Low-concentrated protein compositions for preventing tissue adhesions - Patents.com

Abstract

To provide kits comprising fibrinogen and thrombin, and uses thereof for reducing and preventing tissue adhesion.SOLUTION: A mixture comprises fibrinogen, thrombin, calcium ions, and albumin. The mixture comprises a total protein in a range of about 2.5 mg / ml to about 30 mg / ml, fibrinogen in a range of about 50 wt.% to about 80 wt.% of the total protein, and albumin in a range of more than 0.65 mg / ml to about 3 mg / ml.SELECTED DRAWING: None

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present disclosure relates to kits containing fibrinogen and thrombin and their use for reducing and preventing tissue adhesions.

[0002] (Related Technology) References that are considered relevant as background to the subject matter of this disclosure are listed below. -International Publication No. 9855140 -U.S. Patent No. 6,965,014 -U.S. Patent Application Publication No. 2002 / 001584 -U.S. Patent No. 6,613,325 -Wiseman et al., The effect of tranexamic acid in fibrin sealant on adhesion formation in the rat.J. Biomed.Mater.Res.B Appl. Biomater.(2004); 68(2): 222-30.

[0003] Acknowledgment of the above references herein does not imply that they are to be construed as being relevant in any way to the patentability of the subject matter of this disclosure. [Background technology]

[0004] Tissue adhesions following invasive procedures often result in pain, discomfort, and in some cases can even be life-threatening, and therefore the reduction or prevention of such tissue adhesions is of great importance.

[0005] WO 9855140 discloses a fibrinogen concentrate having a fibrinogen concentration range of less than 80% of total protein and having naturally occurring plasma proteins such as fibronectin, factor VIII, von Willebrand factor, factor XIII, and vitronectin at least 20% of total protein.

[0006] US Patent Application Publication No. 2002 / 001584 discloses a fabric adhesion agent that is stabilized in a liquid or frozen state and stored in a fibrinogen formulation with the addition of a chaotropic substance, and a thrombin preparation that reduces or prevents post-operative fabric adhesion disease.

[0007] U.S. Patent No. 6,965,014 discloses a biodegradable fibrin material obtained by mixing fibrinogen and thrombin, which are reconstituted or diluted in a specific calcium-free high-rigor strength medium. Such fibrin-based biomaterials produce a compact structure with fine fibers and small pore sizes suitable for use as an anti-adhesion barrier.

[0008] US Patent No. 6,613,325 discloses a fibrin polymer film formed by applying a material that most closely resembles natural clotting material to surgical adhesion formation.

[0009] Wiseman D et al. describe that fibrin containing either tranexamic acid or aprotinin reduces the incidence and severity of adhesions. Summary of the Invention [Means for solving the problem]

[0010] According to some embodiments, the present disclosure provides a kit comprising: (i) a first container containing a solution of a fibrinogen-containing component comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and having a total protein concentration range of about 15 mg / mL to about 40 mg / mL; and (ii) a second container containing a solution of a thrombin-containing component.

[0011] According to some embodiments, the present disclosure provides a mixture comprising a fibrinogen-containing component, a thrombin-containing component, calcium ions, and albumin, the mixture comprising total protein in the range of about 10 mg / mL to about 30 mg / mL, fibrinogen in the range of about 50% to about 80% of the total protein, and albumin in the range of greater than 0.65 mg / mL to about 3 mg / mL.

[0012] According to some other aspects, the present disclosure provides a hydrogel material comprising fibrin and calcium ions, wherein the total protein concentration is in the range of about 2.5 mg / mL to about 30 mg / mL, and the fibrin is present in a concentration range of 50 to 80 wt% of the total protein.

[0013] According to some further aspects, the present disclosure provides a hydrogel material comprising cross-linked fibrin obtained by applying onto tissue (i) a fibrinogen-containing solution comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and a total protein concentration range of about 15 mg / mL to about 40 mg / mL, and (ii) thrombin, under conditions that allow for the formation of a clot. In some embodiments, the fibrinogen-containing solution and thrombin are mixed before application onto the tissue. In some other embodiments, the fibrinogen-containing solution and thrombin are applied separately (sequentially or simultaneously) onto the tissue.

[0014] According to some further aspects, the present disclosure provides a two-component composition for use in preventing tissue adhesions, the two-component composition comprising: (i) component A comprising a fibrinogen-containing solution comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and a total protein concentration range of about 15 mg / mL to about 40 mg / mL; and (ii) component B comprising thrombin.

[0015] The kits, mixtures, hydrogels, and two-component compositions each are according to several embodiments for use in reducing tissue adhesions.

[0016] According to some further aspects, the present disclosure provides a method for reducing, preventing, or inhibiting tissue adhesions, the method comprising applying a fibrinogen-containing component and a thrombin-containing component to at least a portion of a tissue of a subject, the fibrinogen-containing component comprising a total protein concentration range of about 15 mg / mL to about 40 mg / mL and fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL, the amounts of fibrinogen-containing component and thrombin-containing component being effective to reduce / prevent / inhibit tissue adhesions when contacted with the tissue. DETAILED DESCRIPTION OF THE INVENTION

[0017] Tissue adhesions are a natural part of the healing process after invasive procedures. However, tissue adhesions often cause inflammation associated with pain and further serious consequences. Therefore, reducing or inhibiting adhesions after invasive procedures is highly desirable.

[0018] The present invention is based on the development of a kit that can be administered to tissue, for example, after an invasive procedure, to reduce, prevent, or inhibit tissue adhesions. The kit includes at least a fibrinogen component and, as described in more detail below, may include an additional thrombin component. Thus, the kit described herein may include two components: a fibrinogen component and a thrombin component.

[0019] Fibrinogen-based kits are known. For example, the FDA-approved kit, EVICEL®, contains a fibrinogen component containing approximately 55-85 mg / mL of fibrinogen, with 80-120 mg / mL of total protein.

[0020] The present inventors have surprisingly found that kits containing low amounts of fibrinogen, and even more surprisingly, low amounts of total protein, are effective in reducing / preventing adhesions after invasive procedures compared to known fibrinogen preparations. Specifically, as shown in the following examples, the formulations of the present disclosure reduce postoperative adhesions and affect the water-retaining capacity of the formed hydrogel. Based on this, the present inventors have suggested that the kits of the present disclosure exhibit superior anti-adhesion properties, for example, in preventing / reducing / inhibiting adhesions, while the amount of plasma components is reduced and the degree of physical barrier formed (e.g., in the form of a hydrogel) is subsequently reduced.

[0021] The kits described herein include small amounts of raw materials, specifically a solution of a fibrinogen-containing component comprising fibrinogen and having a total protein concentration of less than 40 mg / mL. Thus, in its broadest aspect, the present disclosure provides a kit comprising a container containing a solution of a fibrinogen-containing component comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and having a total protein concentration range of about 15 mg / mL to about 40 mg / mL. The kit may include an additional thrombin component.

[0022] Thus, according to some aspects, the present disclosure provides kits comprising: (i) a first container containing a solution of a fibrinogen-containing component, the solution containing fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and having a total protein concentration range of about 15 mg / mL to about 40 mg / mL; and (ii) a second container containing a solution of a thrombin-containing component. In some embodiments, the kit is an anti-adhesion kit, i.e., has adhesion prevention / reduction / inhibition properties.

[0023] As used herein and in the art, the term "fibrinogen" refers to the precursor protein of the blood clotting matrix. Fibrinogen has a molecular weight of approximately 340,000 daltons and consists of three pairs of distinct polypeptide chains, Aα, Bβ, and γ, linked together by disulfide bonds. Typically, fibrinogen has a tripartite structure with two identical D-terminal globular domains and one central E globular domain connected by a coiled-coil α-helix.

[0024] The "fibrinogen-containing component" should be understood as a formulation containing a priori fibrinogen. The fibrinogen-containing component is an aqueous formulation. An "aqueous formulation" encompasses a blend of components in liquid or frozen form that contain water molecules. In some embodiments, the aqueous formulation is in liquid form. When in liquid form, in some embodiments, the liquid carrier is a buffer having an essentially neutral pH, for example, pH 7.0±0.5. In some embodiments, the fibrinogen-containing component is frozen. Prior to use, the fibrinogen-containing component may be thawed, thereby resulting in a liquid form at room temperature. The formulation may be in the form of a solution.

[0025] The term "liquid" refers to a substance that can flow, is not of fixed shape, and is not a solid or a gas. The term "solution" refers to a dispersed or dissolved substance(s) and the medium in which it is dispersed or dissolved, or a single homogeneous liquid phase that is a mixture whose components are uniformly distributed throughout the mixture.

[0026] The term "component" relates to any ingredient that may be present in a product, such as a drug product.

[0027] In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 15 mg / mL to about 40 mg / mL. In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 30 mg / mL to about 40 mg / mL. In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 20 mg / mL to about 25 mg / mL. In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 35 mg / mL to about 40 mg / mL. In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 15 to 36 mg / mL. In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 20 to 36 mg / mL. In some embodiments, the fibrinogen-containing component comprises a total protein concentration range of about 20 to 40 mg / mL.

[0028] Total protein can be determined by any method known in the art, for example, by measuring absorbance at 280 nm (UV range).

[0029] In some embodiments, the fibrinogen-containing component comprises fibrinogen at a concentration of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 mg / mL (including any values and ranges therebetween). In some embodiments, the fibrinogen-containing component comprises fibrinogen in a concentration range of about 8 mg / mL to about 25 mg / mL. In some embodiments, the fibrinogen-containing component comprises fibrinogen in a concentration range of about 11 mg / mL to about 25 mg / mL. In some embodiments, the fibrinogen-containing component comprises fibrinogen in a concentration range of about 11 mg / mL to about 15 mg / mL. In some embodiments, the fibrinogen-containing component comprises fibrinogen in a concentration range of about 20 mg / mL to about 25 mg / mL. In some embodiments, the fibrinogen-containing component comprises fibrinogen in a concentration range of about 11 mg / mL to about 23 mg / mL, hi some embodiments, the fibrinogen-containing component comprises fibrinogen in a concentration range of about 9 mg / mL to about 14 mg / mL.

[0030] In some embodiments, the fibrinogen-containing component comprises one or more additional factors selected from, but not limited to, Factor XIII, Factor VIII, fibronectin, von Willebrand factor (vWF), and vitronectin.

[0031] In some embodiments, the fibrinogen-containing component comprises fibrinogen at a weight concentration ranging from about 50% to about 80% of total protein.

[0032] The fibrinogen in the fibrinogen-containing component can be purified and isolated from plasma. The fibrinogen-containing component can be a biologically active component (BAC), such as a fibrinogen-enriched virus-inactivated cryoprecipitate, from a solution containing human plasma. BAC includes plasma-derived proteins. The term "derived" refers to receiving from a source. For example, "derived from" refers to taking from, obtaining from, or receiving from.

[0033] In some embodiments, fibrinogen comprises plasma cryoprecipitated fibrinogen. In some embodiments, fibrinogen originates (or is derived) from plasma cryoprecipitated fibrinogen. In the context of the present disclosure, the term "cryoprecipitated fibrinogen" refers to fibrinogen obtained from frozen plasma, the latter typically being prepared from whole blood (also called fibrinogen, obtained by plasma cryoprecipitation).

[0034] In some embodiments, cryoprecipitated fibrinogen is obtained when frozen plasma is thawed at low temperatures, typically 0-4° C., resulting in the formation of a precipitate containing primarily the fibrinogen. In some embodiments, the cryoprecipitate is collected, for example by centrifugation, and then dissolved in a suitable buffer, such as a buffer containing 120 mM sodium chloride, 10 mM trisodium citrate, 120 mM glycine, or 95 mM arginine hydrochloride.

[0035] In some embodiments, cryoprecipitated fibrinogen is considered the biologically active component (BAC) of plasma. In some embodiments, the BAC is virally inactive. There are several types of BAC. In some embodiments, the BAC is a biologically active component that contains tranexamic acid as an antifibrinolytic agent.

[0036] In some embodiments, fibrinogen is provided as part of a biologically active component (BAC). Non-limiting routes for preparing BAC are described in U.S. Pat. No. 6,121,232 and / or WO 98 / 033533, the contents of which are incorporated by reference.

[0037] In some embodiments, the BAC composition comprises one or more antifibrinolytic agents (eg, tranexamic acid) and arginine hydrochloride.

[0038] In some embodiments, the concentration range of the antifibrinolytic agent, such as tranexamic acid, in the BAC ranges from about 80 to about 110 mg / mL.

[0039] In some embodiments, the fibrinogen-containing component does not contain tranexamic acid. In some embodiments, the fibrinogen-containing component does not contain aprotinin. In some embodiments, the fibrinogen-containing component is a biologically active component that does not contain tranexamic acid or aprotinin. During preparation of BAC2, plasminogen (the zymogen precursor to plasmin, which degrades fibrinogen and fibrin) and / or plasmin are removed, and therefore BAC2 does not contain tranexamic acid or aprotinin.

[0040] In some embodiments, the BAC is biologically active component 2 (BAC2), i.e., a biologically active component lacking tranexamic acid. BAC2 is a concentrated, virus-inactivated cryoprecipitate of human plasma that is primarily composed of fibrinogen (approximately 85%), depleted of plasminogen (plasminogen removal is typically performed as described in EP 1,390,485), and contains no added antifibrinolytic agents (this cryoprecipitate is typically prepared as described in EP 534,178). Given the removal of plasmin / plasminogen from the cryoprecipitate, the addition of antifibrinolytic agents such as tranexamic acid or aprotinin is not necessary.

[0041] Thus, in some embodiments, the fibrinogen component is derived from BAC2 that is plasmin(ogen) depleted and does not contain tranexamic acid or aprotinin.

[0042] In some embodiments, fibrinogen is the biologically active component of the plasma cryoprecipitate derived from antihemophilic factor preparations. Antihemophilic factors are naturally occurring proteins in blood that assist in the clotting of blood.

[0043] In some embodiments, fibrinogen is purified from aluminum hydroxide precipitate from a by-product in the manufacturing process of factor VIII (FVIII), as disclosed in WO 2013 / 001524(A1).

[0044] The BAC solution may further include stabilizers such as arginine, lysine, and other sealant additives known in the art. In some embodiments, the BAC, and preferably BAC2, may be derived from plasma cryoprecipitate (particularly concentrated cryoprecipitate).

[0045] Examples of fibrinogen sources include, but are not limited to, recombinant fibrinogen, plasma-purified fibrinogen, including the fibrinogen component of EVICEL® (i.e., BAC2), and the fibrinogen component of Tisseel (an antifibrinolytic agent containing aprotinin).

[0046] In some embodiments, the blood-derived fibrinogen concentrate is a by-product of the Factor VIII manufacturing process and may be selected from acid precipitates, cryoprecipitates, aluminum hydroxide precipitates (see, e.g., U.S. Pat. No. 4,455,300, the contents of which are incorporated herein by reference), glycine precipitates (see, e.g., U.S. Pat. No. 4,297,344, the contents of which are incorporated herein by reference), ethanol precipitates, and heparin precipitate pastes.

[0047] In some embodiments, plasma cryoprecipitated fibrinogen refers to fresh frozen plasma precipitate after centrifugation containing, but not limited to, total protein in the range of 30-60 mg / mL; total viable count (TVC) <1000 CFU / mL; factor XIII, 2-9 IU / mL; fibronectin, 0.5-6 mg / mL; and cryoprecipitated fibrinogen, 18-39 mg / mL.

[0048] In yet some other embodiments, the blood-derived fibrinogen concentrate comprises or is a fraction of suspended or precipitated Cohn Fraction I, sometimes referred to as "Paste I."

[0049] In some other embodiments, Cohn fractionation, a process that exploits differences in the isoelectric properties of various plasma proteins, involves a series of purification steps involving changes in pH, ethanol concentration, and temperature to separate proteins into five "fractions" (I-V) by precipitation. The Cohn process, also known as low-temperature ethanol precipitation, is described, for example, in U.S. Pat. No. 2,390,074 and Cohn et al. (J. Am. Chem. Soc. 68:459, 1945; J. Am. Chem. Soc. 72:465-474, 1950). In particular, in the context of the present disclosure, reference to "suspended or precipitated Cohn fraction F" should be understood to encompass not only the Cohn process referred to herein above, but also any product of ethanol fractionation by which at least fibrinogen is precipitated.

[0050] In some embodiments, plasma is subjected to ethanol concentration to obtain suspended or precipitated Cohn Fraction I. Specifically, Cohn Precipitate I (Fraction I) can be obtained from thawed pooled plasma by precipitation at, for example, 8-10% ethanol at -3°C to -5°C and neutral pH.

[0051] Fibrinogen concentrates are also commercially available. Examples of fibrinogen include, but are not limited to, the fibrinogen component of EVICEL® (i.e., BAC2) and the fibrinogen component of Tisseel (an aprotinin-containing antifibrinolytic agent).

[0052] In some embodiments, the fibrinogen-containing component is diluted BAC2. As described above, BAC2 does not contain tranexamic acid or aprotinin. Tranexamic acid and / or aprotinin have been thought to be necessary to reduce tissue anti-adhesion. Surprisingly, it has been found that effective anti-adhesion activity is observed even though the fibrinogen-containing component (i.e., diluted BAC2) does not contain tranexamic acid or aprotinin.

[0053] In some embodiments, the fibrinogen-containing component comprises BAC2 diluted at least 2-fold. In some embodiments, the fibrinogen-containing component comprises BAC2 diluted at least 2-10-fold. In some embodiments, the fibrinogen-containing component comprises BAC2 diluted 2-fold. In some embodiments, the fibrinogen-containing component comprises BAC2 diluted 4-fold. In some embodiments, the fibrinogen-containing component comprises BAC2 diluted 8-fold.

[0054] Various buffers may be used to dilute the fibrinogen-containing component. In some embodiments, the buffer comprises at least one of sodium chloride, trisodium citrate dihydrate, glycine, arginine hydrochloride, and calcium chloride dihydrate. In some embodiments, the buffer comprises about 120 mM sodium chloride, about 10 mM trisodium citrate dihydrate, about 120 mM glycine, about 9.5 mM arginine hydrochloride, and about 1 mM calcium chloride dihydrate. In some embodiments, the buffer has a pH value of 7.0 to 7.2. Exemplary buffers are shown in Table 2 below.

[0055] In some embodiments, the dilution buffer of the fibrinogen-containing component (solution) does not contain albumin.

[0056] As will be appreciated, in the absence of albumin in the dilution buffer, BAC2, the amount of albumin in the fibrinogen-containing component is determined by the dilution factor of BAC2.

[0057] In some embodiments, the fibrinogen-containing component is essentially free of albumin. In such embodiments, the thrombin-containing component may include albumin.

[0058] In some embodiments, the thrombin-containing component is essentially free of albumin. In such embodiments, the fibrinogen-containing component may include albumin.

[0059] In some such embodiments, the fibrinogen-containing component has an albumin concentration range of less than 20% by weight of total protein. In some embodiments, the fibrinogen-containing component has an albumin concentration range of less than 15% by weight of total protein. In some embodiments, the fibrinogen-containing component has an albumin concentration range of less than 10% by weight of total protein. In some embodiments, the fibrinogen-containing component has an albumin concentration range of less than 11% by weight of total protein. In some embodiments, the fibrinogen-containing component has an albumin concentration range of less than 5% by weight of total protein. In some embodiments, the fibrinogen-containing component has an albumin concentration range of about 2% to about 17% by weight of total protein, sometimes about 10% to 15% by weight of total protein, sometimes about 5% to about 15% by weight of total protein, sometimes about 8% to about 10% by weight of total protein, and sometimes about 8% to about 12% by weight of total protein.

[0060] In some embodiments, the fibrinogen-containing component is essentially free of albumin. "Essentially free of albumin" means containing albumin in an amount less than 0.65 mg / mL.

[0061] The fibrinogen-containing component may include plasma proteins other than fibrinogen and albumin. In some embodiments, plasma proteins other than fibrinogen may be present. Non-limiting examples of plasma proteins other than fibrinogen and albumin include naturally occurring plasma proteins such as fibronectin, factor VIII, von Willebrand factor, factor XIII, and vitronectin.

[0062] As described herein, the kit includes a second compartment containing thrombin. Thrombin is a mammalian serine protease that is part of the blood coagulation cascade and converts fibrinogen into insoluble chains of fibrin, as well as catalyzing other coagulation-related reactions. In the context of the present disclosure, the term "thrombin" is also meant to encompass functional analogs of thrombin. The term "functional analog of thrombin" refers to an entity that can at least cleave fibrinogen to form fibrin.

[0063] A "thrombin-containing component" is to be understood as a formulation comprising a priori thrombin and, optionally, additional proteins such as albumin. The thrombin-containing component may be in liquid or frozen form. The thrombin-containing component may be manipulated before use, for example by thawing if present in frozen form.

[0064] In some embodiments, the thrombin-containing component comprises about 100 IU / mL to about 300 IU / mL of thrombin. In some embodiments, the thrombin-containing component comprises about 100 IU / mL to about 200 IU / mL of thrombin. In some embodiments, the thrombin-containing component comprises about 150 IU / mL to about 200 IU / mL of thrombin. As used herein, the term "IU" refers to "International Units" and may be measured by a coagulation assay against an internal reference standard for potent concentration measurement, e.g., calibrated to the World Health Organization (WHO) Second International Standard for Thrombin 01 / 580. Units (U) are equivalent to International Units (IU).

[0065] In some embodiments, the thrombin-containing component comprises a diluted EVICEL® thrombin component. In some embodiments, the thrombin-containing component comprises an EVICEL® thrombin component that is diluted at least 2-fold. In some embodiments, the thrombin-containing component comprises an EVICEL® thrombin component that is diluted at least 2-10-fold. In some embodiments, the thrombin-containing component comprises an EVICEL® thrombin component that is diluted 2-fold. In some embodiments, the thrombin-containing component comprises an EVICEL® thrombin component that is diluted 4-fold. In some embodiments, the thrombin-containing component comprises an EVICEL® thrombin component that is diluted 8-fold.

[0066] Various buffers may be used to dilute the thrombin-containing component. In some embodiments, the buffer comprises at least one of sodium acetate trihydrate, D-mannitol, calcium chloride dihydrate, and sodium chloride. In some embodiments, the buffer comprises about 20 mM sodium acetate trihydrate, about 0.1 mM D-mannitol, about 40 mM calcium chloride dihydrate, and about 90 mM sodium chloride. In some embodiments, the buffer has a pH value of 6.8 to 7.2. Exemplary buffers are shown in Table 3 below.

[0067] In some embodiments, the dilution buffer of the thrombin-containing component does not contain albumin.

[0068] In some embodiments, the thrombin-containing component is essentially free of albumin. "Essentially free of albumin" means containing albumin in an amount less than 0.65 mg / mL.

[0069] As will be appreciated, in the absence of albumin in the dilution buffer, the amount of albumin in the thrombin-containing component will be determined by the dilution factor of the thrombin.

[0070] In some embodiments, the thrombin-containing component comprises albumin. In such embodiments, the thrombin-containing component may comprise albumin. In some embodiments, the thrombin-containing component comprises up to about 6 mg / mL of albumin. In some embodiments, the thrombin-containing component comprises 1 to up to about 6 mg / mL of albumin. Thrombin converts fibrinogen to fibrin, which polymerizes spontaneously. Thus, contacting the two components, the fibrinogen-containing component and the thrombin-containing component, allows for anti-adhesion activity.

[0071] The two components may be mixed prior to application to the tissue, or each one of the two components may be applied separately onto the same tissue such that the two are mixed on the tissue.

[0072] In some embodiments, the fibrinogen-containing component and the thrombin-containing component are mixed in a ratio of 0.8:1.2 to about 1:2:0.8, sometimes about 1:1. In some embodiments, the fibrinogen-containing component and the thrombin-containing component are mixed prior to application onto tissue. Mixing the two components prior to application is referred to herein as an anti-adhesion mixture. It is understood that mixing these two components forms a hydrogel (as described below) and is applied onto tissue within a time frame that allows for use, typically up to 1 or 1.5 minutes. It is also understood that this mixture is applied onto tissue to allow for clot formation.

[0073] In some embodiments, the fibrinogen-containing component and the thrombin-containing component are applied separately onto the tissue and mixed on the tissue to form a gel, and it will be appreciated that application of the two components onto the tissue allows for clot formation.

[0074] The mixture (i.e., mixed prior to application) or the formed hydrogel (i.e., upon mixing or when applied directly onto tissue) of the fibrinogen-containing component and the thrombin-containing (also referred to herein as the "composite component") comprises an albumin concentration of greater than 0.65 mg / mL to about 3 mg / mL, sometimes 1.2 mg / mL or more, sometimes 1.25 mg / mL or more, sometimes 3 mg / mL or less, sometimes 1.5 mg / mL or more, and sometimes 3 mg / mL or less.

[0075] In some embodiments, the albumin in the composite component is less than 20% by weight of the total protein in the composite component. In some embodiments, the composite fibrinogen-containing component and thrombin-containing component contain albumin equal to about 6.5% to less than 20% by weight of total protein, e.g., 6.5% to up to 17% by weight of total protein.

[0076] In some embodiments, the total albumin of the conjugated fibrinogen-containing component and the thrombin-containing component is about 1.5-3 mg / mL.

[0077] In some embodiments, the total albumin of the combined fibrinogen-containing component and thrombin-containing component is about 1.5, 2, 2.5, or 3 mg / mL (including any value and range therebetween).

[0078] In some embodiments, the total albumin of the combined fibrinogen-containing component and thrombin-containing component ranges from greater than 0.65 to 3 mg / mL.

[0079] In some embodiments, the total albumin of the combined fibrinogen-containing component and thrombin-containing component is about 0.7, 1, 1.2, 1.25, 1.5, 2, 2.5, or 3 mg / mL (including any value and range therebetween).

[0080] In some embodiments, the conjugated fibrinogen-containing component and thrombin-containing component are essentially free of albumin, where "essentially free of albumin" means containing albumin in an amount less than 0.65 mg / mL.

[0081] For composite components, whether mixed prior to application or applied directly onto tissue, cross-linking of polymerized fibrin is required to form a stable clot. This cross-linking of polymerized fibrin occurs through the action of factor XIII, which is activated by thrombin, in the presence of divalent ions, such as calcium ions. In other words, the presence of calcium ions is required for the formation of a stable clot. The fibrinogen-containing component and / or the thrombin-containing component contain free calcium ions. In some embodiments, the free calcium ions are ionized, unchelated calcium.

[0082] In some embodiments, the fibrinogen-containing component comprises solubilized calcium in a concentration range of about 0.5 to about 2 μM. In some embodiments, the fibrinogen-containing component comprises solubilized calcium in a concentration range of about 0.5 to about 1 μM. In some embodiments, the fibrinogen-containing component comprises solubilized calcium in a concentration range of about 1 to about 2 μM.

[0083] In some embodiments, the thrombin-containing component comprises solubilized calcium in a concentration range of about 30 to about 50 μM. In some embodiments, the thrombin-containing component comprises solubilized calcium in a concentration range of about 30 to about 40 μM. In some embodiments, the thrombin-containing component comprises solubilized calcium in a concentration range of about 40 to about 50 μM.

[0084] The calcium may be present in or derived from one or more calcium salts, including, but not limited to, calcium chloride, calcium acetate, calcium lactate, calcium oxalate, calcium carbonate, calcium gluconate, calcium phosphate, calcium glycerophosphate, or any combination thereof. In some embodiments, the calcium salt is calcium chloride. In some embodiments, the calcium cation is derived from calcium chloride, i.e., the formulation is prepared using calcium chloride.

[0085] In some embodiments, the calcium is unchelated calcium. As used herein, the term "chelated" or "chelation" in the context of unchelated calcium refers to the formation or presence of one or more bonds or other attractive interactions between a chelator and two or more binding sites within the calcium species. As used herein, the term "chelator" is also meant to encompass calcium precipitating agents. In some embodiments, the precipitating agent forms an insoluble salt of calcium ions. In exemplary embodiments, "unchelated calcium" is meant to refer to calcium ions present in a precipitant-free medium, such as phosphate-buffered saline (PBS). Typically, PBS comprises an aqueous saline solution containing disodium hydrogen phosphate, sodium chloride, and, in some formulations, potassium chloride and potassium dihydrogen phosphate. In exemplary embodiments, the fibrinogen and / or thrombin components are chelator-free, such as PBS.

[0086] The kit according to the present invention includes two components that are separated from each other. In other words, each of the two components is enclosed in a separate container. In some embodiments, the fibrinogen-containing component and / or the thrombin-containing component may be in the form of an applicator. In some embodiments, the applicator includes a barrel that holds the fibrinogen-containing component and / or the thrombin-containing component disclosed herein and a resealable opening for delivering the fibrinogen-containing component and / or the thrombin-containing component therethrough.

[0087] In some embodiments, the applicator is a syringe. Application may be by extruding each of the components from a syringe, e.g., mixed into a common conduit to form a composition (mixture) which is then applied directly to the tissue from the common conduit, or by extruding each of the components from a syringe directly to the desired target site or directly onto the tissue.

[0088] The fibrinogen-containing component and / or the thrombin-containing component may be in liquid, lyophilized, or dry form, the latter being to be moistened (eg, with saline) prior to application.

[0089] In some embodiments, the fibrinogen-containing component and / or the thrombin-containing component may be used, for example, by spraying or dripping, and applied to the tissue to be treated.

[0090] The applicator may be single-use, i.e., disposable after using all or part of the fibrinogen-containing component and / or thrombin-containing component, or may be designed for multiple uses, with the opening resealed between uses.

[0091] When the composite components (fibrinogen and thrombin) are brought into contact (mixed or directly applied), coagulation is initiated and a fibrin-clotting-based tissue adhesive is formed. Thus, application of a fibrinogen-containing component and a thrombin-containing component (mixed or directly applied) results in the formation of a sealant formulation. In the context of the present invention, the term "sealant formulation" should be understood as a formulation having components that react upon contact to subsequently form a tissue adhesive.

[0092] Accordingly, the present disclosure, in some embodiments, relates to a two-component composition for use in preventing tissue adhesions, the two-component composition comprising: a first component (component A) comprising a fibrinogen-containing solution, the fibrinogen concentration range being from about 5 mg / mL to about 30 mg / mL and a total protein concentration range being from about 15 mg / mL to about 40 mg / mL; and a second component (component B) comprising thrombin.

[0093] Application of the fibrinogen-containing component and thrombin-containing composition onto the target tissue (either mixed a priori, as described above, or not) results in the formation of a gel with increased viscosity compared to each of the individual components. This increased viscosity is the result of "setting" and can be achieved by interactions, including polymerization and / or crosslinking, of the protein components (e.g., fibrin) in the composition. The temperature at which the setting reaction can be carried out can be around room temperature (e.g., 15-30°C). In some embodiments, the temperature can range from a few seconds up to 600 seconds. The term "setting time" refers to the time until the end point of setting is reached.

[0094] The term "gel" refers to a substantially liquid dispersed in a solid. Typically, a gel has the properties of a solid state and, under certain conditions (e.g., temperature, pH), exhibits the properties of a liquid state. In some embodiments, the gel is a hydrogel. The term "hydrogel" is intended to refer to a hydrophilic polymer network having a water content.

[0095] In some embodiments, curing occurs spontaneously at ambient conditions.

[0096] In some embodiments, curing is achieved by one or more methods, including, but not limited to, through the use of an activator (such as a catalyst), heat, or a physical activator such as ultraviolet (UV) radiation. Accordingly, the present disclosure provides, according to some aspects, a hydrogel material comprising fibrin and calcium, wherein the total protein concentration ranges from about 2.5 mg / mL to about 30 mg / mL, and the fibrin is present in a concentration range of 50-80% by weight of the total protein.

[0097] In some embodiments, the fibrin in the hydrogel is cross-linked fibrin.

[0098] In some embodiments, the total protein concentration in the hydrogel ranges from about 7.5 mg / mL to about 20 mg / mL.

[0099] In some embodiments, the total protein concentration in the hydrogel ranges from about 5.5 mg / mL to about 10 mg / mL.

[0100] As described herein, the formed hydrogel has anti-adhesion properties.

[0101] In some embodiments, albumin is present in the hydrogel at a concentration of about 1.5 to 3 mg / mL. In some embodiments, albumin is present in the hydrogel at a concentration of at least 1.25 mg / mL and no more than 3 mg / mL. In some embodiments, albumin is present in the hydrogel at a concentration of at least 1.20 mg / mL and no more than 3 mg / mL. In some embodiments, albumin is present in the hydrogel at a concentration of greater than 0.65 mg / mL and no more than 3 mg / mL.

[0102] As shown in the following examples, water retention in hydrogels is affected by the kits of the present invention. Hydrogels formed by the kits of the present invention retain a higher percentage of water than currently available fibrin materials. A higher degree of water retention is particularly beneficial for therapeutic uses of hydrogels. Without being bound by any particular mechanism, a higher degree of water retention indicates a stronger gel or barrier structure. Such gels can retain water even when subjected to high pressure, compression, or impact stress, for example, by adjacent tissue. Water retention is beneficial for controlling the concentration of therapeutic agents that may be contained within the fibrin hydrogel and for the effective release of these therapeutic agents and additives. The ability of fibrin hydrogels to retain water while subjected to compressive forces was tested and compared to the water retention capacity of conventional fibrin materials. Specifically, compression was applied by centrifuging the material at various rotation speeds, and the amount of water retained was measured. A refrigerated centrifuge (Sorvall RT 6000 B) spun the fibrin hydrogel at various speeds.

[0103] In some embodiments, the hydrogel is characterized by retaining at least 70% of its initial water content upon application of a pressure of about 400-500 PSI onto its surface.

[0104] In some embodiments, the hydrogel is characterized by retaining at least 70%-80% of its initial water content upon application of a pressure of about 400-500 PSI onto its surface.

[0105] In some embodiments, the hydrogel is characterized by retaining at least 70% of its initial water content upon application of a pressure of about 400-500 PSI onto its surface.

[0106] In some embodiments, the hydrogel is characterized by retaining at least about 80%-95% of its initial water content upon application of a pressure of about 400-500 PSI to its surface.

[0107] According to some further aspects, the present disclosure provides a hydrogel material comprising cross-linked fibrin obtained by applying a mixture comprising (i) a fibrinogen-containing solution comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and a total protein concentration range of about 15 mg / mL to about 40 mg / mL, and (ii) thrombin onto tissue under conditions capable of forming cross-linked polymerized fibrin in / on the tissue. In some embodiments, the mixture comprises free calcium ions. In some embodiments, the mixture is obtained by combining the fibrinogen-containing solution with thrombin ex vivo.

[0108] As described above, the kit of the present disclosure has significantly improved anti-adhesion effects when applied to target tissues.

[0109] Thus, in yet another aspect, the present invention provides a method for reducing, preventing, or inhibiting adhesions in tissue, the method comprising applying a fibrinogen-containing component and a thrombin-containing component to at least a portion of a tissue of a subject, the fibrinogen-containing component comprising a total protein concentration range of about 15 mg / mL to about 40 mg / mL and fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL, the amounts of fibrinogen-containing component and thrombin-containing component being effective to reduce or prevent tissue adhesions when contacted with the tissue. As used herein, the term "effective amount" is determined by considerations known to those of skill in the art.

[0110] In some embodiments, the tissue is wounded / damaged tissue. Damaged tissue can be due to surgery, trauma, infection, or radiation. Tissue damage can be associated with any physiological process that can trigger macrophages and / or fibroblasts, for example, for the healing process.

[0111] The adhesions reduced / prevented by the kit may occur after an invasive procedure. As used herein, the term "anti-adhesion" refers to preventing or reducing adhesions in or near tissues. An anti-adhesion composition refers to a composition that can prevent or reduce adhesions, such as post-surgical adhesions, which may result from complications that appear after an invasive procedure in or near tissues.

[0112] In some embodiments of the method, an anti-adhesion agent can be applied to a target area and, optionally, used to coat an area of adhesion or potential adhesion to inhibit adhesion, thereby exerting an anti-adhesion effect. In some embodiments, the anti-adhesion agent can be applied to a target area, and other desired areas can be attached to the anti-adhesion agent and then left or pressed for a period of time.

[0113] Typically, for anti-adhesion applications at a target site, such as a surgical site, the disclosed compositions are capable of providing a durable physical barrier between different organs at the surgical site. The term "durable" in the context of anti-adhesion applications means providing such a robust physical barrier for a period of at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, or at least 14 days, for example, 1 to 14 days.

[0114] The term "adhesion" is generally applied to describe the formation of scars that extend from one tissue to another, usually across a virtual space such as the abdominal cavity. Adhesion formation after invasive procedures typically occurs when two damaged surfaces are in close proximity to each other, which can lead to inflammation and fibrin deposition on the damaged tissue. Adhesion formation can often form an interface between tissues that do not join together. While adhesions themselves are painless, adhesion-related complications can occur, causing, for example, pain and obstruction. As described above, the inventors have surprisingly found that the kit of the present invention, which contains a low amount of fibrinogen, is effective in reducing / inhibiting tissue adhesions.

[0115] The present disclosure is not limited to specific damaged / wounded tissue. Non-limiting examples of tissues may be, for example, abdominal tissue, cardiac tissue, thoracic tissue, head tissue, neck tissue, pelvic tissue, and skin tissue. In some embodiments, the present method is for reducing / preventing tissue adhesions after abdominal surgery, cardiovascular surgery, or pelvic surgery.

[0116] In some embodiments, the damaged tissue is not bleeding tissue.

[0117] In some other embodiments, the method is for preventing or reducing tissue adhesions during an invasive procedure.

[0118] The invasive procedure may, according to some embodiments, be a surgical procedure.

[0119] There are many surgical procedures in which the disclosed methods and kits can be used, including, but not limited to, abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, skin and subcutaneous tissue procedures, etc. In some embodiments, the methods are for reducing / preventing tissue adhesions after abdominal surgery, cardiovascular surgery, or pelvic surgery.

[0120] The term abdominal surgery includes surgical procedures that involve opening the abdomen (laparotomy). Non-limiting examples of abdominal surgery can include appendectomy, cesarean section, exploratory laparotomy, or laparoscopy.

[0121] The invasive procedure may, according to some other embodiments, be a diagnostic procedure.

[0122] In some embodiments, the method is for a human subject.

[0123] The fibrinogen-containing component and the thrombin-containing component may be mixed prior to application to the target tissue, or may be administered simultaneously onto the target tissue. In some embodiments, application onto the target tissue includes spraying or dripping, smearing, brushing, or injecting. The terms "inhibition," "mitigation," "reduction," "decrease," or "attenuation" or "prevention" or "diminishment," as referred to herein, should be understood to mean that a process (e.g., tissue adhesions, etc.) is associated with a phenomenon or phenotype by at least about 1% to 100%, about 5% to 95%, about 10% to 90%, about 15% to 85%, about 20% to 80%, about 25% to 75%, about 30% to 70%, about 35% to 65%, about 40% to 60%, or about 45% to 55%. Inhibition, mitigation, attenuation, restriction, prevention, blockage, reduction, decrease, or gradual decrease of a process, phenomenon, or phenotype (such as tissue adhesions) also refers to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, It may be 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or about 100%.

[0124] As used herein, the term "about" refers to values that may deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the stated value, the deviation range including integer values and, where applicable, non-integer values, constituting a continuous range.

[0125] The terms "comprises," "comprising," "includes," "including," "having," and combinations thereof, mean "including but not limited to." The term "consisting of" means "including and limited to." The term "consisting essentially of" means that a composition, method, or composition may include additional components, steps, and / or parts, but only if the additional components, steps, and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method, or composition.

[0126] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and / or to exclude the incorporation of features from other embodiments.

[0127] As used herein, the word "optionally" means "provided in some embodiments and not provided in other embodiments." Any particular embodiment of the present invention may include multiple "optional" features unless such features are inconsistent.

[0128] As used herein, the singular forms "a," "an," and "the" are intended to include plural referents unless the context clearly dictates otherwise. For example, the term "compound" or "at least one compound" may include multiple compounds, including mixtures thereof.

[0129] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the broadness of the range.

[0130] Whenever a numerical range is given herein, it is meant to include any recited number (fractional or integer) within the given range. The terms "ranging / ranges between" a first and second designator number and "ranging / ranges from" a first designator number to a second designator number are used interchangeably herein and are meant to include the first and second designator numbers and all fractional and integer numbers therebetween.

[0131] As used herein, the term "method" means methods, means, techniques, and procedures for accomplishing a given task, including, but not limited to, methods, means, techniques, and procedures that are either known or readily developed as well-known methods, means, techniques, and procedures by practitioners in the chemical, pharmacological, biological, biochemical, and medical arts.

[0132] As used herein, the term "treating" includes arresting, substantially inhibiting, slowing, or reversing the progression of the condition, substantially ameliorating the clinical or cosmetic symptoms of the condition, or substantially preventing the appearance of the clinical or cosmetic symptoms of the condition.

[0133] When notation similar to "at least one of A, B, and C, etc." is used, such syntax is generally intended in the sense that one of ordinary skill in the art would understand the notation (e.g., "a composition having at least one of A, B, and C" includes, but is not limited to, compositions having only A, only B, only C, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Those of ordinary skill in the art will further understand that whether in the specification, claims, or drawings, virtually all disjunctive words and / or phrases presenting two or more alternative terms should be understood to consider the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" would be understood to include the possibilities of "A" or "B" or "A and B."

[0134] Although certain features of the invention are described, for clarity, in the context of separate embodiments, it is understood that they may also be provided in combination in a single embodiment. Conversely, various features of the invention, while for brevity described in the context of a single embodiment, may also be suitably provided separately or in any suitable subcombination, or in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0135] Various embodiments and aspects of the present invention, as delineated hereinabove and as claimed in the claims section below, find experimental support in the following examples.

[0136] The invention will now be illustrated in the following description of experiments conducted in accordance with the present invention. It should be understood that these examples are intended to be illustrative in nature rather than limiting. Obviously, numerous modifications and variations of these examples are possible in light of the above teachings.

[0137] Non-limiting examples Example: Water retention under various conditions This study aimed to quantify the percent water retention in clots formed using various formulations and to evaluate the effect of albumin on percent water retention. Each experiment involved combining two different components (0.5 mL of each component), referred to herein as the fibrinogen component and the thrombin component.

[0138] The two components, BAC2 and thrombin (undiluted), are collectively known as the components of the product EVICEL®. Table 1 summarizes the formulations tested. Fibrinogen component and thrombin component refer to each of these components separately, while hydrogel refers to the albumin concentration (mg / mL) in the two components combined (either before or after application to tissue).

[0139] [Table 1]

[0140] In this experiment, the fibrinogen material used was an FDA-approved commercially available material designated BAC2, diluted as shown in Table 1. BAC2 (biologically active component 2) is a fibrinogen-containing product supplied by Omrix Biopharmaceuticals Ltd. BAC2 contains approximately 70 mg / mL fibrinogen and a total protein concentration of 80-120 mg / mL (approximately 100 mg / mL). For the purposes of this study, BAC-2 samples were thawed and diluted 1:4 (approximately 14 mg / mL fibrinogen) or 1:8 (approximately 8 mg / mL fibrinogen) with either an albumin-based buffer solution or an albumin-free buffer solution. These fibrinogen solutions were then diluted 1:1 with the thrombin component to obtain mixed final solutions of approximately 7 mg / mL (1:4) and 4 mg / mL (1:8). Due to the dilution with albumin-free buffer, the total protein concentrations before mixing with thrombin were approximately 20 mg / mL (1:4) and 11 mg / mL (1:8). After mixing 1:1 with thrombin, the total protein was approximately 10 and 5.5 mg / mL. Table 2 details the components of the albumin-based buffer solution and the albumin-free buffer solution, respectively, used to dilute BAC-2.

[0141] The concentration of albumin in BAC-2 is approximately 12 mg / mL, the concentration of albumin in thrombin is approximately 6 mg / mL (no albumin present in TH04), and for formulations diluted with albumin-free solution, the albumin concentrations are as shown in Table 5.

[0142] [Table 2] * Water for injection (WFI) was added instead of albumin, which is relevant for BACII.

[0143] Two different thrombin materials were used in the study. The first, designated "Thrombin," was supplied by Omrix Biopharmaceuticals Ltd. The thrombin contained 1000 IU / mL thrombin, 6 mg / mL calcium chloride, and 6 mg / mL albumin; for the purposes of this study, the solution was diluted approximately four-fold (1:4) with either an albumin-based buffer solution or an albumin-free buffer solution to obtain the final solutions detailed in Table 1.

[0144] Table 3 details the components of the albumin-based buffer solution and the albumin-free buffer solution, respectively, used to dilute the thrombin.

[0145] [Table 3] * Water for injection (WFI) was added instead of albumin.

[0146] A second source of thrombin, designated herein as "TH04," contains 100-300 IU / mL of thrombin. TH04 is a process fraction in thrombin production where the thrombin source is free of albumin and calcium ions.

[0147] To prepare a formulation that does not contain calcium ions (calcium-free), 5 mM of EDTA, a calcium chelating agent, was added.

[0148] Example 1A: Ambient Conditions The percent water retention was assessed under ambient conditions 1-2 hours after hydrogel formation (hydrogel formation was determined by visual inspection). After 1-2 hours of hydrogel formation, the surface area was approximately 1 cm 2 The amount of retained water in the hydrogel was evaluated by gently centrifuging 1 mL of the hydrogel at 630 g in a Centricon tube for 30 min at 47°C. Table 4 summarizes the results of the water retention capacity of different coagulations.

[0149] [Table 4]

[0150] Example 1B: High Pressure Immediately after hydrogel formation, the water retention rate (%) of the hydrogel was evaluated under high pressure conditions (hydrogel formation was determined by visual inspection). After hydrogel formation, 1 mL of the hydrogel was placed in an Eppendorf tube 0.7-1 cm thick. 2 The hydrogels were centrifuged at 31,514 g (approximately 457 PSI) for 30 minutes in a centrifuge to evaluate the amount of water retained in the hydrogels. Table 5 summarizes the results of the water retention capacity of the different hydrogels.

[0151] [Table 5]

[0152] The results show that a 1:1 mixture of fibrinogen (7 mg / mL and 4 mg / mL) and thrombin (diluted 1:4 and 1:8 to form a 1:1 ratio of fibrinogen and thrombin, with fibrinogen concentrations of 14 mg / mL and 8 mg / mL, respectively) can retain more water than a clot containing 70 or 73% of the total protein (total protein in the mixture is 10 and 5.5 mg / mL, respectively), and albumin greater than 0.67 mg / mL (12% of total protein), or greater than 1.2 mg / mL (12% of total protein), but less than 1.8 mg / mL (18% of total protein).

[0153] The results further show that hydrogels or clots (formed with fibrinogen diluted 1:4 and 1:8, resulting in fibrinogen concentrations of 14 mg / mL and 8 mg / mL, diluted 1:1 with thrombin) can retain the greatest amount of water when fibrinogen is 70 or 73% of the total protein, and albumin is equal to 1.25 mg / mL (6.5% of the total protein) and less than 1.8 mg / mL (18% of the total protein (Example 3)).

[0154] The results further demonstrate the importance of the presence of small amounts of albumin (e.g., less than 45.2 mg / mL, 1.2, 1.25, 1.8 mg / mL) in hydrogels prepared with low concentrations of fibrinogen (e.g., less than about 7 mg / mL (e.g., 3.9 mg / mL) of fibrinogen in a 1:1 mixture of thrombin and fibrinogen).

[0155] The results also show that the presence of EDTA (ethylenediaminetetraacetic acid) or the absence of free calcium did not alter the coagulation's ability to retain water, i.e., the trend in water loss was the same in groups 1-4 (in the presence of free calcium).

[0156] Example 3: Effects of fibrinogen and albumin in a rabbit uterine horn model The purpose of this study was to characterize the efficacy of various formulations with different concentrations of fibrinogen and albumin in a rabbit uterine horn adhesion model.

[0157] Materials and Methods Test System Juvenile / young adult (13–15 weeks old at the time of surgery) female New Zealand White rabbits (Oryctolagus cuniculus) were used in the study. Rabbits were obtained from Western Oregon Rabbit Co. (PO Box 653, Philomath, OR, USA) and individually identified by unique ear tags applied by the supplier. Immediately upon arrival at the testing facility, animals were examined by experienced animal care personnel.

[0158] Animals were acclimated for a minimum of 5 days before the start of the study and monitored daily by experienced animal care personnel. Animals supplied for this study were used only if they appeared generally normal (i.e., had a clean, lint-free coat, clear eyes, no abnormal exudates from body orifices, and exhibited an alert, active posture).

[0159] Environment and husbandry: Animals were housed individually in stainless steel cages, each labeled with an individual card indicating the study number and individual animal number. The room environment was maintained at approximately 68°F (approximately 20°C), 30-70% relative humidity, and a 12-hour / 12-hour light / dark cycle.

[0160] Purina Prolab Rabbit diet 15% protein (LabDiet, St. Louis, MO) and tap water were provided ad libitum to the animals throughout the study, and carrot and alfalfa cubes were provided.

[0161] Pre-Surgical Preparation Adhesion studies were performed using surgery. Animals were weighed on the day of surgery. Anesthesia was induced and maintained by inhalation of isoflurane (5% and 3.5% concentrations, respectively). Hair removal at the surgical site was performed using electric animal clippers. The area was vacuumed to remove clipped hair and debris, then rinsed with 70% isopropyl alcohol. The bladder was expressed (i.e., externally manipulated to expel urine). The entire area was smeared with an aqueous iodophor solution of 1% available iodine (iodine scrub). The area was then wiped with a 70% isopropyl alcohol solution.

[0162] Procedures were performed in a sterile fashion in a room reserved for sterile survival surgery. Sterile towels, drapes, and instruments were used. The anesthetized, surgically prepared rabbit was brought to the operating table and placed in the desired recumbent position with limb restraints. A sterile incise drape was applied to the prepared area. Because there is an approximate correlation between animal weight and uterine horn size, animals were assigned to surgery sequentially based on their weight to avoid surgery on animals with undersized or oversized uterine horns outside the range, and because there were two consecutive surgery days in each of two consecutive weeks. Animals were assigned to one of the study groups according to their consecutive surgery assignment.

[0163] Test System Validity: The rabbit uterine horn simple scraping model was performed essentially as described by Wiseman 1992 (Effect of thrombin-induced hemostasis on the efficacy of an absorbable adhesion barrier. The Journal of Reproductive Medicine, Vol. 37, No. 9, September 1992).

[0164] Six animals per group will be included in the study. Given the historical variance observed, these group sizes are sufficient to provide the study with sufficient power to detect statistical differences between treatment and control groups.

[0165] This model has been widely used to determine the efficacy of putative anti-adhesion agents, with good correlation between animal and clinically obtained data (Wiseman, 1999 Effect of different barriers of oxidized regenerated cellulose (ORC) on cecal and sidewall adhesions in the presence and absence of bleeding. Journal of Investigative Surgery 12:141-146).

[0166] The 42 consecutively assigned animals were randomized by lottery drawing by an unrelated individual into seven blocks, with each block representing one of the study groups.

[0167] The rabbit uterine horn plain scrape model was performed as described by Wiseman et al. (1992).

[0168] Intraoperative procedure: The abdomen was entered through a midline laparotomy incision approximately 6 cm in length. The bladder and uterus were incised.

[0169] Only animals with two uterine horns measuring 9 or more and 16 French or less in diameter were included in the study. Using a No. 10 scalpel blade, 5 cm long uterine horns approximately 1 cm from the uterine bifurcation were scraped 40 times on each side. Hemostasis was controlled by tamponade with gauze. Small blood vessels were ligated as necessary. Organs were anatomically repositioned. During the procedure, animals were carefully observed, and any animals with unexpected responses to the anesthetic procedure were excluded. Controls consisted of animals that underwent all surgical procedures but without the administration of test materials.

[0170] Tested formulations were used in 5 mL vials of each component per animal and thawed at room temperature prior to induction of anesthesia. Each product was handled according to the manufacturer's instructions and delivered by spraying using an Omrix® Application airless sprayer. A sufficient amount of product was applied to create a thin film covering the traumatized surface. The applied volume was recorded.

[0171] Postoperative Procedures The abdominal incision was closed using continuous Vicryl 4-0 sutures. The fascia was closed loosely with 4-0 Vicryl and the skin was closed using subcutaneous sutures with unstained 4-0 Vicryl (cutting needle).

[0172] Three doses of buprenorphine (Buprenex) (0.03 mg / kg, 0.4 mL x 0.3 mg / mL) were administered by subcutaneous injection: one on the morning of surgery, one 6-8 hours later, and one the following morning. Additional doses were given if deemed necessary by the veterinarian.

[0173] Clinical signs in animals: All animals were carefully observed until they resumed lying upright when returned to their cages. During this initial postoperative period, rabbits were placed under a heat lamp or on a heating blanket.

[0174] Following surgery, each rabbit was observed daily to determine its health status based on general posture and appearance, food intake, weight loss, fecal and urinary output, and the presence of any abnormal fecal matter. Animals were observed daily for incision integrity, excessive bruising and infection, and assessment of pain and / or discomfort, and were given analgesics in addition to the standard doses listed above. If deemed necessary by the attending veterinarian, animals were euthanized and excluded from analysis.

[0175] Daily monitoring records were maintained. Any abnormal findings were reported to the study supervisor and / or veterinarian, who made a decision to treat the animal or, in the case of moribund animals, to euthanize them. Moribund animals were euthanized and recorded.

[0176] Animal caretakers were blinded to group assignment.

[0177] Detection of adhesions and adhesion strength: Adhesion assessment was performed 11 to 16 days after surgery, and animals were euthanized by intravenous injection of 1.5 mL of Beuthanasia-D (pentobarbital sodium 390 mg / mL; phenytoin sodium 50 mg / mL) (Merck Animal Health, Madison, NJ). Animal weights were recorded.

[0178] The abdomen was opened and the surgical site was examined. Adhesion was graded by a blinded observer.

[0179] Tests conducted Degree and incidence of adhesions: The length of each uterine horn with adhesions is estimated. Results are expressed as the incidence of adhesions (number of horns with adhesions / total number) and the degree of adhesions (% length of horn with adhesions).

[0180] Adhesion Severity: Adhesion severity is graded as 0 (none), 1 (membranous adhesions), 2 (strong adhesions, requiring sharp dissection).

[0181] Degree of uterine rotation: The degree of uterine rotation, which is a measure of the anatomical distortion of the organs due to adhesions, is recorded according to the following scale: No rotation: The linear length of the attached or unattached uterine horns is clearly visible. Partial rotation: The uterine horns are convoluted, intertwining 50% to 75% of their length and preventing the identification of straight segments. Completely rotated uterus: The horns are completely interdigitated, making it impossible to see the uterine anatomy.

[0182] Data analysis of adhesions: For each animal, the mean extent of adhesions in the two uterine horns is calculated. This mean is used to calculate the mean extent of adhesions (percent of uterine length involved) for the group (±SEM) and is expressed to one decimal point. Comparison of all groups with the control group (no treatment) is performed using Dunnett's test (Dunnett, 1964). The incidence of adhesions in each group is compared with the control group using Fisher's exact test, and adhesion strength and degree of uterine rotation are compared using chi-squared tests. For all tests, the level of statistical significance is p<0.05.

[0183] Exclusion from analysis: Upon examination of the animal at necropsy, but prior to examination of the surgical site and assessment of adhesions, a decision is made as to whether the animal should be excluded from the primary analysis. Animals should be excluded from the primary analysis if there are signs of abnormal development that may affect the results. Such signs usually include the presence of intraperitoneal infection or excessive weight loss (>10% of body weight). Any decision to exclude an animal is made without knowledge of group assignment or the presence or extent of adhesions.

[0184] Table 6 below summarizes the experimental details and Table 7 below provides a summary of the results regarding the evaluation of the effect of fibrin-based formulations on adhesion formation.

[0185] [Table 6]

[0186] [Table 7] 1 % length of uterine horn with adhesions, average of right and left uterine horns 2 Student's t-test p-values versus control 3. % of uterine horns free of adhesions (number of uterine horns free of adhesions / total number) 4 Number of uterine horns with no adhesions / grade 1 adhesions / grade 2 adhesions 5 Number of uterine horns with no rotation / partial rotation / complete rotation * p<0.05 Dunnett's t-test vs. control # p-value χ2 test vs. control). Only values <0.1 are shown.

[0187] Improved anti-adhesion activity was observed in Experiment 3, indicating that the kit described herein has superior anti-adhesion activity.

[0188] Tables 8 and 9 show summary individual data statistics for the 6-8 samples used in the anti-adhesion experiments tested for each composition (fibrinogen component - Table 8; and fibrinogen and thrombin in Table 9).

[0189] [Table 8] * FGBACII = fibrinogen in BACII ** FG Pure Fibrinogen = Fibrinogen in Pure Fibrinogen *** adhesion%

[0190] [Table 9]

[0191] The most effective adhesion prevention effect was obtained in Samples 3 and 4. The total protein in Samples 3 and 4 was approximately 18 and 10 mg / mL, respectively, with fibrinogen accounting for 67% and 55% of the total protein, and albumin accounting for 3 mg / mL (16.7% of the total protein) and 1.5 mg / mL (15% of the total protein).

[0192] Comparative statistical analysis further confirmed that the highest anti-adhesion activity was observed in experiment 3 when the adhesion extent threshold was set at >35%, with no samples exceeding the threshold.

[0193] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0194] [Embodiment] (1) A kit comprising: (i) a first container containing a solution of a fibrinogen-containing component comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and having a total protein concentration range of about 15 mg / mL to about 40 mg / mL; (ii) a second container containing a solution of a thrombin-containing component. (2) The kit according to embodiment 1, wherein the fibrinogen-containing component does not include tranexamic acid or aprotinin. (3) The kit according to embodiment 1 or 2, wherein the fibrinogen-containing component comprises total protein in a concentration range of about 20 mg / mL to about 40 mg / mL. (4) The kit according to any one of embodiments 1 to 3, wherein the fibrinogen-containing component contains fibrinogen in a concentration range of about 8 mg / mL to about 25 mg / mL. (5) A kit according to any one of embodiments 1 to 4, wherein the fibrinogen-containing component comprises fibrinogen in a concentration range of about 10 mg / mL to about 25 mg / mL and a total protein concentration of about 20 mg / mL to about 40 mg / mL.

[0195] (6) The kit according to any one of embodiments 1 to 5, wherein the thrombin-containing component contains thrombin in a concentration range of about 100 IU / mL to about 300 IU / mL. (7) The kit according to any one of embodiments 1 to 6, wherein the fibrinogen-containing component contains albumin in a concentration range of less than 20% by weight. (8) The kit according to any one of embodiments 1 to 7, wherein the fibrinogen-containing component and / or the thrombin-containing component contains free calcium ions. (9) The kit according to any one of the preceding embodiments, wherein the fibrinogen is obtained by plasma cryoprecipitation. (10) The kit according to any one of embodiments 1 to 8, wherein the fibrinogen is derived from biologically active component 2 (BAC2).

[0196] (11) The kit according to any one of embodiments 1 to 9, wherein the fibrinogen is a biologically active component of a plasma cryoprecipitate derived from an antihemophilic factor preparation. (12) The kit according to any one of embodiments 1 to 11, wherein the fibrinogen-containing component and / or the thrombin-containing component are in liquid or frozen form. (13) The kit according to any one of embodiments 1 to 12, comprising instructions for use in preventing / reducing tissue adhesions. (14) A kit according to any one of embodiments 1 to 12 for use in the preparation of an anti-adhesion mixture. (15) A kit according to any one of embodiments 13 and 14 for use in preventing / reducing tissue adhesions after invasive procedures.

[0197] (16) The kit of embodiment 15, wherein the invasive procedure is a surgical procedure or a diagnostic procedure. (17) The kit according to any one of embodiments 1 to 16, wherein the solution is administered by spraying or dropping onto tissue. (18) The kit according to any one of embodiments 1 to 17, wherein the first container and / or the second container is an applicator. (19) The kit according to embodiment 18, wherein the applicator is a syringe. (20) A mixture comprising fibrinogen, thrombin, calcium ions, and albumin, the mixture comprising total protein in the range of about 2.5 mg / mL to about 30 mg / mL, fibrinogen in the range of about 50% to about 80% by weight of the total protein, and albumin in the range of more than 0.65 mg / mL to about 3 mg / mL.

[0198] (21) The mixture of embodiment 20, wherein the albumin is in the range of about 1.20 mg / mL to about 3 mg / mL. (22) A hydrogel material comprising fibrin and calcium ions, wherein the total protein concentration is in the range of about 2.5 mg / mL to about 30 mg / mL, and the fibrin is present in a concentration range of 50 to 80 wt% of the total protein. 23. The hydrogel material of claim 22, further comprising albumin in the range of greater than 0.65 mg / mL to about 3 mg / mL. (24) The hydrogel material of embodiment 22 or 23, wherein the albumin is in the range of about 1.2 mg / mL to about 3 mg / mL. (25) The hydrogel material according to any one of embodiments 22 to 24, wherein the total protein concentration range is from about 7.5 mg / mL to about 20 mg / mL.

[0199] (26) The hydrogel material of embodiment 22 or 25, wherein the fibrin is cross-linked fibrin. (27) The hydrogel material according to any one of embodiments 22 to 26, having anti-adhesion properties. (28) A hydrogel material comprising cross-linked fibrin obtained by applying, under conditions that allow the formation of a hydrogel, a mixture comprising: (i) a fibrinogen-containing solution comprising fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and a total protein concentration range of about 15 mg / mL to about 40 mg / mL, wherein the fibrinogen accounts for about 50% by weight to about 80% by weight of the total protein; and (ii) a thrombin-containing solution onto tissue. 29. The hydrogel material of claim 28, wherein the mixture is obtained by combining the fibrinogen-containing solution and the thrombin-containing solution ex vivo. (30) A two-component composition for use in preventing tissue adhesions, comprising: A two-component composition comprising: component A, which contains a fibrinogen-containing solution containing fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL and a total protein concentration range of about 15 mg / mL to about 40 mg / mL, wherein the fibrinogen accounts for about 50% to about 80% by weight of the total protein; and component B, which contains thrombin.

[0200] (31) The two-component composition of embodiment 30, wherein the fibrinogen-containing solution comprises fibrinogen in a concentration range of about 10 mg / mL to about 25 mg / mL and a total protein concentration in a range of about 20 mg / mL to about 40 mg / mL. (32) The two-component composition according to any one of the preceding claims, wherein component B comprises thrombin in a concentration range of about 100 IU / mL to about 300 IU / mL. (33) The two-component composition according to any one of embodiments 30 to 32, wherein component A and / or component B comprises free calcium ions. (34) A method for preventing or reducing tissue adhesions, the method comprising applying a fibrinogen-containing component and a thrombin-containing component to at least a portion of the tissue of a subject, the fibrinogen-containing component comprising a total protein concentration in the range of about 15 mg / mL to about 40 mg / mL and fibrinogen in a concentration range of about 5 mg / mL to about 30 mg / mL, and the fibrinogen is in the range of about 50% to about 80% by weight of the total protein. (35) The method of embodiment 34, comprising simultaneously applying the fibrinogen-containing component and the thrombin-containing component to at least a portion of the tissue of the subject.

[0201] (36) The method of any one of embodiments 34 and 35, comprising combining the fibrinogen-containing component and the thrombin-containing component ex vivo prior to said application to said tissue. (37) The method of any one of embodiments 34 to 36, wherein applying comprises spraying or dripping, smearing, brushing, or pouring. (38) The method according to any one of embodiments 34 to 37, wherein the tissue is wound tissue and the method is for preventing tissue adhesions during an invasive procedure. (39) The method of embodiment 38, wherein the invasive procedure is a surgical procedure. (40) The method of embodiment 39, wherein the surgical procedure is at least one of abdominal surgery, cardiovascular surgery, thoracic surgery, head and neck surgery, pelvic surgery, and skin and subcutaneous tissue procedures.

[0202] (41) The method of any one of embodiments 34 to 40, wherein the tissue is not a bleeding tissue. (42) The method according to any one of embodiments 34 to 41, wherein the subject is a human subject. (43) The method according to any one of embodiments 34 to 42, wherein the fibrinogen-containing component comprises total protein in a concentration range of about 15 mg / mL to about 40 mg / mL and fibrinogen in a concentration range of about 8 mg / mL to about 25 mg / mL. (44) The method according to any one of embodiments 34 to 43, wherein the thrombin-containing component contains thrombin in a concentration range of about 100 IU / mL to about 300 IU / mL. (45) The method according to any one of embodiments 34 to 44, wherein the fibrinogen-containing component and / or the thrombin-containing component contains free calcium ions.

[0203] (46) The method according to any one of embodiments 34 to 45, wherein the fibrinogen is obtained by plasma cryoprecipitation or is derived from biologically active component 2 (BAC2) or a biologically active component of a cryoprecipitate-derived antihemophilic factor preparation.

Claims

1. A mixture comprising fibrinogen, thrombin, calcium ions, and albumin, the mixture comprising total protein in a concentration range of 2.5 mg / mL to 30 mg / mL, fibrinogen in a concentration range of 50% to 80% by weight of the total protein, and albumin in a concentration range of greater than 0.65 mg / mL to 3 mg / mL.

2. 2. The mixture of claim 1, wherein the albumin is in the concentration range of 1.20 mg / mL to 3 mg / mL.

3. A hydrogel material comprising fibrin and calcium ions, wherein the concentration of total protein ranges from 2.5 mg / mL to 30 mg / mL, and the fibrin is present in a concentration range of 50-80% by weight of the total protein.

4. 4. The hydrogel material of claim 3, further comprising albumin in a concentration range of greater than 0.65 mg / mL to 3 mg / mL.

5. 5. The hydrogel material of claim 4, wherein the albumin is in the concentration range of 1.2 mg / mL to 3 mg / mL.

6. 6. The hydrogel material of claim 3, wherein the concentration range of the total protein is from 7.5 mg / mL to 20 mg / mL.

7. The hydrogel material of claim 3 or 6, wherein the fibrin is cross-linked fibrin.

8. The hydrogel material according to any one of claims 3 to 7, having anti-adhesion properties.

9. A hydrogel material comprising cross-linked fibrin obtained by applying onto tissue, under conditions that allow the formation of a hydrogel, a mixture comprising: (i) a fibrinogen-containing solution comprising fibrinogen in a concentration range of 8 mg / mL to 30 mg / mL and total protein in a concentration range of 15 mg / mL to 40 mg / mL, wherein the fibrinogen is in a concentration range of 50% to 80% by weight of the total protein; and (ii) a thrombin-containing solution.

10. 10. The hydrogel material of claim 9, wherein the mixture is obtained by combining the fibrinogen-containing solution and the thrombin-containing solution ex vivo.

11. 1. A two-component composition for use in preventing tissue adhesions, comprising: A two-component composition comprising: component A comprising a fibrinogen-containing solution containing fibrinogen in a concentration range of 8 mg / mL to 30 mg / mL and total protein in a concentration range of 15 mg / mL to 40 mg / mL, wherein the fibrinogen is in a concentration range of 50% to 80% by weight of the total protein; and component B comprising thrombin.

12. 12. The two-component composition of claim 11, wherein the fibrinogen-containing solution comprises fibrinogen in the concentration range of 10 mg / mL to 25 mg / mL and total protein in the concentration range of 20 mg / mL to 40 mg / mL.

13. 13. The two-component composition of claim 11 or 12, wherein component B comprises thrombin in a concentration range of 100 IU / mL to 300 IU / mL.

14. The two-component composition according to any one of claims 11 to 13, wherein component A and / or component B comprises free calcium ions.

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