辣根过氧化物酶)连接的兔抗猪IgG抗体(Sigma-Aldrich公司)用PBS以1 :20稀释应用于所有孔,在室温下孵育1小时, 并且用PBS洗涤5次。 Rabbit anti-pig IgG antibody (Sigma-Aldrich Corporation) (horseradish peroxidase) connected with PBS 1:20 dilution applied to all wells, incubated at room temperature for 1 hour and washed 5 times with PBS. 通过将基质(2, 2' -连氮基-双(3-乙基苯并噻唑-6-磺酸)二铵盐,IOmg/胶囊,Sigma-Aldrich公司)的一个胶囊溶解于100mL磷酸盐-梓檬酸盐缓冲液(pH 5.0)以及添加H2O2(0.25ml的3%溶液)制备基质。 By the matrix (2, 2 '- azino - bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt, IOmg / capsule, Sigma-Aldrich Corporation) was dissolved in 100mL of one capsule of phosphate - Zi citric acid buffer (pH 5.0) was prepared and added H2O2 (3% solution of 0.25ml) matrix. 接着,在室温下孵育10分钟,使用BioTek的EX800酶标仪确定405nm的光密度。 Next, incubated for 10 minutes at room temperature using a BioTek plate reader EX800 determining the optical density of 405nm.

[0135] 每个目标组分用磷酸盐缓冲盐水(PBS (pH 7.4)以1:100稀释,包被于微量滴定板孔中,并且在4°C下孵育过夜。这些孔用PBS(封闭缓冲液)中的0.25% (wt/vol)脱脂奶粉/0. 2% Tween 20封闭,然后用封闭缓冲液中的1:10稀释的动物血清50yL在37°C下孵育1小时。结合的IgG和IgM作为二次抗体的标准ELISA系统进行检测。 [0135] Each of the target component with phosphate buffered saline (PBS (pH 7.4) at 1: 100 dilution in microtiter plates coated wells and incubated overnight at 4 ° C for these holes with PBS (blocking buffer. solution) 0.25% (wt / vol) skim milk powder / 0. 2% Tween 20 is closed, then 50yL at 37 ° C for incubation with animal serum diluted 1:10 in blocking buffer for 1 hour. IgG and bound normal IgM ELISA system to detect the secondary antibody.

[0136] 用5份正常动物血清来确定正常范围。 [0136] with 5 parts of normal animal serum to determine the normal range. 升高的抗体水平被定义为大于正常平均值以上的两个标准偏差。 Increased levels of antibodies is defined as greater than two standard deviations above the normal average value. 每个板包括用除了稀释血清的所有试剂孵育的孔,该孔提供了从所有结果中减去的背景吸收。 Each plate comprises all wells except reagent incubated serum dilution, which holes provide all results from subtracting the background absorption. 纯化的凝血酶的抗体由描述为凝血酶原的抗体的确定,除了纯化的凝血酶(5 μ g/mL)用于包被微孔滴定板。 Purified thrombin thrombin antibodies described by the determination of antibodies, in addition to purification of thrombin (5 μ g / mL) was used to coat microtiter plate. 抑制性抗凝血因子(anti-factor) V抗体结合于因子V C2结构域,这与出血临床表象相关。 Inhibitory anti-coagulation factor (anti-factor) V antibody binds to Factor V C2 domain, which is associated with the appearance of clinical bleeding. 人凝血因子(human factor) V抗体通过用鼠单克隆抗凝血因子V抗体6A5 (50 μ L 2. 5 μ g/mL,4°C整夜)包被微量滴定板孔识别。 Human coagulation factor (human factor) V by treatment with murine monoclonal antibody anti-factor V antibody 6A5 (50 μ L 2. 5 μ g / mL, 4 ° C overnight) were coated microtiter plate wells identified. 洗涤和封闭各孔,之后,将用50 yL 5 μ g/mL人凝血因子V孵育各孔。 Washing and blocking the wells, after which coagulation with 50 yL 5 μ g / mL human Factor V were incubated in each well. 用1:10稀释的动物血浆在37°C下孵育各孔1小时。 Animal plasma diluted 1:10 with incubation at 37 ° C for 1 hour in each well. 如上所述地检测结合的IgG(标准夹心ELISA)。 IgG binding is detected as described above (standard sandwich ELISA).

[0137] 非常重要的是要考虑到,然后提出的是在大鼠和猪中测试的对于人体成分的跨物种模型产酶系统。 [0137] It is very important to take into account, then made a cross-species enzyme production system model in rats and pigs tested for body composition. 即使动物产生针对任何ClotBlock组分的抗体,这仍不能肯定结果可以外推至人类。 Even animals produce antibodies against any ClotBlock components, it is still not certain results can be extrapolated to humans. 在临床前阶段没有同源实验系统。 No homology to the experimental system in preclinical stages.

[0138] 统计分析 [0138] Statistical Analysis

[0139] 将使用Kruskal I-Wal I is测试执行两个治疗武器内的6个随机小组之间定量变量的比较。 [0139] using Kruskal I-Wal I is comparison test performed quantitative variables between randomized groups within six two treatment arms. Mann-Whitney U测试将用于评价每个随机组之间的差异。 Mann-Whitney U test will be used to evaluate the difference between each group randomly. 统计分析采用Stata, 9. 0版本(Stata,学院站,TX)进行,P〈0. 05认为有统计学显著差异。 Statistical analysis was performed using Stata, 9. 0 version (Stata, College Station, TX) were, P <0. 05 was considered statistically significant.

[0140] 结果与结论:当针对纤维蛋白或凝血酶测试时,从对照和ClotBlock处理的猪在第0天、第7天或第21天收集的血清未观察到OD读数的显著差异(图9和10)。 Significant difference (FIG. 9 when for fibrin or thrombin test, from control and pigs ClotBlock treated on day 0, day 7 or day 21 sera collected was not observed OD readings: [0140] Results and conclusion and 10). 我们的结论是实验猪未产生可检测的抗ClotBlock抗体。 We conclude that the experimental pigs did not produce detectable antibodies anti ClotBlock.

[0141] 5.灭菌 [0141] 5. Sterilization

[0142] 对clotcake的无菌制备进行研究。 [0142] clotcake aseptically prepared for study.

[0143] 使用Nalg-Nunc公司500mL装置(目录#450-0045,硝化纤维素膜,0· 45m的过滤器)在生物安全柜中无菌过滤酸性纤维蛋白单体。 [0143] Using apparatus 500mL Nalg-Nunc Company (catalog # 450-0045, nitrocellulose membrane, 0 · 45m filter) in a biological safety cabinet sterile filtered acidic fibrin monomer.

[0144] 生长研究:一般实验方案包括制备样品溶液,随后在培养皿中的马铃薯葡萄糖琼脂(PDA,Sigma-Aldrich公司,目录号P2182)和胰蛋白酶大豆琼脂(TSA,Sigma-Aldrich公司,目录号T4536)凝胶上涂样品溶液以用于生长。 [0144] Growth studies: experimental protocol generally includes preparing a sample solution, followed by potato dextrose agar petri dishes (PDA, Sigma-Aldrich, Catalog No. p2182) and Tryptic Soy Agar (TSA, Sigma-Aldrich, Catalog No. T4536) for application of the sample was grown on a gel. 在菌落生长(模具和/或细菌)的指示时间周期孵育并观察PDA和TSA凝胶。 TSA and incubated and observed PDA gel growth (mold and / or bacteria) indicating a time period colonies.

[0145] 样品在37°C下孵育30分钟,并且用肉眼在结果和讨论部分所指示的时间周期评估菌落生长。 [0145] Samples were incubated for 30 minutes at 37 ° C, colonies were grown and evaluated in the Results and Discussion section the time period indicated by the naked eye. 该样品以二分裂或三分裂形式生长,在数据表中用1、2和3指示标示多个样本。 The samples were grown two or three divisions in the form of division, the data table designated by indicating a plurality of samples 3 and 1,2. 用于评估的评分如下: Scores used to assess the following:

[0146] 表2.菌落计数键 [0146] Table 2. Colony counts bond

[0147] [0147]

Figure CN105007841AD00161

[0148] 表3示出FIBRIN_ClotFoam的无菌组分的PDA和TSA上的微生物生长分析的研究的结果。 Growth analysis results of the study [0148] Table 3 shows the PDA FIBRIN_ClotFoam sterile component and microorganisms on TSA.

Figure CN105007841AD00162

[0150] **无菌C用于SUNY中的动物研究,在4°C下存储一周 [0150] C ** for sterile SUNY in animal studies, stored at 4 ° C for one week

[0151] 增长数据表明,无菌组分即使在11天后也没有产生显著的生长。 [0151] growth data indicate that even sterile component no significant growth after 11 days. 此外,下列技术可以用于灭菌。 In addition, the following techniques may be used for sterilization.

[0152] 6.生物相容性 [0152] 6. Biocompatibility

[0153] 两个ClottBlock制剂在无菌条件下制备和测试。 [0153] ClottBlock two formulations are prepared under sterile conditions and tested. 用人成纤维细胞(HF)和人上皮细胞(A549细胞系,ATCC)测试这些制剂的生物相容性。 Human fibroblasts (HF) and human epithelial cells (A549 cell line, ATCC) testing the biocompatibility of these formulations.

[0154] 正常人成纤维细胞(HF)由商业购买得到并且在60mm组织培养平板中以Dulbecco 改良的Eagle培养基(补充有10%胎牛血清)构建培养物,并且在潮湿的5% CO2气氛(CO2培养箱)中保持在37°C。 [0154] Normal human fibroblasts (HF) obtained from the commercially available and 60mm tissue culture plates in Dulbecco's modified Eagle medium (supplemented with 10% fetal bovine serum) culture construct, and in an atmosphere of 5% CO2 humid (CO2 incubator) maintained at 37 ° C. 人上皮细胞系A549保持在补充有10%胎牛血清和2mM谷氨酰胺的最低必需培养基中。 A549 human epithelial cell line maintained in minimal essential medium supplemented with 10% fetal bovine serum and 2mM glutamine. 当成纤维细胞和上皮细胞的培养物达到亚融合时,对照和苯甲酸钠ClotFoam制剂混合,并且立即传递到单独的皿器。 When as fibroblasts and epithelial cells subconfluent cultures reached, and sodium benzoate ClotFoam control formulation were mixed and immediately transferred to a separate and dish. 将培养物返回到CO2培养箱并且每日检查共五天。 The cultures were returned to the CO2 incubator and checked daily for five days. ClotFoam材料和介质从所有培养物中除去,并且贴壁细胞用结晶紫(0.1%,在2%乙醇中)染色。 ClotFoam materials and media was removed from all cultures, and the adherent cells with crystal violet (0.1%, 2% in ethanol) staining.

[0155] 结果:主要观察是对细胞完全没有损伤或毒性,并且没有任何细菌或真菌污染。 [0155] Results: The main observation is that no cell damage or toxicity, and without any bacterial or fungal contamination.

[0156] 图11 (a,b)中的暴露于ClotBlock制剂的人成纤维细胞培养物中,细胞比对照未处理的培养物显得稍微更大或更分散。 ClotBlock exposed to the formulation [0156] FIG. 11 (a, b) of the human fibroblast cell culture, the cells appear slightly larger than the dispersion or control untreated cultures. 并且图12中(a,b)中的在人上皮细胞中也是。 And FIG. 12 in the human (a, b) is in the epithelial cells.

[0157] 结论:ClotBlock是生物相容的,并且不抑制,而是刺激,细胞的生长和分化;这是伤口愈合剂的重要属性。 [0157] Conclusion: ClotBlock is biocompatible, and does not inhibit, but rather stimulated, cell growth and differentiation; this is an important attribute of a wound healing agent.

Claims (13)

  1. 1. 一种用于在最小按压下控制出血的纤维蛋白封闭剂组合物,所述组合物包括: a) 冻干的desAB纤维蛋白II聚合物的层,以及b) 冻干的凝血酶的层。 1. A method for controlling bleeding in a minimum of pressing the fibrin sealant composition, said composition comprising: a) a layer desAB lyophilized fibrin II polymer, and b) layer lyophilized thrombin .
  2. 2. 根据权利要求1所述的组合物,其中,通过纤维蛋白II单体和中和缓冲液的反应来制备所述纤维蛋白聚合物,以1:1的体积比加入所述中和缓冲液。 2. The composition according to claim 1, wherein said fibrin polymer is prepared by reaction of fibrin II monomer and neutralization buffer to 1: 1 volume ratio and added to the buffer .
  3. 3. 根据权利要求2所述的组合物,还包括基于体积-体积添加的约1 %至约5 %的甘油。 3. The composition according to claim 2, further comprising a volume basis - from about 1% to about 5% by volume of glycerol added.
  4. 4. 根据权利要求2所述的组合物,其中,所述中和缓冲液包括: 150mM NaCl, 50mM HEPES, 3mM CaCl2, 0. 12g/mL Activa (非妈依赖的转谷氨酰胺酶);以及每mL中和缓冲液中的21Lowey单位的XIII因子。 4. The composition according to claim 2, wherein the neutralizing buffer comprising: 150mM NaCl, 50mM HEPES, 3mM CaCl2, 0. 12g / mL Activa (non mother-dependent transglutaminase); and factor XIII per mL of buffer and 21Lowey units.
  5. 5. 根据权利要求4所述的中和缓冲液,其中,所述中和缓冲液的pH为8. 5。 According to claims 4 and the buffer, wherein, pH and the buffer is 8.5.
  6. 6. 根据权利要求2所述的组合物,其中,使用混合到所述中和缓冲液内的非钙依赖的转谷氨酰胺酶来交联所述纤维蛋白聚合物。 6. The composition according to claim 2, wherein the calcium-dependent mixed in the neutralization buffer transglutaminase to crosslink the fibrin polymer.
  7. 7. 根据权利要求1所述的组合物,其中,所述组合物的特征为是生物相容的。 7. The composition according to claim 1, wherein said composition is biocompatible.
  8. 8. 根据权利要求1所述的组合物,当将所述组合物应用于出血伤口组织时,所述组合物吸收血液从而形成凝胶,所述凝胶用于在伤口上形成稳定的纤维蛋白凝块。 8. The composition of claim 1, wherein, when the composition is applied to a bleeding wound tissue, said composition absorbs the blood to form a gel, the gel was used to form a stable fibrin in the wound clot.
  9. 9. 根据权利要求8所述的组合物,其中,在按压或不按压的情况下,在将所述组合物应用于伤口组织的一分钟内实现止血。 9. The composition of claim 8, wherein, in a case where the pressing or not pressing, to achieve hemostasis within the composition is applied to the wound tissue for one minute.
  10. 10. -种控制出血的设备,其中,根据权利要求1所述的组合物可以放置在粘合支撑件上或在没有支撑件的情况下使用。 10. The - method of controlling bleeding device, wherein composition according to claim 1, the adhesive may be placed on the support member or in the absence of the support member.
  11. 11. 根据权利要求10所述的设备,其中,所述粘合支撑件是可生物降解且去除的。 11. The apparatus of claim 10, wherein the adhesive support is removed and biodegradable.
  12. 12. 根据权利要求10所述的设备,其中,所述粘合支撑件是非可生物降解的粘合绷带。 12. The apparatus according to claim 10, wherein said support member is non-adhesion biodegradable adhesive bandage.
  13. 13. 根据权利要求所述的组合物,其中,所述纤维蛋白聚合物是在具有各种形状和尺寸的模具中冻干的。 13. The composition according to claim wherein said fibrin polymer is in various shapes and sizes of molds lyophilized.
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