HK1148488B - Gelatin-transglutaminase hemostatic dressings and sealants - Google Patents

Description

Gelatin-transglutaminase hemostatic dressings and sealants

Technical Field

The present invention relates to hemostatic dressings, devices, and agents comprising absorbable or non-absorbable materials and/or coagulating proteins. The hemostatic devices are useful for the treatment of wounded tissue.

Background

Controlling bleeding (bleeding) is a critical step in emergency and battlefield wound treatment. Unfortunately, excessive bleeding or fatal bleeding at accessible sites often occurs (J.M. Rocko et al (1982) J.Trauma 22: 635). Mortality data from the vietnam war indicates that 10% of combat deaths were due to uncontrolled limb bleeding. Up to one third of deaths due to blood loss during the vietnam war could have been avoided by using effective battlefield hemorrhage control methods. (SAS/STAT use guide, 4 th edition (Cary, N.C.: SAS Institute Inc; 1990)).

Although civilian trauma mortality statistics do not provide an exact number of pre-hospital deaths due to limb bleeding, examples and anecdotal reports indicate similar incidence (j.m. rocko et al (1982) j. trauma 22: 635). These data indicate that significant increases in survival can be achieved by pre-hospital use of simple and effective methods of bleeding control. Unfortunately, this approach has not been successfully demonstrated through the use of commercially available hemostatic devices.

Individually, surgical wound closure is currently accomplished by sutures and staples that facilitate healing by pulling the tissues together. However, they often fail to produce a sufficient seal necessary to avoid fluid leakage. Accordingly, there is a significant and unmet need for an apparatus and method that avoids post-surgical leakage, including leakage that often occurs along the line of staples and sutures. Such devices and methods are needed as an adjunct to sutures and staples to achieve hemostasis or other Fluid stagnation in peripheral vascular reconstruction, epidural reconstruction, thoracic, cardiovascular, pulmonary, neurological, and gastrointestinal surgical procedures.

Most high pressure hemostatic devices currently on the market are nominal if truly adhesive. A good example of such a device isAC S^TM(Z-Medica, Wallington, CT) and HemCon^TMBandages (HemCon, Portland, OR), two of these hemostatic devices currently available to members of the U.S. armed forces. The mineral zeolite crystals in the QuikClot sponge cause absorption of water molecules in the blood, thus concentrating the coagulation factors and accelerating blood coagulation. The chitosan mixture that makes up the HemCon bandage has a positive charge and attracts red blood cells that have a negative charge. The red blood cells are drawn into the dressing, forming a seal over the wound and stabilizing the wound surface.

The HemCon bandage products mentioned above were developed to provide pre-hospital bleeding control and have shown some success in the art. However, the chitosan network that makes up the HemCon bandage can be saturated with blood and rapidly fail in the face of rapid blood flow or moderate blood flow with trauma encountered after 1-2 hours (B.S kheiiralabi et al (2005). j. trauma.59: 25-35; a.e. pusateri et al (2006). j. trauma.60: 674-682). Furthermore, HemCon bandage patches can only be used as rigid patches that cannot easily accommodate irregular wounds, further limiting their utility.

Chinese character already listedAnother polysaccharide-based hemostatic device known for hemorrhage control is RDH^TM(AcetylGlucosamine)、TraumaDEX^TM(MPH) and Chitoskin^TM(Chitosan&Gelatin). However, none of these types of bandages consistently showed avoidance of failure in the face of significant blood flow. Other recently introduced hemostatic devices include Celox^TM(Chitosan Crystals) and WoundStat^TM(TraumaCure inc., MD) (particulate mixture of montmorillonite mineral and superabsorbent polymer). However, both products expand rapidly to fill the wound site, making them suitable only for accelerating blood clotting in certain types of wounds and bringing the risk of reducing or even eliminating blood flow in the surrounding blood vessels.

QuikClot ACS also mentioned above^TMAlso has demonstrated efficacy against moderate levels of bleeding hemostasis. However, the water absorption mechanism of mineral zeolites cannot occur without significant heat release. Thus, QuikClot ACS^TMCauses high temperature and severe burns at the wound site, which damages the surrounding tissue area and makes the subsequent medical care much more complicated (a.e.pusateri et al (2006). j.trauma.60: 674-682). Clearly, hemostatic solutions without such significant side effects are more desirable. While QuikClot has developed a mineral mix that releases less heat when applied, the effectiveness of the cooler mix is not sufficient for severe trauma treatment. In addition, neither the initial nor the colder mineral mixture can stop the rapid arterial bleeding.

All of the above mentioned products rely on the natural coagulation cascade to control fluid exudation from the wound site. Thus, they are all only useful for stopping blood flow and each is only useful under conditions appropriate for that particular device. Typical wound site seals, especially when the wound site exudes non-blood fluids, are beyond the scope of these products.

Summary of The Invention

It would be desirable and useful to have available a non-toxic adhesive material that can be used in a wide range of applications, including but not limited to surgical applications, hemostasis control, and wound bleeding control. It would also be desirable and useful to have a non-toxic adhesive material that can be used as part of a hemostatic bandage. It would also be desirable and useful to have a non-toxic adhesive material that could be used as a surgical sealant.

The present invention overcomes the deficiencies of the background art by providing an adhesive material comprising a cross-linkable protein and a non-toxic material that induces cross-linking of the cross-linkable protein. Preferably, the cross-linkable protein comprises gelatin and any gelatin variant or variant protein as described herein. Alternatively and preferably, the non-toxic material comprises Transglutaminase (TG), optionally comprising microbial transglutaminase (mTG). In accordance with certain embodiments of the present invention, the adhesive material is provided in a bandage, which is preferably suitable for use as a hemostatic bandage. According to other embodiments, it is provided as a sealing material, which is preferably suitable for use as a sealing material for surgical procedures.

When acted upon by transglutaminase, the denatured form of collagen gelatin undergoes rapid crosslinking to form a fibrillating gel (vibrant gel). The gelation process that occurs is very similar to the natural late coagulation cascade that fibrin undergoes when it comes into contact with factor XIII and calcium. And the resulting gel exhibited a binding capacity very similar to, if not higher than, fibrin glue ((m.k. mcdermott, biomacromolecules.2004, months 7-8; 5 (4): 1270-9).

The invention utilizes the similarity of gelatin-TG crosslinking and fibrin coagulation cascade to simulate the excellent hemostatic performance of high-grade fibrin dressing. The result of replacing the fibrin-thrombin laminate of the fibrin bandage with a gel-TG laminate is the creation of a new, inexpensive and stable dressing that can control bleeding without significant side effects. This new bandage maximizes the adhesion of gelatin-TG mixtures by allowing the controlled application of large amounts of the mixture to the wound site in a manner that prevents TG from spreading to areas not contacted by the bandage. This collaborative technology therefore represents an advance in both the advanced fibrin dressing field and the gelatin-TG bonding field.

Unlike coagulated fibrin networks, gelatin-TG networks have additional benefits in that they can be specifically solubilized using specific proteases otherwise devoid of physiological activity (T.Chen, biomacromolecules.2003, months 11-12; 4 (6): 1558-63). Thus, while the gelatin-mTG hemostatic laminate dressing may replicate the properties of a fibrin-thrombin hemostatic laminate, it may also be removed as needed without difficulty.

In addition to its application as a battlefield dressing for hemostasis in wound treatment, the gelatin-TG based hemostatic device of the present invention has great potential in controlling rapid arterial bleeding during surgery, bleeding after intravascular catheterization (catherization), or exudation of other body fluids after injury or after surgery.

To date, although the gelling properties of crosslinked gelatin-TG and the adhesive ability of gelatin-TG crosslinks have been studied separately, no effort has been made to utilize both properties simultaneously to form a hemostatic or tissue-sealing composition.

Adhesive use of gelatin-TG compounds has been demonstrated in vivo in a rat retina model, where one drop of gelatin-TG mixture was used for retinal attachment (T.Chen, J Biomed Mater Res BAppl Biomate.2006, month 5; 77 (2): 416-22).

The use of gelatin-TG gel as a scaffold for cell therapy has also been tested (U.S. Pat. No. 5,834,232, also Ito A, J Biosci & Bioeng.2003; 95 (2): 196-99, also BroderickEP, J Biomed Mater Res B Appl Biomate.2005, 1/15 days; 72 (1): 37-42).

These studies emphasize the safety of physiological use of gelatin-TG mixtures, but they each use only one of the properties of gelatin-TG cross-linking and fail to teach or show the advances in hemostasis and tissue sealing proposed by the present invention.

The use of gelatin-TG mixtures for hemostasis or fluid stasis also marks a significant advancement in numerous evidentially sufficient attempts to use transglutaminase, especially tissue TG, independently as a surgical adhesive (USP 5736132, USP 61908196, and others). The use of gelatin as a substrate for TG adds a mechanical scaffold to TG activity, providing a number of advantages over TG alone. TG together with gelatin can be administered more precisely than TG alone, can be precisely adapted to the wound site and allows for controlled bioabsorbability.

The present invention overcomes the deficiencies in the background art. Previously attempted solutions have used many forms of modified and unmodified gelatin networks for mild to moderate hemostasis. However, methods of forming strongly cross-linked gelatin networks in situ that can control rapid bleeding arterial bleeding or other significant fluid exudation are lacking. One approach, such as gelatin-TG crosslinking, which can form a strong gelatin network in vivo, increases the mechanical strength of the gelatin matrix and makes it suitable for controlling high pressure arterial bleeding and other fluid exudation. In addition to the improved cross-linking process, the present invention relates to a number of other innovations which provide it with advantages over existing gelatin-based hemostatic materials. The following provides a partial list of non-limiting, exemplary illustrations:

1) in situ cross-linking between gelatin chains and collagen in the tissue ECM (extracellular matrix) creates a strong hemostatic fluid barrier.

2) Gelatin and TG may be more effective in achieving hemostasis or fluid stasis by being administered in lyophilized form and reconstituted with blood or other bodily fluids.

3) The lyophilized form of the gelatin-TG mixture has increased shelf life.

4) The layered lyophilized forms of gelatin and TG provide for more rapid reconstitution, which aids in a high pressure fluid flow environment.

5) The addition of a mechanical liner to the base gelatin-TG mixture increases the hemostatic or fluid control ability of the mixture by slowing down the fluid and allowing more time for the gelatin-TG to crosslink and block fluid seepage.

According to certain preferred embodiments of the present invention, the gelatin-mTG mixture is partially cross-linked prior to administration to the wound site or prior to lyophilization. In another embodiment, uncrosslinked gelatin or mTG is present with partially crosslinked gelatin-mTG.

Hemostatic bandages that are adhesive in nature are known in the art, and their use has many difficulties and drawbacks. For example, widespread hemostatic use of fibrinogen and thrombin has been common in the last year of world war II, but abandoned due to the spread of hepatitis (D.B. Kendrick, blood program in world war II (Washington DC: the office of the General health administration, the Department of the land and the military (Department of arms); 1989), 363-.

The fibrinogen dressing was first used by the trauma surgeon during the first world war when Grey and his colleagues made pre-polymerized fibrin flakes and powders. During world war ii, the U.S. military and the U.S. red cross use pre-polymerized styrofoam-like flakes of fibrin and fibrin films to produce fibrin glue. Fibrin-based dressings show significant differences in controlling bleeding time and reducing blood loss when compared to controls (Jackson, M., et al (1996) J.of Surg.Res.60: 15-22; and Jackson, M.et al (1997) Surg.Forum. XL, VIII: 770-772).

Despite the efficacy of fibrinogen dressings in controlling bleeding, the use of fibrinogen dressings has been discontinued when diseases involving blood and serum-derived sources (e.g., hepatitis and HIV) are often transmitted, as the dressings contain purified human or animal fibrinogen or other purified blood products (Holcomb, J.B. et al (1997). Surgical Clinics of North America.77: 943-.

Over the past few years, however, plasma purification techniques have greatly reduced the risk of blood and serum-borne diseases, and there has been a renewed interest in fibrin-based products for the treatment of wounds.

The american red cross has described a hemostatic laminate dressing comprising a thrombin layer sandwiched between fibrinogen layers (see, e.g., PCT/US99/10952, U.S. patent nos. 6054122, 6762336). Such hemostatic dressings have shown great success in treating potentially fatal wounds (e.m. acheson. (2005): j. trauma.59 (4): 865-74; discussion 874-5; b.s. kheirabadi. (2005): j. trauma.59 (1): 25-34; discussion 34-5; a.e. purasteri. (2004). j.biomed. mater.res.b appl. biometer.15; 70 (1): 114-21). Indeed, in these studies on pigs, fibrin laminate dressings performed well beyond the HemCon and QuikClot products on potentially lethal wounds, showing > 75% survival after 2 hours relative to 0% survival using standard military field bandages, the HemCon bandage or QuikClot powder.

Although these dressings may be used in methods for treating wounded tissue, these conventional laminate dressings may delaminate, whereby the edges of the layers of the dressing no longer adhere to each other. Such delamination can lead to reduced interaction of the constituent layers of the dressing, as well as reduced efficacy of the dressing in preventing bleeding.

An improved fibrin-based hemostatic laminate dressing has been described that includes multiple layers containing absorbable materials and/or coagulated proteins. In particular, the dressing (see PCT/US03/28100, U.S. patent application No. 0060155234) includes a thrombin layer sandwiched between first and second layers of fibrinogen, wherein the thrombin layer is not co-extensive with the first and/or second layers of fibrinogen.

Despite advances in fibrin wound dressings, these bandages suffer from a number of drawbacks. The freeze-dried fibrinogen used to make bandages must be purified from human plasma. Since this is an expensive and carefully handled method, the resulting fibrinogen bandage is extremely expensive to produce and has only a very short shelf life at normal temperature. The more fibrinogen added to the pad, the better the bandage will produce when bleeding stops. However, the more fibrinogen added to the pad, the more expensive the bandage. In addition, the large amount of fibrinogen on the bandage pad can increase the brittleness of the bandage, making it brittle and difficult to handle. As a result of these limitations, no effective fibrin bandage is commercially available.

Thus, while advanced fibrin dressings can control bleeding without significant side effects and compensate for the previously mentioned deficiencies of active wound therapy hemostasis, price and stability limitations introduce significant disadvantages to the use of this type of dressing.

Liquid fibrin sealants or glues have been used for many years as an operating room aid for bleeding control (J.L.Garza et al (1990) J.Trauma.30: 512-513; H.B.Kram et al (1990) J.Trauma.30: 97-101; M.G.Ochsner et al (1990) J.Trauma.30: 884-887; T.L.Matthew et al (1990) Ann.Thorac.Surg.50: 40-44; H.Jakob et al (1984) J.Vase.Surg.1: 171-180). Moreover, single donor fibrin sealants have been used extensively in clinical settings in a variety of surgical situations. (W.D.Spotnitz. (1995). Thromb.Haemost.74: 482-485; R.Lerner et al (1990). J.Surg.Res.48: 165-181)

Although a number of absorbable surgical hemostats are currently used in the surgical field, existing products are not powerful enough to provide the mechanical and biological support necessary to control severe bleeding or the forceful flow of other biological fluids.

Currently available hemostatic bandages such as collagen wound dressings (INSTAT)^TMEthicon, Somerville, NJ and AVITENE^TMCR Bard, Murray Hill, NJ) or dry fibrin thrombin wound dressings (TACHOCOMB)^TMHafslund Nycomed Pharma, Linz, Austria) is limited to use in surgical applications and is not limited toSufficient to resist dissolution in large blood streams. They do not yet possess sufficient adhesive properties to play any practical role in hemostasis of severe blood flow. These currently available surgical hemostatic bandages also require careful handling and therefore they are prone to failure when subjected to bending or bearing pressure damage. They also readily dissolve in hemorrhagic bleeding. This dissolution and disruption of these bandages can be catastrophic as it can lead to loss of adhesion to the wound and result in continued unabated bleeding.

Administration of oxidized cellulose (SURGICEL, Ethicon, Somerville, NJ) or gelatin sponge (SURGIFOM, Ethicon, Somerville, NJ) absorbable hemostats also does not treat arterial bleeding. These products are intended to control the low pressure bleeding that permeates through the bone and epidural veins. Gelfoam is not suitable for high pressure, fast flowing arterial bleeding because they do not form a tight bond with the source of the bleeding and are therefore easily removed. Oxidized cellulose is also not suitable for controlling arterial bleeding because it swells and needs to be removed from the site of application when hemostasis is achieved. When blood flow is too great, excessive swelling occurs before hemostasis is complete (m.sabel et al (2004).

For reasons generally associated with mild toxicity and inability to be readily prepared and applied in the art, the most widely used tissue adhesives are generally not suitable for use as hemostatic or in vivo fluid stasis devices. A good example of such an adhesive is a topical skin adhesive of the cyanoacrylate family, for example Dermabond^TM、Indermil^TM、Liquiband^TMAnd the like. The rapidly activating nature of cyanoacrylates when exposed to air makes cyanoacrylate-based products unsuitable for use in active hemostatic field dressings, and their inability to bind to wet surfaces makes them unsuitable for in vivo hemostasis or fluid stasis use.

Existing products intended for in vivo fluid stagnant use also have significant problems. BioGlue^TM(Cryolife Inc.) is a strong adhesive and sealant material but contains albumin crosslinked by glutaraldehydeWhite, a toxic and highly neurotoxic substance. This toxicity greatly limits their use. Another encapsulant is coseal (baxter), which contains polyethylene glycol (PEG). Although it is non-toxic, it has only weak adhesion, greatly limiting its application.

Gelatin has been used in a variety of wound dressings. Gelatin gels are not very stable at body temperature, since they have a relatively low melting point. It is therefore important to stabilize these gels by establishing cross-links between protein chains. In practice, this is usually achieved by treating the gelatin with glutaraldehyde or formaldehyde. The cross-linked gelatin can thus be made into a dry sponge useful for inducing hemostasis in bleeding wounds. Commercially available examples of such sponges include Spongostan (Ferrosan, Denmark), Gelfoam (Upjohn, USA) and Surgifoam (ethicon. The main disadvantage of these sponges is that the crosslinking agents used (glutaraldehyde or formaldehyde) are toxic to the cells. The negative effects of glutaraldehyde crosslinking are exemplified, for example, by the findings of de Vries et al (abstracts collection of WHS second annual meeting, Richmond, USA, p 51, 1992). These authors suggest that the glutaraldehyde-crosslinked collagen network structure is toxic to cells, while the uncrosslinked species is non-toxic. Thus, even though they have beneficial hemostatic properties, these products are not very desirable as wound dressings for the treatment of problematic wounds. Therefore, a gelatin-based wound dressing using different less toxic crosslinking techniques is highly desirable.

In addition to potential toxicity, the gelatin network alone does not provide the mechanical properties necessary for controlling rapid bleeding. They are more suitable for wound treatment applications that require only a small amount of fluid absorption. In one study, it was concluded that thin sheets of glutaraldehyde cross-linked gelatin are more suitable as a dressing for sustained wound healing, especially of dystrophic tissue which requires a longer period of time. Alternatively, they can be used as scaffolds for cell attachment, where they can excite a poorly reactive microenvironment (MG Tucci. (2001). J.Bioactive & Comp.polymers.16 (2): 145-157) throughout the extended in situ presence.

Gelatin networks cross-linked with polysaccharides have also been proposed for controlling bleeding. These hemostatic compounds are not limited by the potential toxicity of glutaraldehyde cross-linked gelatin sponges. However, gelatin-polysaccharide materials often lack mechanical strength and are primarily intended to control small amounts of penetrating fluid during surgery or to limit wound penetration during extended post-medical care periods.

An example of a gelatin-polysaccharide compound is an in situ cross-linked gelatin-alginate wound dressing. Such dressings have no adhesive function and are primarily used to control moisture at the wound site. The dressing expands to 90% of its original size, which greatly reduces its mechanical strength (B Balakrishnan et al (2005): biomaterials.26 (32): 6335-42).

Another more widespread example is cross-linked gelatin-chitosan wound dressings (examples in us patents 6,509,039, 4,572,906). Although some have suggested using such dressings for wound treatment (Chitoskin)^TM) However, the hemostatic properties of this material are simply insufficient to control high pressure bleeding. Moreover, the material expands significantly in the face of large volumes of body fluids. The dressing is more suitable for treating chronic wounds and burns.

Yet another example is mentioned (us patent 6,132,759) in which solubilised (solubilised) gelatin is cross-linked with oxidised dextran. This material is suggested for use in wound coverage and long-term treatment due to its high absorption capacity and advantageous controlled release properties for the delivery of therapeutic substances, especially to wounds.

No substance is currently involved in the crosslinked gelatin network or networks of other substances crosslinked with gelatin that independently provide hemostasis for rapid in vivo bleeding, even with the addition of thrombin. Studies have been conducted to compare the hemostatic capabilities of FloSeal gelatin matrix (BioSurgery, Fremont, CA) and GelFoam gelatin matrix soaked in active thrombin solution. None of the enhanced hemostatic devices was able to stop bleeding of the flow characteristics in patients beyond 2/3 after 5 minutes. Pulsatile arterial bleeding is much more rapid than ambulatory bleeding and certainly causes problems with these thrombin-soaked matrices (FAWeaver et al (2002): ann. vase. surg.16 (3): 286-93).

In either instance, there are different deficiencies in wound treatment because there are no new active hemostatic battlefield dressings available that can control bleeding without significant side effects. Similarly, there are different drawbacks in surgical care, since there are no nontoxic sealing materials available that can withstand rapid bleeding and can seal wound sites exuding non-blood bodily fluids.

According to certain embodiments of the present invention, there is provided a method of treating a wounded tissue comprising administering to said tissue a composition comprising gelatin and a non-toxic cross-linking agent.

Optionally, the non-toxic crosslinking agent comprises transglutaminase. Preferably, transglutaminase is included as part of the transglutaminase composition, and the weight ratio of gelatin to transglutaminase composition is in the range of from about 1: 1 to about 300: 1. More preferably, the transglutaminase composition has a specific activity level of at least about 40U/gm. Most preferably, the transglutaminase composition has a specific activity level of at least about 800U/gm.

Alternatively and preferably, the activity of transglutaminase in the gelatin-transglutaminase composition is from about 25 to about 400U/g gelatin. More preferably, the activity is from about 40 to about 200U/g gelatin.

Alternatively, the transglutaminase comprises a plant, recombinant animal or microorganism-derived transglutaminase other than blood-derived factor XIII. Preferably, the composition has a pH in the range of from about 5 to about 8.

Alternatively, gelatin is produced from an animal source, a recombinant source, or a combination thereof. Preferably, the animal source is selected from the group consisting of fish and mammals. More preferably, the mammal is selected from the group consisting of swine and cattle.

Alternatively, the gelatin is type a (acid treated) or type B (base treated). More preferably, the gelatin comprises a high molecular weight gelatin.

Alternatively, the traumatized tissue is selected from the group consisting of surgically cut tissue, surgically repaired tissue and traumatized tissue.

Optionally, the method further comprises reducing bleeding of the tissue or exudation of other body fluids. Alternatively, the bodily fluid is selected from the group consisting of cerebrospinal fluid, intestinal fluid, air (air), bile and urine. Preferably, the method further comprises inducing stasis of hemostasis or other exuding body fluids in the tissue.

Alternatively, the wound is bleeding or oozing another body fluid, and treating the tissue of the wound comprises applying the composition to the wound site to promote in situ cross-linking between gelatin chains and endogenous collagen of the tissue extracellular matrix to create a barrier to fluid exudation or bleeding.

Optionally, the method further comprises forming a biomimetic blood clot.

Optionally, administering the composition comprises: mixing gelatin and transglutaminase to form a mixture; and applying the mixture to the tissue.

In accordance with other embodiments of the present invention, there is provided a method for inducing hemostasis in a wound of a mammal, the method comprising applying to the wound a composition comprising gelatin and transglutaminase.

In accordance with other embodiments of the present invention, there is provided a method of inducing biomimetic clot formation at a site of an injured blood vessel comprising applying to the wound a composition comprising gelatin and transglutaminase.

In accordance with other embodiments of the present invention, there is provided a composition comprising a combination of gelatin and transglutaminase, wherein the ratio of the amount of gelatin to the amount of transglutaminase is selected to induce the formation of a biomimetic blood clot in a mammal.

In accordance with other embodiments of the present invention, there is provided a composition comprising a combination of gelatin and a non-toxic cross-linking agent, wherein the ratio of the amount of gelatin to the amount of non-toxic cross-linking agent is sufficient to reduce bleeding in a wound of a mammal.

Preferably, the non-toxic crosslinking agent comprises transglutaminase. More preferably, transglutaminase is added as part of the transglutaminase composition and the weight ratio of gelatin to transglutaminase composition is in the range of from about 1: 1 to about 300: 1. More preferably, the ratio is in the range of from about 1: 1 to about 100: 1. Most preferably, the transglutaminase composition has a specific activity level of at least about 40U/gm. Also most preferably, the transglutaminase composition has a specific activity level of at least about 80U/gm. Also most preferably, the transglutaminase composition has a specific activity level of at least about 200, 400, or 800U/gm.

Alternatively, the activity of transglutaminase in the gelatin-transglutaminase composition is from about 25 to about 400U/g gelatin. Preferably, the activity is from about 40 to about 200U/g gelatin.

Alternatively, the transglutaminase comprises a plant, recombinant, animal or microorganism-derived transglutaminase other than blood-derived factor XIII. Preferably, the composition further comprises a stabilizer or filler. Also preferably, the composition has a pH in the range of from about 5 to about 8.

Alternatively, gelatin is produced from an animal source, a recombinant source, or a combination thereof. Preferably, the animal source is selected from the group consisting of fish and mammals. More preferably, the mammal is selected from the group consisting of swine and cattle. Most preferably, the gelatin comprises porcine skin or porcine bone or a combination thereof. Also most preferably, the gelatin is form a (acid treated) or form B (base treated). Also most preferably, the gelatin comprises a high molecular weight gelatin.

Alternatively, the gelatin has at least about 250 bloom. Preferably, the fish comprises cold water species of fish.

Alternatively, recombinant gelatin is produced using bacterial, yeast, animal, insect or plant systems or any type of cell culture.

Optionally, the gelatin is purified to remove salts.

Optionally, the gelatin has at least one property that is adjusted, tailored, or predetermined. Alternatively, gelatin does not undergo thermoreversible gelation.

In accordance with other embodiments of the present invention, hemostatic or humoral sealants are provided that comprise a combination of gelatin and a non-toxic cross-linking agent. Optionally, the non-toxic crosslinking agent comprises transglutaminase. Preferably, the combination comprises aggregated gelatin and transglutaminase.

A method or composition as described herein, wherein the transglutaminase is optionally extracted from one or more of Streptoverticillium bardahliae (Streptomyces baldacci), Streptomyces Hygroscopicus (Streptomyces Hygroscopicus) or Escherichia Coli (Escherichia Coli).

In accordance with other embodiments of the present invention, there is provided a method of inducing hemostasis in and/or sealing a wound tissue comprising administering to the tissue a composition comprising a cross-linked protein substrate and a non-toxic cross-linking agent. Optionally, the non-toxic crosslinking agent comprises transglutaminase. Preferably, the substrate comprises one or more synthetic polymer sequences characterized by a transglutaminase crosslinking site. More preferably, the substrate comprises a modified polypeptide comprising at least one transglutaminase crosslinking site.

In accordance with other embodiments of the present invention, there is provided a composition for inducing hemostasis and/or sealing wounds, comprising a mixture of gelatin and transglutaminase, wherein the mixture is modified such that the gelatin and transglutaminase form a solution below the normal sol-gel transition temperature of standard animal gelatin.

Alternatively, gelatin has been modified to have a reduced sol-gel transition temperature. Preferably, the composition further comprises an additive to increase the stability of the gelatin in the mixture. More preferably, the composition further comprises an additive to lower the sol-gel transition temperature of the gelatin. Most preferably, the composition further comprises a plasticizer. Alternatively and most preferably, the plasticizer is selected from the group consisting of polyols, glycerol (glycerine), glycerol (glycerol), xylitol, sucrose, sorbitol, triethanolamine, resorcinol, thiodiglycol, sodium salts of toluene sulfonic acid (toluene sulfonic acid), butylene glycol, urea nitrate, thiourea, urea, glutamic acid, aspartic acid (aspartic acid), valine, glycine, KSCN, KI and LiBr.

Alternatively, the concentration ratio of glycerol ranges from about 0.5: 1 to about 5: 1 glycerol to gelatin, weight to weight. Preferably, the concentration ratio ranges from about 1: 1 to about 2: 1 glycerol to gelatin, weight to weight. Alternatively, the concentration ratio of sorbitol ranges from about 0.5: 1 to about 5: 1 sorbitol to gelatin, weight to weight. Preferably, the concentration ratio ranges from about 1: 1 to about 3: 1 sorbitol to gelatin, weight to weight.

Alternatively, the urea concentration ratio ranges from about 1: 2 to about 2: 2 urea to gelatin, weight to weight.

Optionally, the composition further comprises a conditioning agent selected from the group consisting of a pH adjusting agent and an ionic concentration adjusting agent. Preferably, the pH adjusting agent provides a pH in the range of from about 1.5 to about 5.0 or from about 7.0 to about 9.0.

Optionally, the composition further comprises a salt.

Optionally, the composition further comprises trehalose saccharides, mannitol saccharides or other saccharides for stabilization by spray drying, freeze drying or other protein drying.

Optionally, the composition further comprises a denaturant. Preferably, the denaturant is selected from the group consisting of guanidine hydrochloride and urea. More preferably, the concentration ratio ranges from about 1: 2 to about 2: 2 GuHCl: gelatin, weight/weight. Also more preferably, the concentration ratio ranges from about 0.5: 1 to about 1: 1 urea to gelatin, weight to weight.

Optionally, the composition further comprises a reducing agent. Preferably, the reducing agent is selected from the group consisting of magnesium chloride and hydroquinone. More preferably, hydroquinone is present in solution in the mixture in a concentration of from about 0.2 to about 0.5M. Most preferably, the concentration is from about 0.3 to about 0.4M.

Alternatively, the magnesium chloride is present in the solution of the mixture at a concentration of from about 2 to about 4M. Preferably, the concentration is from about 2.5 to about 3.5M.

Optionally, the composition further comprises an exothermic agent. Preferably, the exothermic agent comprises one or more of calcium chloride, other calcium salts, magnesium chloride, metal oxides/zeolites, or combinations thereof. More preferably, the calcium chloride is present in the solution in an amount from about 0.2 to 0.7g calcium chloride per mL of the mixture for each degree celsius temperature increase above room temperature.

Optionally, the composition further comprises a gelatin-specific protease.

Optionally, the composition further comprises a protease inhibitor.

Optionally, the composition further comprises an additional hemostatic agent. Preferably, the additional hemostatic agent further comprises one or more of albumin, collagen, fibrin, thrombin, chitosan, ferric sulfate, or other metal sulfate.

According to other embodiments of the present invention, there is provided a hemostatic or sealing dressing comprising: (i) a first gelatin layer; (ii) a transglutaminase layer adjacent to the first gelatin layer; and (iii) a second gelatin layer adjacent to the transglutaminase layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the first gelatin layer and/or the second gelatin layer.

According to other embodiments of the present invention, there is provided a hemostatic or sealing dressing comprising: (i) a layer of absorbable or non-absorbable material; (ii) a first gelatin layer adjacent to the material layer; (iii) a transglutaminase layer adjacent to the first gelatin layer; and (iv) a second gelatin layer adjacent to the transglutaminase layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the first gelatin layer and/or the second gelatin layer.

According to other embodiments of the present invention, there is provided a hemostatic or sealing dressing comprising: (i) a gelatin layer; (ii) a transglutaminase layer adjacent to the gelatin layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the gelatin layer.

According to other embodiments of the present invention, there is provided a hemostatic or sealing dressing comprising: (i) a layer of absorbable or non-absorbable material; (ii) a gelatin layer adjacent to the material layer; (iii) a transglutaminase layer adjacent to the gelatin layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the gelatin layer.

According to other embodiments of the present invention, there is provided a hemostatic or sealing dressing comprising: (i) a gelatin layer; (ii) an absorbable or non-absorbable material layer adjacent to the first gelatin layer; (iii) a transglutaminase layer adjacent to the material layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the gelatin layer.

Optionally, the dressing further comprises a backing material.

According to other embodiments of the present invention, there is provided a hemostatic or sealing device comprising: (i) an absorbable or non-absorbable matrix; (ii) gelatin; (iii) transglutaminase, wherein the gelatin and the transglutaminase are contained in a matrix.

According to other embodiments of the present invention, there is provided a hemostatic or sealing device comprising: (i) a porous, absorbable or non-absorbable matrix; (ii) gelatin; (iii) transglutaminase, wherein the gelatin and the transglutaminase are bound to a substrate.

According to other embodiments of the present invention, there is provided a medical device for insertion into the body of a human or lower mammal, comprising a haemostatic or sealant agent or composition as described herein. Preferably, the device comprises a vascular catheter.

According to other embodiments of the present invention, there is provided a medical device for topical application on the body of a human or lower mammal, comprising a haemostatic or sealant agent or composition as described herein. Alternatively, the device comprises a pressurized spray or foam.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are intended to provide further explanation of the present invention, as claimed.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, and publications mentioned herein are incorporated by reference.

As used herein, a transglutaminase layer that is described as "non-coextensive" with a gelatin layer is one in which the spatial boundary of the transglutaminase layer in two dimensions is less than the spatial boundary of one or both gelatin layers such that the transglutaminase layer is independently coextensive with only about 5% to about 95% of the surface area of the first gelatin layer of the hemostatic dressing and/or coextensive with only about 5% to about 95% of the surface area of the second gelatin layer of the hemostatic dressing. For example, the transglutaminase layer can be independently coextensive with about 10, 20, 30, 40, 50, 60, 70, 75, 80, or 90% of the surface area of each of the first and second gelatin layers. The transglutaminase layer "co-extensive" with the gelatin layer provides complete coverage of the gelatin layer and is co-extensive with 100% of the surface area of the gelatin layer. For example, by using gelatin layers having different total surface areas or shapes, the transglutaminase layer may be non-coextensive with the first gelatin layer but coextensive with the second gelatin layer, or vice versa.

As used herein, "patient" refers to a human or animal subject in need of medical care and/or treatment.

As used herein, "wound" refers to any damage to any tissue of a patient that results in the loss of blood from the circulatory system or any other bodily fluid from its physiological pathway. The tissue may be in vivo tissue (e.g., an organ or a blood vessel), or in vitro tissue (e.g., skin). The loss of blood or body fluid may be in vivo (e.g., from a ruptured organ), or in vitro (e.g., from a laceration). The wound may be in soft tissue (e.g., organs) or in hard tissue (e.g., bone). The damage may be caused by any agent or any source, including traumatic injury, infection, or surgical intervention. The damage may be life threatening or non-life threatening.

As used herein, "absorbable material" refers to a material that breaks down spontaneously and/or via the mammalian body into components that are consumed or eliminated in a manner that does not significantly interfere with wound healing and/or tissue regeneration, and that does not cause any significant metabolic disturbance.

As used herein, "stability" refers to the retention of those material properties that determine activity and/or function.

As used herein, a "binding agent" refers to a compound or mixture of compounds that enhances the adhesion of one layer of a hemostatic dressing to one or more different layers and/or the adhesion of a component of a given layer to other components of that layer.

As used herein, "solubilizing agent" refers to a compound or mixture of compounds that increases the solubilization of a protein in an (preferably) aqueous solvent.

As used herein, "filler" refers to a compound or mixture of compounds that provides bulk and/or porosity to one or more layers of the hemostatic dressing.

As used herein, "release agent" refers to a compound or mixture of compounds that aids in the removal of the hemostatic dressing from the manufacturing mold.

As used herein, "blowing agent" refers to a compound or mixture of compounds that generates a gas when hydrated under suitable conditions.

"TG" refers to any type of transglutaminase; depending on the context, "mTG" may also refer to microbial transglutaminase and/or any type of transglutaminase (in the specific experimental examples below, the term refers to microbial transglutaminase)

The term "mammal", especially when referring to methods of treatment and/or use and application with devices and/or compositions, refers to humans and lower mammals, unless specifically stated otherwise.

As used herein, "about" means plus or minus about ten percent of the indicated value.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

Brief Description of Drawings

The invention is herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the details of the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only, and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for a fundamental understanding of the invention, the description taken with the drawings making apparent to those skilled in the art how the several forms of the invention may be embodied in practice.

In the drawings:

FIG. 1 is a schematic block diagram of an exemplary bandage in accordance with the present invention;

FIG. 2 shows a front view of an exemplary bandage according to the present invention covered with an optional absorbent pad and an optional plastic envelope;

FIG. 3 is a schematic block diagram of an exemplary hemostatic device according to the present disclosure, the device comprising a porous matrix;

FIG. 4 is a graph showing the effect of different percentages of gelatin tested on bond strength;

FIG. 5 shows the effect of temperature on transglutaminase activity (temperature range tested was 32-150 ℃ F.; optimal range was 122-131 ℃ F. (50-55 ℃ C.));

FIG. 6 shows representative burst pressure measurements for tissue adhesives based on composition A;

FIG. 7 shows representative burst pressure measurements for tissue adhesives based on composition B;

FIG. 8 is a photograph showing the formation of a gel and also the induction of hemostasis (FIG. 8A shows the full area and FIG. 8B shows a portion of the area enlarged for further detail);

fig. 9A shows the lack of clot formation after administration of the control solution, while fig. 9B shows gelation and hemostasis of the experimental solution.

FIGS. 10A-D show photographs of an artery (10A) while the artery is being cut; a photograph (10B) of a heavily bleeding cut artery; photograph (10C) of a cut artery administered with the composition of the present invention; and a photograph of hemostasis with biomimetic clot formation (10D);

FIGS. 11A and 11B show an example of a two-syringe application of a two-component hemostatic sealing material; and

figures 12A and 12B show an example of vascular insertion point closure in which the catheter is covered with a composition described herein.

Detailed Description

The present invention relates to an adhesive material comprising a cross-linkable protein and a non-toxic material inducing cross-linking of the cross-linkable protein. Preferably, the cross-linkable protein comprises gelatin and any gelatin variant or variant protein as described herein. Alternatively and preferably, the non-toxic material comprises Transglutaminase (TG), which may optionally comprise any type of calcium dependent or independent transglutaminase (mTG), which may for example optionally be a microbial transglutaminase. According to certain embodiments of the present invention, the adhesive material is provided in a bandage, which is preferably suitable for use as a hemostatic bandage. Various embodiments of the present invention are described in more detail below under the section headings, which are provided for purposes of clarity only and are not intended to be limiting in any way.

Gelatin and transglutaminase

According to a preferred embodiment of the present invention, there is provided a composition for hemostasis and tissue sealing, wherein the cross-linking substance comprises transglutaminase and the cross-linkable protein comprises gelatin.

According to a preferred embodiment, transglutaminase is present in the composition having a specific activity level of at least about 100U/gm, although lower activity levels may alternatively be used, e.g., by alternatively adjusting the ratios described above. Such optionally lower activity levels of the composition preferably comprise at least about 20U/gm, more preferably at least about 40U/gm, even more preferably at least about 60U/gm and most preferably at least about 80U/gm.

Transglutaminase is preferably added to the gelatin (either alone or as part of the composition) in an amount such that the resulting transglutaminase activity in the mixture is preferably from about 25 to about 100U/g gelatin, and more preferably from about 40 to about 60U/g gelatin.

Suitable gelatins and transglutaminase can be obtained by any of the methods known and available to those skilled in the art. Gelatin optionally comprises any type of gelatin including proteins known in the art, preferably including but not limited to gelatin obtained by partial hydrolysis of animal tissue including but not limited to animal skin, connective tissue (including but not limited to ligaments, cartilage and the like), deer horn or keratin and the like, and/or bone, and/or fish scales and/or bone or other components, and/or collagen obtained from animal tissue; and/or recombinant gelatin produced using bacterial, yeast, animal, insect or plant systems or any type of cell culture.

According to a preferred embodiment of the invention, the gelatine from animal origin preferably comprises gelatine from mammalian origin and more preferably comprises one or more of pork skin, pork and beef bones, or double layer cattle hide, or any other porcine or bovine origin. More preferably, such gelatin comprises porcine gelatin because of its lower allergic response rate. Gelatin from animal sources may alternatively be of type a (acid treated) or type B (base treated), but it is preferably of type a.

Preferably, the gelatin from animal origin comprises gelatin obtained during the first extraction, which is generally carried out at a lower temperature (50-60 ℃, although this range of extraction temperatures is not necessarily limiting). Gelatin produced in this manner will be in the range of 250-300 bloom and have a high molecular weight of at least about 95-100 kDa. Preferably, 300 bloom gelatin is used.

A non-limiting example of a producer of such gelatin is PB Gelatins (Tessenderlo Group, Belgium).

According to certain embodiments of the invention, the gelatin from an animal source optionally comprises gelatin from fish. Alternatively, any type of fish may be used, preferably cold water species of fish such as carp, cod, or mullet or tuna. The pH of such gelatins (measured in a 10% solution) preferably ranges from 4 to 6.

Cold water fish gelatin forms a solution in water at 10 ℃ and therefore all cold water fish gelatin is considered to be 0 bloom. For the purposes of the present invention, high molecular weight cold water fish gelatin is preferably used, more preferably comprising a molecular weight of at least about 95-100 kDa. This corresponds to the molecular weight of 250-300 bloom animal gelatin. As a result of their lower proline and hydroxyproline levels, cold water fish gelatin undergoes thermoreversible gelation at much lower temperatures than animal gelatin. Cold water fish gelatin has 100-130 proline and 50-75 hydroxyproline groups per 1000 amino acid residues compared to 135-145 proline and 90-100 hydroxyproline groups in animal gelatin (Haug IJ, Draget KI,O.(2004).Food Hydrocolloids.18：203-213)。

a non-limiting example of a producer of such gelatin is Norland Products (Cranbury, NJ).

In a preferred embodiment of the invention, the gelatin is purified to remove salts. This may be done according to the techniques described previously. One such technique involves forming a 20% w/v gelatin solution in water and heating it to 60 ℃ with stirring. The mixture was then allowed to stand overnight. The gel obtained was dialyzed against repeated changes of deionized water to eliminate salts, stirred and heated to 50 ℃ to disaggregate the physical network. The final solution was filtered and freeze-dried (Crescenzi V, France scangeli A, Taglienti A. (2002). biomacromolecules.3: 1384-. Alternatively, gelatin may be desalted through a size exclusion column.

According to certain embodiments of the invention, recombinant gelatin is used. Recombinant gelatin is currently produced commercially by FibroGen (San Francisco, Calif.). The presently preferred method is to use a recombinant yeast system (Pichia Pastoris) to express specific fragments of type I, alpha 1 human sequence collagen.

In an alternative but preferred embodiment of the invention, the recombinant gelatin is a completely synthetic molecule, not comprising adulterated components from human or any animal. By "synthetic" is meant that the gelatin is preferably produced according to a method selected from chemical synthesis, cell-free protein synthesis, cell tissue culture, any type of bacterial, insect or yeast culture or in plants. The use of synthetic gelatin eliminates many of the variables and deficiencies that tissue-derived materials have, including eliciting unwanted immune responses. For example, fish gelatin exhibits a high degree of allergenicity while animal gelatin exhibits a low degree of allergenicity, whereas recombinant gelatin may be non-allergenic. In human safety studies, no adverse events associated with recombinant gelatin were found.

Methods of producing recombinant gelatin and the benefits of their use are fully described in U.S. patents 6,413,742 and 6,992,172, which are incorporated herein by reference as if fully set forth herein.

Recombinant gelatin can be produced as highly (99%) purified. Recombinant gelatin production allows for the alternative production of gelatin having at least one defined and predetermined property, including but not limited to defined molecular weight, pI (isoelectric point), guaranteed batch-to-batch reproducibility, and the ability to adjust molecular weight to match a particular application.

An example of adjusting molecular weight to match a particular application has been described previously in which gelatin is produced to be highly hydrophilic (Werten MWT et al (2001). Protein engineering.14 (6): 447- & 454). Alternatively and preferably, the gelatin according to the present invention comprises a gelatin having at least one adjusted, tailored or predetermined property.

Non-limiting examples of other types of properties that may optionally be so tailored according to the invention include undergoing or not undergoing thermoreversible gelation. Recombinant gelatin may be produced to undergo thermoreversible gelation or not. Gelatin that has one or more of the beneficial properties of natural animal gelatin but that has not undergone thermoreversible gelation has great utility in enabling gelatin to be crosslinked by other means at temperatures at which it would normally undergo thermoreversible gelation. Such gelatin is also encompassed by certain embodiments of the present invention.

Animal (bovine, porcine, etc.) gelatins, warm water fish gelatins, and recombinant gelatins (gelling type) can undergo thermoreversible gelation at some point between 35-40 degrees, especially at high molecular weight and/or high concentration (> 20%) and/or with modifications and/or one or more additional substances (described below). At room temperature, they are in the form of a gel and cannot be easily mixed with mTG. Various modifications of the compounds according to certain embodiments of the invention to maintain the gelatin solution in liquid form at room temperature are described below.

Cold water fish gelatin and recombinant gelatin (non-gelling type) do not form thermoreversible gels at room temperature, even if no further modifications and/or one or more additional substances are present. They have a transition point well below room temperature. At room temperature, they remain in solution and can react with mTG without further modification

According to a preferred embodiment of the invention with respect to recombinant gelatin, a suitable in vitro culture system is used to produce recombinant gelatin. In addition to the use of recombinant methylotrophic yeast systems for recombinant gelatin production, other microorganisms are used.

Recombinant gelatin-like proteins have been expressed in E.coli, but the expression levels typically obtained in E.coli are rather low and purification of the proteins produced intracellularly can be difficult. Bacillus brevis (Bacillus brevis) has been used for expression of gelatin-like proteins, in which a sequence segment is selected from natural collagen genes and polymerized to form semi-synthetic gelatin (Werten MWT et al, Secreted production of a custom-made gelatin, high-purity hydrophyllic gelatin in Pichia pastoris (secretory production of highly hydrophilic gelatin specifically designed in Pichia pastoris). protein engineering, Vol.14, No. 6, 447-454, month 6 2001).

Other successful efforts in producing recombinant gelatin have included the production of recombinant gelatin using mammalian and insect cells. Collagen and gelatin are also expressed in transgenic tomato plants, transgenic mice. A transgenic silkworm system has been used to produce a fusion protein comprising a collagen sequence. These systems lack endogenous prolyl hydroxylase activity sufficient to produce fully hydroxylated collagen, which can be overcome by overexpression of prolyl hydroxylase (Olsen D et al Recombinant collagen and gelatin for Drug delivery). Adv Drug Deliv Rev.2003, 11/28; 55 (12): 1547-67). Plant-based systems may also optionally be used; for example, the lowa state university is working with fibrigen to develop gelatin expression in transgenic corn.

The gelatin used in the hemostatic dressing may be a gelatin complex or any gelatin, or a derivative or metabolite thereof, or a gelatin produced according to a single process or multiple processes. For example, gelatin may alternatively comprise gelatin type a or gelatin type B or a combination thereof.

Transglutaminase optionally comprises any plant, animal or microorganism derived transglutaminase, preferably other than blood derived factor XIII. Preferably, microbial transglutaminase from Streptoverticillium mobaraensis is used.

The transglutaminase may alternatively be in a composition comprising at least one further substance, such as a stabilizer or filler. Non-limiting examples of such materials include maltodextrin, hydrolyzed skim milk protein or any other proteinaceous material, sodium chloride, safflower oil, trisodium phosphate, sodium caseinate or lactose or combinations thereof.

Although the optimum pH for the native transglutaminase activity is 6.0, it also functions with high activity in the range of pH5.0 to pH 8.0. Thus, the composition according to the invention for use in hemostasis preferably has a pH value in the range of from about 5 to about 8.

Transglutaminase is characterized by a negative temperature coefficient. Within the temperature range of transglutaminase activity, higher temperatures require shorter reaction times and lower temperatures require longer times to become effective. The following table shows the different reaction times at different temperatures comparing the same reaction grade as the reaction that takes place at 50 ℃ and pH6.0 over ten minutes:

TABLE 1 reaction temperature of transglutaminase

The temperature is 5 ℃, 15 ℃,20 ℃, 30 ℃ and 40 DEG C

Time (minutes) 240105703520

Non-limiting examples of commercially available transglutaminase products include those produced by ajinomoto co (Kawasaki, Japan). A preferred example of such a product from this company is Activa TG-TI (in Europe: Activa WM) -composition: mTG and maltodextrin; activity: 81-135U/gActiva. Other non-limiting examples of suitable products from this company include Activa TG-FP (ingredient: hydrolyzed skim milk protein, mTG; activity: 34-65U/g Activa TG-FP); activa TG-GS (ingredients: sodium chloride, gelatin, trisodium phosphate, maltodextrin, mTG and safflower oil (processing aid), activity: 47-82U/g Activa TG-GS); activa TG-RM (in Europe: Activa EB) -composition: sodium caseinate, maltodextrin and mTG; activity: 34-65U/g Activa; activa MP (ingredient: mTG, lactose and maltodextrin; activity: 78-126U/g Activa).

Other non-limiting examples of commercially available transglutaminase Products include those produced by Yiming Biological Products Co. A preferred example of such a product from this company is TG-B (composition: 1% mTG, 99% helper protein; activity: 80-130U/g TG-B). Other non-limiting examples of suitable products from this company include TG-A (ingredient: 0.5% mTG, 99.5% helper protein; activity: 40-65U/g TG-A).

For all examples, preferred transglutaminase products are those with high specific activity and simplest adjunct ingredients, as they are believed (without wishing to be limited by a single hypothesis) to have optimal reactivity upon administration and a lower probability for undesired side reactions.

In another embodiment, transglutaminase may optionally be extracted From Streptoverticillium dahliae or Streptomyces hygroscopicus strains to produce enzyme variants that have been shown to work best at lower temperatures (about 37 ℃ and 37 ℃ -45 ℃ respectively) (Negus SS. A Novel microbial Transglutaminase Derived From Streptoverticillium baldachi. A doctor paper of biological and biological science.Griffith University, Queelantd, Australia and Cui L et al Purification and characterization of transglutaminase From a new strain of Streptomyces anophylogenicus 105: 618). Higher specific activity at lower temperatures is desirable in order to obtain faster and stronger gelatin crosslinking at ambient conditions.

According to certain embodiments, the transglutaminase may be used in the form of any of the compositions described above, optionally including any of the commercially available transglutaminase-containing mixtures.

In another embodiment, any of the transglutaminase mixtures hereinbefore may optionally be purified by gel filtration, cation exchange chromatography, hollow fiber filtration or tangential flow filtration to remove their carrier proteins and/or carbohydrates. Some of these methods have been described previously (Bertoni F, Barbani N, Giusti P, Ciardeli G.: Transglutaminase reactivity with a protein: application in tissue engineering) Biotechnol Lett (2006) 28: 697 protein 702) (Broderick EP et al Enzymatic Stabilization of Gelatin-Based Scaffolds J Biomed Mater Res 72B: 37-42, 2005). The filter pore size used for filtration is preferably about 10 kDa.

In any case, the activity of transglutaminase is preferably measured with a transglutaminase reactivity assay prior to use and/or manufacture of the composition according to the invention. Such assays may alternatively include, but are not limited to, hydroxamic acid methods, Nessler assays, colorimetric assays, or any other assay of transglutaminase activity (see, e.g., Folk JE, Cole PW. Transglutaminase: mechanical characteristics of the active site determined by kinetics and inhibitor studies. BiochimBiophys acta.1966; 122: 244-64; or Bertoni F, Barbani N, Giusti P, Ciarddelig. transglutaminase reactivity with gelatin: experimental applications in tissue engineering) biological assay 2006: experimental applications in tissue engineering (Lett) technique 702).

Generally, the purity and/or quality of the gelatin and/or transglutaminase used in the hemostatic composition will be of an appropriate purity known to one of ordinary skill in the relevant art to result in the efficacy and stability of the protein.

Cross-linked protein substrates other than gelatin

As indicated above, the cross-linkable protein preferably comprises gelatin but may additionally or alternatively comprise another type of protein. According to certain embodiments of the invention, the protein is also a substrate of transglutaminase and is preferably characterized by transglutaminase-specific polypeptide and polymer sequences. These proteins may alternatively include, but are not limited to, synthetic polymer sequences that independently have the property of forming a bioadhesive, or polymers that have been more preferably modified with a transglutaminase-specific substrate that enhances the ability of the material to be cross-linked by the transglutaminase. Non-limiting examples of each of these types of materials are described below.

Synthetic polypeptide and polymer sequences with suitable transglutaminase cross-linking targets have been developed, which have transition points preferably from about 20 to about 40 ℃. Preferred physical properties include, but are not limited to, the ability to bind tissue and the ability to form fibers. Like gelatin of the gelling type (described above), these polypeptides may alternatively be used in compositions also characterized by one or more substances that lower their transition point.

Non-limiting examples of such peptides are described in U.S. patent nos. 5,428,014 and 5,939,385, both of which are filed by ZymoGenetics Inc, both of which are incorporated herein by reference as if fully set forth herein. U.S. patent No. 5,428,014 describes biocompatible, bioadhesive, transglutaminase-crosslinkable polypeptides, wherein transglutaminase is known to catalyze an acyl transfer reaction between the γ -formamide group of a protein-bound glutaminyl residue and the epsilon-amino group of a Lys residue, resulting in the formation of epsilon- (γ -glutamyl) lysine isopeptide bonds.

For example, polypeptides having 13-120 amino acid residues comprising fragments of the formula S1-Y-S2 are described, wherein: s1 is Thr-Ile-Gly-Glu-Gly-Gln; y is a 1-7 amino acid spacer peptide or absent; and S2 is Xaa-Lys-Xaa-Ala-Gly-Asp-Val. Alternatively, the spacer peptide Y is Gln-His-His-Leu-Gly, Gln-His-His-Leu-Gly-Gly, or His-His-Leu-Gly-Gly. Also alternatively, Xaa (amino acid 1) of S2 is Ala or Ser. Alternatively, the spacer peptide comprises His-His-Leu-Gly. Alternatively and preferably, at least one of Y and S2 is free of Gln residues. Alternatively, the carboxy terminal amino acid residue of the polypeptide is Pro or Gly. Specific non-limiting examples of polypeptides include the following: Thr-Ile-Gly-Glu-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, Thr-Ile-Gly-Glu-Gly-Gln-Gln-His-His-Leu-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val, Thr-Ile-Gly-Glu-Gly-Gln-His-His-Leu-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val or Leu-Ser-Gln-Ser-Lys-Val-Gly. The patent also describes high molecular weight, biocompatible, bioadhesive, transglutaminase crosslinkable copolymers and homopolymers related to these peptides.

U.S. patent No. 5,939,385 describes biocompatible, bioadhesive, transglutaminase crosslinkable polypeptides. These polypeptides preferably have about 9-120 amino acid residues and comprise a fragment of the formula S1-Y-S2, wherein: s1 is selected from the group consisting of Ile-Gly-Glu-Gly-Gln, Glu-Gly-Gln and Gly-Gln; y is His-His-Leu-Gly-Gly or His-His-Leu-Gly; and S2 is selected from the group consisting of Ala-Lys-Gln-Ala-Gly-Asp, Ala-Lys-Gln-AIa-Gly, Ala-Lys-Gln-Ala, Ala-Lys-Gln, Ala-Lys-Ala-Gly-Asp-Val, Ala-Lys-Ala and Ala-Lys, wherein the polypeptide has an amino terminus and a carboxy terminus and is crosslinkable by transglutaminase. A preferred polypeptide is Gly-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln. Also preferred are polypeptides wherein either or both of the amino-terminus and the carboxy-terminus of the polypeptide are flanked by elastomeric polypeptides. Further provided are elastomeric polypeptides (wherein the elastomeric polypeptide is a pentapeptide or a tetrapeptide), especially a flanked polypeptide (wherein the flanking elastomeric polypeptide is Val-Pro-Gly-Val-Gly, Ala-Pro-Gly-Val-Gly, Gly-Val-Pro, Val-Pro-Gly, or any portion thereof, preferably such that the amino terminus of the flanking polypeptide is Val and the carboxy terminus of the flanking polypeptide is Gly). The patent also describes high molecular weight, biocompatible, bioadhesive, transglutaminase crosslinkable copolymers and homopolymers related to these peptides.

These patents recognize the utility of the described peptides and polymers as tissue adhesives for use in wound closure and various other medical applications. However, both patents show that the desired transition point for these peptides and polymers is 20-40 ℃ and recognize that the transition point needs to be lowered in order for the peptide/polymer to be able to react with transglutaminase in the wound site. All two patents claim: the transition temperature of a "polymer" can be adjusted by the number of polypeptide monomers that can be cross-linked by transglutaminase. As will be appreciated by those skilled in the art, for clinical applications, a reduction in the transition temperature upon application will facilitate rapid solidification of the matrix at the wound site. "

Of course, it is often not possible to remove the crosslinkable monomer in order to ensure maximum cohesive and adhesive strength of the crosslinked polymer intended for use as a bioadhesive. In fact, to maximize the cohesive and adhesive strength of such adhesives, it is generally preferred to add more crosslinkable monomer substrates. Thus, the bioadhesive potential of the polymers described in these patents is significantly limited by the transition temperature of the polymer solution.

Preferred embodiments of the present invention significantly enhance the utility of these polypeptides or polymers for use in hemostatic, adhesive and tissue sealant applications. For example, for reducing the transition point of a polymer that gels at room temperature, alternative embodiments are described below. These strategies can be used to reduce the transition point of the peptide sequences and polymers described in these patents.

Also as described in more detail below, it is preferred for embodiments of the present invention that the amount of these polymers be different with respect to the initial use described in the above patents (which do not teach nor suggest any use of the invention described herein). For example, the polymer concentration used in the tissue adhesive kit taught in these patents ranges from 5 to 100mg/ml and preferably 35-50 mg/ml. Certain embodiments of the invention include higher polymer concentrations, for example in the range of 150-250 mg/ml. Higher transglutaminase concentrations are also preferred for certain embodiments of the invention.

According to certain embodiments of the invention, additional synthetic substrates may also optionally be provided. Preferably, a short transglutaminase substrate is synthesized and then linked and/or bound to a large polymer molecule. The transglutaminase substrates are usually very short (< 20 amino acid residues). The solubility and transition point of such substrates is dependent on the polymer to which the substrate is attached. For example, if the substrate is attached to gelled gelatin, a solution of this newly synthesized molecule would require the addition of another substance to remain in fluid form at room temperature.

Non-limiting examples of this type of material are described in U.S. patent 7,208,171, which is incorporated herein by reference as if fully set forth herein, which describes the rational design of transglutaminase substrate peptides. The design strategy is based on maximizing the number of available acyl acceptor lysine peptide substrates and available acyl donor glutaminyl peptide substrates for transglutaminase cross-linking. In addition, Lys and Glu substrate peptides were designed to have the characteristics of known biomacromolecules and synthetic peptide substrates of transglutaminase. For example, based on increasing Glu repeat length, peptides became better transglutaminase substrates (Gorman, J.J.; Folk, J.E.J.biol.chem.1980, 255, 419-427 & Kahlem, P.; Terre, C; Green, H.; Djian, P.Proc.Natl.Acad.Sci.U.S.A.1996, 93, 14580-14585) and proteins known to contain two or more adjacent Glu residues were good substrates (Etoh, Y.; Simon, M.; Green, H.biochem.Biophys.Res. Commun.1986, 136, 51-56 & Hohennadl, C; Mann, K.; Mayer, U.; Tiulmson, R.; Aeschlimann, D.D.2345. J.S.S.J.2342. peptide residues). In several peptides, the Leu residue was placed adjacent to Glu near the C-terminus, as this has been shown to result in a significant increase in Glu specificity (Gross, M.; Whetzel, N.K.; Folk, J.E.J.biol.chem.1975, 250, 4648-. With regard to the Lys substrate peptide, it has been shown that the amino acid composition and sequence adjacent to lysine residues in peptide and protein substrates has an effect on amine specificity (Groenen, P.; Smulders, R.; Peters, R.F.R.; Grootjans, J.J.; Vanderijssel, P.; Blemendal, H.; Dejong, W.W.Eur.J.biochem.1994, 220, 795-. Finally, in all peptides, a Gly residue was added to the C-terminal side to act as a spacer between the peptide and the polymer in the peptide-polymer conjugate, so that the peptide in the conjugate is more accessible to the enzyme.

The transglutaminase specific assays of the peptides described in this patent show that they successfully produced acyl acceptor and acyl donor substrates with high transglutaminase binding specificity. It is suggested in the patent that these substrates can be covalently conjugated with PEG, dendrimers, chitosan, gelatin, soluble collagen, hyaluronic acid, alginate and albumin. The patent continues to show that these polymer-peptide conjugates in solution or liquid form can be used as surgical sealants and/or medical adhesives.

Although this patent describes highly specific transglutaminase crosslinkable peptide substrates, it does not teach or suggest the advanced methods of administration or material modification described as part of the present invention, nor does it teach or suggest the compositions of the present invention. However, the substrates taught by the patents may alternatively be used to enhance bioadhesives produced by transglutaminase crosslinking or otherwise producing such bioadhesives from non-transglutaminase specific polymers. These substrates will need to be combined with one or more of the other proteins or scaffolds described herein for use in the present invention.

Cross-linked materials other than transglutaminase

As indicated above, the cross-linked material preferably comprises transglutaminase, but may additionally or alternatively comprise another type of cross-linked material.

Non-limiting examples of such crosslinking agents include carbodiimides such as N, N- (3- (dimethylamino) propyl) -N-Ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) with EDC, or carbodiimides used with poly (L-glutamic acid) (PLGA) and polyacrylic acid. In another embodiment, such a cross-linking agent may comprise tyrosinase or tyrosinase together with chitosan. In another embodiment, the crosslinking (polymerization) is initiated with ultraviolet light or gamma-ray light. In another embodiment, the cross-linking agent may comprise alkylene (alkylene), citric acid (carbonic acid), or Nano-hydroxyapatite (n-HA) + poly (vinyl alcohol) (PVA).

In another embodiment, the cross-linking agent is a polyphenol of plant origin such as (i.e., hydrolyzed cinnamic acids such as caffeic acid (3, 4-dihydroxycinnamic acid), chlorogenic acid (its quinic acid esters), caftaric acid (its tartaric acid esters), and flavonoids (i.e., such as quercetin and rutin)). In another embodiment, the additional cross-linking agent is an oxidized mono-or disaccharide, an oxolactose, or a dialdehyde based on a sugar moiety (galactadiuronic acid) (GALA). In another embodiment, genipin or other iridoid glycoside derivatives or secoiridoid, preferably oleuropein, constitute the cross-linker. In another embodiment, the crosslinking agent is a thiol-reactive poly (ethylene glycol). In another embodiment, the crosslinking agent is dextran, oxidized dextran, dextran dialdehyde. In another embodiment, the crosslinking agent is a multicopper oxidase, such as laccase or bilirubin oxidase.

Illustrative compositions

The crosslinking substrate and crosslinking material described above may optionally be combined with one or more other materials to form various compositions according to the present invention. According to certain embodiments, the adhesive material may optionally and preferably comprise: (i) gelatin; (ii) transglutaminase; wherein the gelatin and transglutaminase are formed into a particle, either separately or together. More preferably, gelatin and transglutaminase are provided in amounts sufficient to be used as a sealing, hemostatic agent.

The different amounts of each and their ratios have been described previously. The transglutaminase content can optionally be increased to increase the reaction rate, or decreased to increase safety. According to certain embodiments of the invention, it is preferred to administer a 15-30% solution of gelatin followed by a 15-30% solution of transglutaminase.

In addition, the hemostatic product may also comprise one or more ofVarious supplements, for example drugs such as growth factors, polyclonal and monoclonal antibodies and other compounds. Illustrative examples of such supplements include, but are not limited to: antibiotics, such as tetracycline and ciprofloxacin, amoxicillin and metronidazole; anticoagulants, e.g. activated protein C, heparin, Prostacyclin (PGI)₂) Prostaglandins, leukotrienes, anti-transglutaminase III, ADP enzyme, and plasminogen activators; steroids, such as dexamethasone, inhibitors of inflammation, prostacyclin, prostaglandins, leukotrienes and/or kinins; cardiovascular agents such as calcium channel blockers, vasodilators, and vasoconstrictors; a chemoattractant; local anesthetics such as bupivacaine; and antiproliferative/antineoplastic drugs such as 5-fluorouracil (5-FU), paclitaxel and/or taxotere (taxotere); antiviral agents, such as ganciclovir (ganciclovir), zidovudine, amantadine (amantidine), vidarabine, ribavirin (ribavavin), trifluridine, acyclovir, dideoxyuridine, and antibodies to antiviral components or gene products; cytokines such as alpha-or beta-or gamma-interferon, alpha-or beta-tumor necrosis factor and interleukins; a colony stimulating factor; erythropoietin; antifungal agents, such as, for example, hibitane, ketoconazole (ketoconazole), and nystatin; antiparasitic agents, such as pentamidine; anti-inflammatory agents, such as alpha-1-antitrypsin and alpha-1-antichymotrypsin; anesthetics, such as bupivacaine; an analgesic; a preservative; and hormones. Other exemplary supplements include, but are not limited to: vitamins and other nutritional supplements; a glycoprotein; fibronectin; peptides and proteins; carbohydrates (mono and/or complex); a proteoglycan; anti-angiogenin (antiangiogenins); an antigen; a lipid or liposome; and oligonucleotides (sense and/or antisense DNA and/or RNA).

According to certain preferred embodiments of the present invention, compositions characterized by gelatin that has undergone thermoreversible crosslinking (as described above, some, but not all types of gelatin have undergone thermoreversible crosslinking without modification and/or using one or more additional materials) are provided. Thermoreversible gelation of animal gelatin occurs when a gelatin solution is cooled to below about body temperature (37 ℃). In a gelatin-mTG mixture at room temperature, this gelation traps the mTG in the thermoreversible gel and avoids it reacting with gelatin to form an irreversible sealing gel. Since operating room temperature is usually maintained at 22 ℃, in a clinical setting thermoreversible gelation of gelatin solution will occur rather rapidly if it is not heated continuously. This poses a problem in the haemostatic application of gelatin-mTG mixtures, as the gelatin solution must be heated before it is mixed with the mTG. Since, for example, heaters need to be added to sensitive operating room environments, it is undesirable from a logistical and safety standpoint to have to heat the gelatin immediately prior to application; this need for heating is even more problematic in emergency and/or urgent medical care situations outside the operating room.

In addition to its barrier to tissue adhesion and hemostasis, the inability of gelatin to form a solution at room temperature and to mix with microbial transglutaminase presents difficulties for other possible applications of gelatin-mTG mixtures. When, for example, gelatin-mTG gels have been used as scaffolds for cell encapsulation in tissue engineering, special care has to be taken to ensure that the gelatin solution is sufficiently cold before cell encapsulation. Furthermore, the implantation of encapsulated cells is rather complicated, since the thermoreversible gelation of gelatin takes place before the gelatin-mTG mixture is safely implanted in vivo. Similar problems exist with respect to local drug delivery, where the efficacy of certain drugs can be compromised by contact with heated gelatin and implantation of gelatin-mTG gels containing certain drugs can be hindered by thermoreversible gelation of gelatin.

Fortunately, crosslinking of gelatin via mTG occurs by linking Lys and Gln amino groups (Chen et al Biomacromolecules, vol.4, phase 6, 2003), while the amino acid group in gelatin responsible for its thermoreversible gelation is Pro & Hyp (Haug et al food hydrocolloids 18(2004) 203-. Thus, there is a potential to reduce the tendency of gelatin to thermoreversibly gel without compromising its ability to form a crosslinked gel by crosslinking of mTG. In other words, the solubility of the gelatin used in the gelatin-mTG mixture can be increased and its melting point lowered so that it forms a room temperature solution with the mTG without adversely affecting the cross-linked gelatin-mTG gel formed.

According to certain embodiments of the present invention, there is provided a composition of matter of a gelatin-mTG mixture, wherein the mixture is modified by one of a number of methods to increase the solubility of gelatin and allow gelatin to form a solution with mTG at a temperature below the normal melting point of standard animal gelatin. These compositions include (i) gelatin-mTG mixtures made using standard gelatin that has been modified to reduce its melting point; (ii) gelatin-mTG mixtures comprising additives that increase the solubility of gelatin in the gelatin-mTG mixture itself; (iii) gelatin-mTG mixtures made using commercially available gelatin products that have been processed to have lower transition temperatures; and (iv) a gelatin-mTG mixture that forms a solution under specific, carefully controlled environmental conditions (temperature, pH, ionic concentration, etc.) that lower the melting point of gelatin.

These new compositions greatly increase the utility of gelatin-mTG gels and allow for a wide range of applications (particularly in the medical field) where gelatin-mTG gels that can be formed by mixing gelatin and mTG at room temperature can be utilized. Furthermore, in many cases, gelatin and gelatin-mTG solutions formed with gelatin solutions comprising lower melting point gelatin will have the added benefit of lowering the initial viscosity of the solution, making the mTG more free to move, and increasing the rate at which the gelatin-mTG reaction occurs.

According to certain embodiments of the present invention, gelatin-mTG mixtures are also provided with additional enhancements that have the potential to improve the properties of gelatin-mTG based products. For example, the invention also features a method of further stabilizing mTG in a gelatin-mTG mixture to increase its shelf life.

In another embodiment of the invention, the plasticizer is added to the gelatin-mTG solution. Plasticizers have been shown to lower the melting point of gelatin so that it forms a solution at lower temperatures without undergoing thermoreversible gelation. One or more plasticizers are preferably added to the gelatin particles or to the gelatin solution prior to mixing the gelatin solution with the mTG or mTG solution to lower its melting point. In the case where the gelatin and mTG solutions are lyophilized, one or more plasticizers are added to the gelatin solution prior to its lyophilization. In an alternative embodiment, one or more plasticizers are added to the gelatin solution, as described previously, so that the mTG can be added at lower temperatures where it is not highly active. The gelatin-mTG-plasticizer solution may then be lyophilized or otherwise dried in already mixed form.

In a preferred embodiment, polyols or polyols are used as plasticizers. Such polyols include glycerol, xylitol, sucrose and sorbitol. Sorbitol makes the gelatin-mTG gel more elastic and more viscous. Glycerol makes the gelatin-mTG gel harder and accelerates mTG crosslinking of the gelatin. Preferred glycerol concentration ratios range preferably from about 0.5: 1 to about 5: 1 glycerol to gelatin, more preferably from about 1: 1 to about 2: 1 glycerol to gelatin, weight to weight. Preferred sorbitol concentration ratios range preferably from about 0.5: 1 to about 5: 1 sorbitol to gelatin, more preferably from about 1: 1 to about 3: 1 sorbitol to gelatin, weight to weight.

Polyols have higher boiling points when compared to monohydric alcohols of similar size. The water solubility of the polyols is higher when compared to monohydric alcohols of similar size, because there are more hydroxyl groups that attract water molecules. In the food industry, polyols such as glycerol are used to increase the water solubility of gelatin as described in U.S. patent 2,558,065 (which is hereby incorporated by reference as if fully set forth herein) and U.S. patent 3,939,001 (which is hereby incorporated by reference as if fully set forth herein where gelatin is allowed to absorb the polyol for a period of time sufficient to swell but not dissolve the gelatin capsule), these techniques and variations of these techniques described in those patents will be considered as preferred embodiments for the use of the polyols described as part of the present invention.

The effect of different concentrations of the plasticizers glycerol, xylitol, sorbitol, sucrose and trehalose on the lowering of the Transition point of Gelatin is well documented (D' Cruz NM, Bell LN. thermal unfolding of Gelatin in solutions as Affected by the Glass Transition J Food Science 2005: 70(2), Kozlov PV, Burdygina GI. structure and properties of solid Gelatin and the principles of their modification Polymer, 1983 (24): page 651-.

Although the effect of polyols on the melting point of gelatin has been well documented, they have never been used with gelatin or gelatin solutions prior to their mixing with mTG or with gelatin-mTG solutions prior to the present invention.

In embodiments where a polyol plasticizer is added, the preferred range of weight ratio of plasticizer to gelatin is preferably from about 0.5: 1 to about 1: 1 plasticizer to gelatin.

In another embodiment where a gelatin plasticizer is used to lower the melting point of gelatin in solution, the types of plasticizers used may include triethanolamine, resorcinol, thiodiglycol, the sodium salt of toluene sulfonic acid, butanediol, urea nitrate, thiourea, urea, glutamic acid, aspartic acid, valine, glycine, KSCN, KI, and LiBr.

The addition of urea to gelatin solutions has been previously investigated and demonstrated its ability to prevent the formation of thermoreversible gels at 25 ℃ in high molecular weight gelatin (99kDa) (Otani Y, Tabata Y, Ikada Y. Effect of additives on gelation and tissue adhesion of gelatin-poly (L-glutamic acid) mixtures. Biomaterials 19(1998) 2167-2173).

In a preferred embodiment, where urea is used to prevent thermoreversible gelation of gelatin or gelatin-mTG solutions at temperatures below their normal melting point, urea is added to the solution at a urea to gelatin ratio of between 0.25 and 0.2 weight/weight. Even more preferably, urea is added to the solution in a urea to gelatin ratio of from about 1: 2 to about 2: 2 weight/weight.

In another embodiment of the invention, the pH level and ionic concentration of the aqueous solvent are adjusted to increase the solubility of gelatin dissolved in the solvent. The further the product pH is from the plasma pH, the better the solubility of the gelatin will be. A preferred aqueous solvent used in this technique is Phosphate Buffered Saline (PBS). Other suitable buffers include borates, phosphates, HEPES (N- [ 2-hydroxyethyl ] piperazine-N' - [ 2-ethylsulfonic acid (ethanesulfosulfonic acid) ]), and the like.

In general, in a mixture to be used for a living organism, it is preferable to dissolve gelatin in an aqueous solvent buffered at pH5.0-9.0 and having a low to moderate ionic strength (equivalent to about 1 to 1000mM NaCl, preferably 100 to 150mM NaCl). More preferably, the pH of the solution is about 6.0 to 8.0, more preferably about 7.4. Although gelatin is soluble at these pH and ionic concentrations, its solubility can be increased by increasing the difference between the solution pH and the plasma pH of the gelatin.

One or more salts may optionally be added to lower the transition temperature of the gelatin. Preferably, the salt is added in a suitable concentration range to lower the transition temperature, more preferably to below room temperature. For example, the following salts in the indicated concentration ranges were found to lower the transition point of gelatin to below room temperature: sodium bromide (1-2M), sodium nitrate (1-2M), sodium thiocyanate (0.5-1.5M), sodium iodide (0.5-1.5M), sodium benzenesulfonate (0.5-1.5M), sodium salicylate (0.25-1M), sodium dichloroacetate (1-2M), sodium trichloroacetate (0.5-1.5M), sodium dibromoacetate (0.5-1.5M), sodium tribromoacetate (0.25-1M), sodium diiodide acetate (0.5-1.5M), sodium acetyltryptophanate (0.5-1.5M), sodium acetylenedicarboxylate (1-2M), Lithium salicylate (1-2M), Lithium diiodisalicylate (0.2-1M) (see, for example, Bello J, Riese HCA, vinylid modulus of Gelation of Gelatin (chemical gelatine of Gelatin and Gelatin of Gelatin electrolysis of Gelatin gels Influence of the melting point of the gel) Am Chem soc.1956 month 9 (60) 1299-1306).

Optionally and preferably, one or more acidic substances are added to the composition to lower the pH. Lowering the pH of the gelatin solution lowers its transition point. Generally, lowering the gelatin transition point is useful for reconstituting gelatin in vivo at 37 degrees. For certain preferred classes of gelatin used herein, lowering the pH provides better results for the transition point of the gelatin when, for example, from a mammalian source. However, for certain types of gelatin, raising the pH may alternatively provide better results.

In another embodiment, which increases the pH difference between the gelatin solution and the gelatin plasma spot, the gelatin itself is modified. This can be accomplished by treating it to produce electrostatically charged gelatin prior to dissolving it in a solution as in U.S. patent 6,863,783 (which is hereby incorporated by reference as if fully set forth herein) or by controlling the isoelectric point of gelatin as in U.S. patent 2,398,004 (which is hereby incorporated by reference as if fully set forth herein)

If the pH of the gelatin solution is raised rather than lowered, the pH of the solution will be closer to the plasma point of the gelatin and the transition point will be raised. This modification can alternatively be used to maintain the uncrosslinked gelatin as a thermoreversible gel after implantation in vivo.

In another embodiment of the invention, gelatin, mTG or both have undergone drying after mixing with trehalose saccharides or other saccharides to stabilize the protein or enzyme in its active form so that it can be easily reconstituted. Different embodiments of drying are freeze drying, spray drying, roller drying, air drying, heat drying, vacuum drying or any other method of drying a gelatin-trehalose or gelatin-mTG-trehalose solution.

The excellent stabilizing ability of trehalose in air drying and freeze drying has been well documented. It has been shown that dried material undergoes more rapid reconstitution when dried after trehalose has been added to a particular material or solution (crown LM, Reid DS, crown jh. isotuhose Special for preference Dry Biomaterials.

Drying a protein solution containing trehalose at ambient temperature and atmospheric pressure is well described in U.S. Pat. No. 4,891,319. In the examples described there, the functionality of the dried protein is preserved.

In a particular example of Gelatin, the addition of trehalose to a Gelatin Solution has the additional benefit of increasing the strength of the Gelatin gel (Norie N, Kazuhiro M, Masami N, Yusuke O, Takasio, Keiko N.factors influencing the Gelation of a Gelatin Solution in the presence of Sugar. Journal of Home Economics of Japan.55 (2): page 159-166 (2004)).

In addition, the gelatin-mTG cross-linking reaction has many similarities to the cross-linking reaction of natural blood factors. Trehalose drying to stabilize blood factors has recently been demonstrated (U.S. Pat. Nos. 6,649,386 and 7,220,836) and is in the process of being commercially used to prepare products with readily reconstitutable blood proteins (ProFibrix)^TM，Leiderdorp)。

In another embodiment of the invention, the gelatin is dried in the presence of a sugar. This may include the nebulization of gelatin on different supports (e.g. sugar, maltodextrin or starch).

In an alternative embodiment of the invention, a so-called Cryogel produced by PB Gelatins (Tessenderlo Group, Belgium) is used^TMThe commercial gelatin product of (1). Cryogel is lower than an equivalent but untreated gelatinIs soluble at a temperature of 5-6 ℃. The precise processing for Cryogel production is proprietary.

In yet another embodiment of the present invention, the composition may optionally be characterized by one or more additional substances. For example, the composition may optionally comprise a denaturant, including but not limited to one or more of guanidine hydrochloride or urea. The composition may also alternatively, alternatively or additionally comprise a reducing agent, including but not limited to one or more of magnesium chloride or hydroquinone. The composition may also alternatively, alternatively or additionally comprise a substance such as isopropanol in order to increase hydrogen bond formation. The composition may alternatively, alternatively or additionally also comprise a protic polar solvent, preferably a solvent capable of structurally interacting with proteins and preventing helix formation in gelatin, such as DMSO (dimethyl sulfoxide). The composition may also alternatively, alternatively or additionally comprise a desiccant that releases heat upon entering solution, such as calcium chloride.

According to certain embodiments, the invention also features a gelatin-specific protease that includes an enzyme or mixture of enzymes that can rapidly break down gelatin molecular chains but do not adversely affect the natural fiber-based coagulation network.

Gelatin-specific proteases may alternatively be used to remove bandages/dressings/absorbable hemostats/sealants from a wound site without damaging the native endogenous fibrin clot and causing re-bleeding. This feature is an additional benefit of the present invention compared to existing products and addresses the technical problem that hemostatic dressings that are viscous enough to adhere well to a wound site and stop bleeding cannot be removed without removing or disrupting the fibrin clot. While at least certain embodiments of the bandage according to the present invention are absorbable, there may be a need to remove it from a wound or to place the bandage in a different location if the surgeon wishes to perform a procedure on the wound site.

An exemplary, non-limiting protease is proteinase K (Chen, et al, Biomacromolecules 2003, 4, 1558-. Alternatively, however, other proteases, especially faster acting enzymes, may be used.

According to other embodiments, one or more protease inhibitors, including but not limited to aprotinin, tranexamic acid, alpha-2 plasmin inhibitor, alpha-1 antitrypsin, or Pittsburgh mutant of alpha-1 antitrypsin (Arg-358 alpha-1 antitrypsin; see Owen et al, N.Engl. J.Med.309: 694-. In a preferred embodiment, aprotinin is included in an amount sufficient to provide a final working concentration of 1500-20,000 KIU/mL.

According to other embodiments, the hemostatic material of the invention may further comprise an additional hemostatic substance in addition to the gelatin and TG. Such a substance may be biological or synthetic in nature and may include, but is not limited to, one or more known hemostatic agents, such as albumin, collagen, fibrin, thrombin, chitosan, ferric sulfate, or other metal sulfates.

According to yet some further embodiments, the hemostatic material of the present invention may further comprise an accelerant for accelerating crosslinking when the crosslinking material (such as transglutaminase) is combined with gelatin. Such promoters may optionally include, for example, calcium.

Calcium is the preferred component of the transglutaminase/gelatin crosslinking reaction. Different studies have demonstrated that different calcium concentrations and/or the addition of calcium-mobilizing drugs, including but not limited to, sargassum (MTX), can accelerate the transglutaminase coagulation reaction. Thus according to an embodiment of the invention, calcium and/or a calcium mobilization drug is included, but optionally not used. These modifications using calcium were used for calcium dependent transglutaminase and not for calcium independent transglutaminase.

According to yet some further embodiments, the hemostatic material of the invention may further comprise a material for inducing an exothermic reaction, preferably upon combining a cross-linking material (such as transglutaminase) with gelatin. Induction of the exothermic reaction can selectively and preferably support crosslinking even under ambient conditions, where "ambient" can alternatively be defined as any environment having a temperature of less than about 30 ℃. Such exothermic agents may optionally comprise, for example, one or more of calcium, chlorine-containing molecules (e.g., calcium chloride or magnesium chloride), metal oxides/zeolites, or combinations thereof.

Preparation of the composition

The compositions described herein may alternatively be prepared according to one or more of the various methods of the various embodiments of the present invention. In one embodiment of the invention, the gelatin in the gelatin-mTG mixture is subjected to a very specific drying process involving the use of heat prior to its mixing with the mTG. These drying methods increase the solubility of gelatin by lowering its melting point (preferably to below operating room temperature). The drying process can increase the solubility of gelatin without any additives and without changing the environmental conditions under which the gelatin or gelatin-mTG solution is formed. However, the addition of certain additives, such as plasticizers or stabilizers, or the modulation of certain environmental factors, such as the temperature, ionic strength, and osmotic pressure of the gelatin or gelatin-mTG solution, can be used to further enhance the properties of a gelatin-mTG mixture that already contains gelatin dried using techniques that lower its melting point.

In a preferred embodiment of heat dependent gelatin drying to produce gelatin that can form a solution with mTG at reduced temperatures, a pure gelatin solution having a water content of at least 35% is sprayed onto an excess of finely divided solid gelatin particles comprising less than 8% water at a temperature above the gelation and setting temperatures. The granules are then dried in a fluidized bed to a water content of 8-13%. This process, and variations of this process that are also included within the scope of the present invention, are described in detail in U.S. Pat. No. 4,889,920, which is hereby incorporated by reference as if fully set forth herein.

In yet another preferred embodiment of heat dependent gelatin drying to produce gelatin that can form a solution with mTG at a reduced temperature, gelatin having a water content of more than 8% by weight based on the total weight of gelatin and water is subjected to microwave heating to remove at least 35% of said water content to obtain gelatin having a water content of no more than 16% by weight based on the total weight of gelatin and water. This process, and variations of this process that should also be considered part of the present invention, is described in detail in U.S. patent 4,224,348, which is hereby incorporated by reference as if fully set forth herein.

In yet another preferred embodiment of heat dependent gelatin drying to produce gelatin that can form a solution with mTG at reduced temperature, the gelatin is dried at 100 ℃ under reduced pressure as described in U.S. patent 2,803,548 (which is hereby incorporated by reference as if set forth fully herein). This process changes the gelatin chains themselves so that they cannot be thermoreversibly gelled. Although mTG crosslinking of gelatin is not dependent on the ability of gelatin to form thermoreversible gels, this drying process results in weakening of the gelatin chains and, therefore, any gels produced by the use of such gelatin in gelatin-mTG crosslinking.

In another embodiment of the invention, the gelatin in the gelatin-mTG mixture is subjected to a very specific drying process involving the use of freeze-drying prior to its mixing with the mTG. These drying methods increase the solubility of gelatin by lowering its melting point (preferably to below operating room temperature). The drying process can increase the solubility of gelatin without any additives and without changing the environmental conditions under which the gelatin or gelatin-mTG solution is formed. However, the addition of certain additives, such as plasticizers or stabilizers, or the modulation of certain environmental factors, such as the temperature, ionic strength and osmotic pressure of the gelatin or gelatin-mTG solution, can be used to further enhance the properties of a gelatin-mTG mixture that already contains gelatin dried using freeze-drying techniques that lower its melting point.

In a preferred embodiment of freeze drying gelatin to produce gelatin that can form a solution with mTG at reduced temperature, gelatin dissolved in water at a concentration of 0.1-2% by weight is freeze dried under reduced pressure. This process, and variations of this process that should also be considered part of the present invention, is described in detail in U.S. patent 2,166,074, which is hereby incorporated by reference as if fully set forth herein.

In another embodiment of the invention, once the gelatin and mTG have been mixed in solution, the gelatin-mTG mixture is subjected to freeze-drying. This lowers the melting point of the gelatin to produce a homogeneously mixed freeze-dried gelatin-mTG mixture in which the gelatin in dry form is contacted with the mTG in dry form. In this embodiment, the gelatin and mTG are reconstituted simultaneously from a freeze-dried state and immediately form a solution at the site of reconstitution. This technique can be preferentially used with gelatin or gelatin mixtures that already have a lower melting point than standard gelatin, since the activity of mTG decreases exponentially at lower temperatures (below about 37 ℃).

Thus a solution consisting of the reduced melting gelatin and mTG can be formed at low temperature without rapid crosslinking and gelation occurring. This solution can then be freeze dried, resulting in a dry mixture of homogeneously distributed gelatin and mTG. This mixture can be rapidly reconstituted to form a gel upon contact with a warmer solvent. This technique can be used preferentially in wound dressings, where the body fluid can reconstitute the gelatin and mTG at its normal temperature of 37 ℃.

Preferably, according to certain embodiments of the present invention there is provided a gelatin-mTG particle mixture for hemostatic or tissue sealing purposes, wherein the gelatin and mTG are spray dried together to produce a well-dispersed powder comprising gelatin and mTG in concentrations suitable for hemostatic or tissue sealing material application.

In another embodiment of the invention, in order to increase its solubility. The gelatin used as part of the gelatin-mTG mixture has been hydrolyzed, partially hydrolyzed, or a percentage of the gelatin mixture has been hydrolyzed or partially hydrolyzed. An example of such a technique has been successfully demonstrated in a process involving coating standard gelatin particles with a hydrolyzed gelatin film (U.S. patent 4,729,897, which is hereby incorporated by reference as if fully set forth herein). This embodiment may include the use of gelatin that has been hydrolyzed or partially hydrolyzed in the presence of a plasticizer, which may include polyols, sugars, or other plasticizers as previously described.

In another embodiment of the invention, a solution comprising premixed mTG and gelatin or other protein hydrolysates is lyophilized to increase the stability of the compound. This technique, as used in preparing compositions for use in food processing, is described in U.S. patent 6,030,821, which is hereby incorporated by reference as if fully set forth herein.

In another embodiment of the invention, the gelatin used as part of the gelatin-mTG mixture is spray dried after mixing with an acid to form a dilute acidic gelatin solution (where the acid is maintained at 5-20% of the gelatin level) so that fine droplets are formed for further drying. This process is described in canadian patent 896,965, which is hereby incorporated by reference as if set forth fully herein.

In yet another embodiment of the invention, one or more of the techniques described above for strengthening a product comprising gelatin and mTG are used together or sequentially. This may preferably involve the use of two or more plasticisers together in the gelatin or gelatin-mTG solution before it is dried using one of the described drying methods. It may also comprise drying the gelatin or gelatin-mTG using a drying technique, dissolving the dried gelatin or gelatin-mTG in a solution, and then drying the gelatin or gelatin-mTG again.

These methods may also optionally be used for compositions that undergo thermoreversible gelation, preferably including, for example, compositions for containing various combinations of non-gelatin proteins and optionally other cross-linking agents (other than transglutaminase).

Bandage

An exemplary embodiment of the invention is directed to a hemostatic dressing (e.g., a hemostatic dressing for treating a wound tissue in a patient) comprising gelatin and transglutaminase, preferably separately, until their interaction is required or desired for the activity of the bandage. The bandage may optionally feature a non-absorbent pad, such as a plastic pad. The bandage may optionally feature a layer of absorbable material.

Another exemplary embodiment of the present invention is directed to a hemostatic dressing for treating a wound tissue in a patient, which may optionally and preferably comprise: (i) a gelatin layer; (ii) a transglutaminase layer adjacent to the gelatin layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the gelatin layer.

Another exemplary embodiment of the present invention is directed to a hemostatic dressing for treating a wound tissue in a patient, which may optionally and preferably comprise: (i) a layer of absorbable or non-absorbable material; (ii) a gelatin layer adjacent to the material layer; (iii) a transglutaminase layer adjacent to the gelatin layer, wherein the transglutaminase layer is co-extensive or non-co-extensive with the gelatin layer.

Another exemplary embodiment of the present invention is directed to a hemostatic dressing for treating a wound tissue in a patient, comprising: (i) a first gelatin layer; (ii) an absorbable material layer adjacent to the first gelatin layer; (iii) (iii) a transglutaminase layer adjacent to the absorbable material layer, and (iv) a second gelatin layer adjacent to the transglutaminase layer, wherein the transglutaminase layer is non-coextensive with the first and/or second gelatin layers.

According to certain embodiments, the present invention provides a hemostatic dressing (e.g., a bandage) comprising a transglutaminase layer sandwiched between first and second gelatin layers, wherein the transglutaminase layer is co-extensive or non-co-extensive with the first and/or second gelatin layers. Such hemostatic dressings are useful for treating wounds. The non-coextensive model provides the advantage of layering of the inhibition layer compared to dressings in which the transglutaminase layer is coextensive with the entire first and second gelatin layers. However, the hemostatic properties of the co-expanded model may be superior to those of the non-co-expanded model

According to other embodiments of the present invention there is provided a dressing of the present invention, which may optionally and preferably comprise: (i) an absorbable or non-absorbable matrix; (ii) gelatin; (iii) transglutaminase, wherein a gelatin and a transglutaminase layer are contained in the matrix.

In another embodiment, a hemostatic device comprises: (i) a porous, absorbable or non-absorbable matrix; (ii) gelatin; (iii) transglutaminase wherein the gelatin and transglutaminase layers are bound to said substrate.

In various embodiments, the transglutaminase layer may be shaped in any of a variety of shapes and patterns. For example, and without limitation, the transglutaminase layer may be shaped into a lattice comprising transglutaminase, or into a single spot comprising transglutaminase. Alternatively, the transglutaminase layer may be shaped into a plurality of lines comprising transglutaminase.

Each layer of the hemostatic dressing may also optionally include one or more suitable fillers, binders, and/or solubilizers. In addition, each hemostatic dressing optionally further comprises a release layer comprising a release agent and/or a backing material.

According to a preferred embodiment, each layer of the hemostatic dressing optionally comprises one or more suitable fillers such as sucrose. Each layer of the hemostatic dressing may also optionally contain one or more suitable binders such as sucrose. Each hemostatic dressing may also optionally further comprise a release layer comprising a release agent. An exemplary release agent is sucrose. Each layer of the hemostatic dressing may also optionally contain one or more suitable solubilizing agents, such as sucrose.

Without wishing to be bound by a single hypothesis, the properties of sucrose as part of the invention may alternatively be determined at least in part by the amount added. At relatively high concentrations (20-30% sucrose solution), it can be sprayed onto a surface (e.g., a bandage) to prepare the surface for application of another solution to be bonded (e.g., gelatin or mTG solution). Sucrose may be added to the gelatin or mTG solution at a lower concentration (e.g., around 2%) to help adhere such solution to a surface (e.g., a bandage).

Each layer of the hemostatic dressing may also optionally contain one or more suitable foaming agents such as a mixture of citric acid and sodium bicarbonate.

Each hemostatic dressing may also further include a backing material on a side of the dressing opposite the side facing the wound when the dressing is in use. The gasket material may be secured with a physiologically acceptable adhesive or may be self-adhering (e.g., by having a surface static charge). The backing material may be an absorbable or non-absorbable material, such as a silicone or plastic sheet, and/or may alternatively be inserted into a bodily device such as a vascular catheter and/or other type of medical device.

The transglutaminase layer can be applied to the first gelatin layer such that it is non-coextensive with the first gelatin layer and/or non-coextensive with the second gelatin layer when the second gelatin layer is applied. For example, the transglutaminase layer can comprise from about 5% to about 95% of the surface area of the first gelatin layer and/or from about 5% to about 95% of the surface area of the second gelatin layer. The transglutaminase can be applied to the gelatin layer as a single spot or a series of spots on the gelatin layer such that the total surface area of the transglutaminase spots comprises from about 5% to about 95% of the surface area of the first gelatin layer and/or from about 5% to about 95% of the surface area of the second gelatin layer.

Such a single point or a plurality of points of transglutaminase may have any geometric shape, such as filled or unfilled circles, rectangles, triangles, lines, irregular shapes, or combinations thereof. Such dots may be applied to the first gelatin layer in an ordered or random pattern. Provided that the total surface area of the transglutaminase is from about 5% to about 95% of the surface area of the first gelatin layer and/or from about 5% to about 95% of the surface area of the second gelatin layer, the plurality of dots can form any of a variety of shapes and patterns, such as an array, a grid, a series of concentric dots (e.g., concentric circles or squares), a staggered series of dots (e.g., staggered circles), a spoke radiating from one axis, or any other configuration. Generally, a large number of small dots is preferred over a small number of large dots. For example, a 20 by 20 lattice is generally preferred over a 10 by 10 lattice for the same total surface area. However, the dots can have any size provided that the total surface area of the transglutaminase is from about 5% to about 95% of the surface area of the first gelatin layer and/or from about 5% to about 95% of the surface area of the second gelatin layer. For example, depending on the overall size of the dressing, the dots may be (without limitation) at least about 0.01, 0.1, 0.5, 1, 2,3, 4,5, 6, 7, 8, 9, 10mm or more in diameter, width, or length. In one embodiment, for example, 4 circular spots having a diameter of 2-3mm may each occupy one square centimeter of the dressing. Various other configurations are within the scope of the present invention and are readily available to those skilled in the art.

The dressing may alternatively be manufactured in any of a variety of sizes and shapes. Typically the dressing is of a size and shape that is easily handled by those skilled in the art, typically less than 12 "in length along either side, for example 1" x1 ", 1" x2 ", 4" x4 "and the like. The moisture level of the dressing is typically less than 8% (e.g., less than 7, 6,5, 4,3, 2, or 1%).

Any of a variety of absorbable materials known to those skilled in the art may alternatively be used in the present invention. For example, the absorbable material may be a proteinaceous material such as fibrin, keratin, collagen and/or gelatin, or a saccharide material such as alginate, chitin, cellulose, proteoglycans (e.g., poly-N-acetylglucosamine), glycolic acid polymers, lactic acid polymers or glycolic acid/lactic acid copolymers. For example, the absorbable material may be a carbohydrate. Illustrative examples of absorbable materials are sold under the trade names vicryl.

In general, the different layers of the hemostatic dressing may be secured to one another by any means known and available to those skilled in the art. For example, the gelatin layer and/or transglutaminase layer may alternatively and preferably be applied as a series of rapidly frozen aqueous solution layers and subsequently lyophilized or freeze-dried (e.g., after application of each layer and upon assembly of the overall dressing). The layer can be applied by any of a variety of techniques including spraying, pipetting (e.g., with a multi-channel pipette), spraying, using a mask, electrostatic deposition, using a microinjection dot matrix system, or using a dispersion manifold with wells to produce a high density dot matrix.

In certain embodiments of the invention, when the dressing is prepared using a mold, a release agent, such as sucrose, is applied to the mold prior to application of the first layer of the dressing. In such embodiments, the hemostatic dressing further comprises a release layer comprising the release agent.

Alternatively, a physiologically acceptable adhesive may be applied to the absorbable material and/or the liner material (when present) and the gelatin layer and/or transglutaminase layer which are subsequently immobilized thereto.

In one embodiment of the dressing, the physiologically acceptable adhesive has a shear strength and/or structure such that the absorbable material and/or backing material can be separated from the gelatin layer after application of the dressing to the wound tissue. In another embodiment, the physiologically acceptable adhesive has a shear strength such that the absorbable material and/or the backing material is not separable from the gelatin layer after application of the dressing to the wound tissue.

The concentration of gelatin per wound area depends on many factors including, but not limited to, the final structure of the bandage, the materials used, and the like.

According to other embodiments of the present invention, there is provided a method of making a hemostatic dressing by optionally and preferably providing a first gelatin layer, applying a transglutaminase layer to the first gelatin layer, and applying a second gelatin layer to the transglutaminase layer, wherein the transglutaminase layer is non-coextensive with the first gelatin layer and/or non-coextensive with the second gelatin layer.

Similarly, other embodiments of the present invention include a method of making a disposable absorbent article by providing an absorbent or non-absorbent backing layer having a first gelatin layer adhered thereto; applying a transglutaminase layer to the first gelatin layer on a side of the gelatin layer opposite the side attached to the absorbable or non-absorbable liner layer; and a method of preparing a hemostatic dressing by applying a second gelatin layer to a transglutaminase layer, wherein the transglutaminase layer is non-coextensive with the first gelatin layer and/or non-coextensive with the second gelatin layer.

Hemostatic device

Another exemplary embodiment of the present invention is directed to a hemostatic device (e.g., for hemostasis in a surgical environment of a rapidly bleeding patient) comprising: (i) a porous absorbable or non-absorbable matrix; (ii) gelatin in powder, granule or other solid form; (iii) transglutaminase in powder, granule or other solid form; wherein gelatin and transglutaminase are contained in the matrix.

Another embodiment of the invention is directed to a hemostatic device (e.g., for hemostasis in a surgical environment of a rapidly bleeding patient) comprising: (i) a porous absorbable or non-absorbable matrix; (ii) gelatin; (iii) transglutaminase; wherein gelatin and transglutaminase are bound to the substrate.

Other embodiments of the invention include the application of the hemostatic/sealant mixture by methods that have accepted the application of sealants. Such methods may optionally include the application of the mixture as part of a gelatin, foam or spray. The application of the hemostatic/sealant mixture using these methods may optionally be accomplished, for example, by storing the mixture components separately and mixing them immediately prior to application; and/or for example optionally by storing the components together in an inactive form and activating them immediately prior to administration. The inactive form of the sealant component can optionally be provided as one or more of a frozen solution, a lyophilized powder that requires reconstitution, a spray dried powder that requires reconstitution, and/or any other suitable form of inactive sealant compound.

Hemostatic device preparation

According to certain embodiments of the present invention, freeze-drying and/or lyophilization techniques may optionally be applied to adhere or secure the seal material composition according to the present invention to the surface of any catheter, trocar, or implant, or indeed any other such medical device. This optionally promotes hemostasis of the penetrated wound and its closure, which optionally may be useful for, for example, arterial catheters/devices. Hemostasis after arterial surgery is critical for patients who have been treated with anticoagulant drugs and are more prone to bleeding complications. The hemostatic compositions of the present invention are independent of blood clotting, thus providing additional assistance in avoiding excessive bleeding.

Use of devices, compositions or bandages

During use of the hemostatic dressing, device, or agent, the patient's endogenous fluids (e.g., blood, gas, bile, intestinal fluid) that leak through a bleeding or exuding wound when the dressing, device, or particle mixture is applied to the wound tissue may be activated by gelatin and transglutaminase. Alternatively, where the fluid lost from the wound tissue is insufficient to provide sufficient hydration of the protein layer, the gelatin and/or transglutaminase may be activated by applying a physiologically acceptable liquid (e.g., water, buffer, saline), optionally containing any necessary co-factors and/or enzymes, prior to applying the hemostatic dressing, device, or agent to the wound tissue.

The principles and operation of the present invention may be better understood with reference to the drawings and the accompanying description.

Referring now to the drawings, FIG. 1 is a schematic block diagram of an exemplary bandage in accordance with the present invention. As shown, bandage 100 preferably features at least one and preferably a plurality of gelatin layers 102 (shown as two such layers for purposes of illustration only and not in any limiting sense). Also preferably provided is at least one transglutaminase layer 104; in this example, the transglutaminase layer 104 is shown sandwiched between a plurality of gelatin layers 102 for illustrative purposes only and not for any limiting purpose. Also shown is a suitable pad 106 that preferably provides mechanical strength to the bandage 100. The liner 106 may alternatively be formed as a polyol-acid web or sheet, such as, for example, as Dexon^TMOr Vicryl^TMProvided is a method for preparing a composite material.

FIG. 2 shows a front view of an exemplary bandage according to the present invention covered with an optional absorbent pad and an optional plastic envelope;

fig. 3 is a schematic block diagram of an exemplary hemostatic device according to the present invention, the device comprising a porous matrix. As shown, the hemostatic device 300 preferably features at least one and preferably a plurality of gelatin layers 302 (shown as two such layers for illustrative purposes only and without any limiting intent). Also preferably provided is at least one transglutaminase layer 304; in this example, for purposes of illustration only and not for any limiting purpose, transglutaminase layer 304 is shown sandwiched between gelatin layers 302. Also shown is a suitable liner 306 that preferably provides mechanical strength to bandage 300. The pad 306 may alternatively be made of any type of biodegradable material.

Referring now to the examples below, various compositions according to the present invention were constructed and tested for their ability to reduce bleeding and induce hemostasis. In experimental animals, the tested compositions were found to be very powerful and capable of stopping bleeding even arterial bleeding.

Example 1

Preparation of the exemplary illustrative adhesive

This example relates to the preparation of an exemplary illustrative, non-limiting adhesive according to the present invention. This example used a calcium-independent microbial transglutaminase having a specific activity level of 100U/gm (batch No. L-04207, Ajinomoto USA, Chicago, IL). Also, the gelatin tested was type a gelatin from pig skin, 300 bloom (Sigma-Aldrich, st.

The following methods were used to prepare the exemplary illustrated adhesives: 20% w/w gelatin in PBS was prepared (phosphate buffered saline; 20g gelatin was added to 80g PBS). Next, 20% w/v mTG in PBS was prepared (1 mg of mTG was added to 5mL of PBS). Thereafter 5g of gelatin solution was mixed with 0.5mL of mTG solution (in other words, a ratio of 10: 1).

Figure 4 shows the effect of different percentages of gelatin on the bond strength of the adhesive. The bond strength was measured by bonding a pigskin sample to a second such sample, placing a weight of 47.5g on the joint, and immediately thereafter immersing it in water for 120 minutes. After the immersion period, a pull force was applied at 5 mm/min to determine the maximum bond strength (Mcdermott et al, Biomacromolecules2004, 5, 1270-.

As confirmed in FIG. 5, the optimum reactivity level of microbial transglutaminase is in the range of 50-55 ℃. At physiological levels of 37 ℃, the level of reactivity is only about 60% of the optimal level. Thus, the use of an exothermic agent to raise the temperature of the reaction will raise the reactivity level and thereby accelerate the crosslinking of the gelatin. Thus, alternatively and preferably, an exothermic agent is part of the present invention.

Calcium may preferably be used as part of such an agent because calcium chloride releases heat when dissolved but not hot enough to damage tissue. Also, as previously mentioned, calcium may help to accelerate the reaction in other ways that do not rely on its exothermic dissolution.

Alternatively, a non-toxic exothermic material may be included in a bandage having one or more cross-linking factors. Alternatively or additionally, one or more non-absorbable exothermic agents may optionally be added after the bandage pad, as previously described.

Example 2

In vitro burst pressure test

This example demonstrates the ability of the composition according to the invention to resist rupture as a representative of its resistance to high pressure arterial blood flow. As described below, to simulate high pressure blood flow, a burst pressure system was developed to apply pressure to the wound on the pigskin sample with hot PBS instead of blood. Since physiological blood pressure is almost always less than 200mmHg, a resistance to a pressure of 200mmHg for 2 minutes is considered a successful criterion. These burst test results demonstrate that the compositions according to the invention are suitable for use in the treatment of blood flow, including high pressure arterial flow.

Most samples (8/10) were resistant to pressure of 200mmHg for 2 minutes. Those samples that failed may be associated with human or systematic errors, with an average burst pressure of 320 ± 50mmHg, but this figure is conservative, since samples that failed to burst are assigned a value of 354mmHg, since 354mmHg is the maximum pressure that can be measured by laboratory equipment. These results demonstrate the ability of adhesive compositions according to certain embodiments of the present invention to be used for hemostatic purposes, even under stringent test conditions.

Material

Gelatin (from porcine type a, bloom 300) obtained from Sigma-Aldrich (st. louis, MO). The calcium-independent microbial transglutaminase (mTG) mixture TG-TI was obtained from Ajinomoto and used without further purification. The manufacturer reports that these enzymes have a specific activity of 100U/gm. The pigskin tissue was purchased from a local grocery store.

Sample preparation

The pigskin was treated with dilute NaOH for one hour before being cut into disc shapes with a diameter of about 6-6.5 cm. The skin was degreased with a scalpel. A2 mm hole was made in the center of the skin section to simulate a wound. The skin was washed with a large amount of water and PBS buffer, and stored in a petri dish with about 1mL of PBS buffer to moisturize the skin until use. For all experiments described herein, Dulbecco's phosphate buffered saline with a pH of 7.4 was used as PBS buffer.

Gelatin solutions in PBS buffer (25% w/w) were freshly prepared daily and stored at 65 ℃ before use. A stock of mTG (20% w/w) in PBS buffer was prepared and dispensed in 2ml vials, stored at-18 ℃. The enzyme solution was thawed at room temperature before use.

The skin surface was wiped dry with a laboratory tissue wipe prior to application of the adhesive. The adhesive was prepared by mixing 1ml of gelatin and 0.5ml of mTG in a 2ml vial. Two different compositions were prepared. Composition "A" used transglutaminase from Ajinomoto, while composition "B" used transglutaminase from (Yiming Biological Products Co. (Jiangsu, China; preferred Products as described above.) 0.6ml of the resulting mixture (exemplary tissue adhesive according to the invention) was applied on the pig skin surface, covering the wells.

Burst test

The self-made device was equilibrated in warm buffer (44 ℃) prior to assembly. After the incubated skin was quickly loaded into the device, about 50ml of 42 ℃ PBS buffer was poured into the device onto the skin tissue. The nitrogen flow was manually controlled to increase the pressure. The overall procedure for the burst test is as follows:

step 1-increase pressure to 200mmHg and hold for 2 minutes;

step 2-increase pressure to 300mmHg and hold for 2 minutes;

step 3-increase pressure to > 354mmHg (maximum pressure measurable).

As a control, a pure gelatin solution was applied on the skin and allowed to gel (i.e. set) at room temperature for 30 minutes by forming a physical gel. Gelatin-warm refers to the use of a buffer solution at 42 ℃ that dissolves the physical gelatin gel.

Results

Fig. 6 shows representative burst pressure measurements for tissue adhesives based on composition a. Data for samples #4 and #5 are shown in figure 6. A summary of the burst test results for composition a is given in table 2, while a complete list of samples is shown in table 3.

TABLE 2 summary of rupture test results for Compound A

^*The "unbroken" sample takes a value of 354mmHg (maximum pressure measurable).

TABLE 3 rupture test results for samples of composition A

^aAt 200mmHg the device began to leak gas. Tightening the device increases the pressure but may also deform the skin, causing the adhesive to rupture at 232 mmHg.

FIG. 7 shows representative burst pressure measurements for tissue adhesives based on composition B; data for samples #4 and #5 are shown in figure 7. The results for the complete list of samples are shown in table 4.

TABLE 4 rupture test results for samples of composition B.

Sample #	200 mmHg	300 mmHg	Burst pressure (mmHg)	Type of rupture	Remarks for note
						1*		2 minutes	Maximum of
2	2 minutes	2 minutes	Maximum of
						3*			Maximum, 2 minutes
4	2 minutes	-	315	Cohesive	44℃PBS
						5	2 minutes	2 minutes	Maximum of

^*The pressure of these samples was set above 200mmHg inadvertently because the pressure was manually controlled and there was no pressure relief valve.

Example 3

Hemostasis in rat model

This example provides an initial in vivo demonstration of a gelatin-mTG composition for achieving hemostasis in a living animal according to the present invention. The rats were adult female Syrian rats.

Material

A gelatin solution characterized by 25% w/w gelatin in pBS (porcine, type a, 30 bloom, from Sigma-Aldrich (st. louis, MO)) was used. The solution was mixed by mixing heated (50 ℃) PBS into the gelatin powder while stirring manually with a spatula. Prior to administration, the gelatin solution was stored in a capped 5mL syringe immersed in a water bath at 50 ℃ to maintain its liquid phase.

Transglutaminase (mTG) solution was 20% w/w microbial transglutaminase (Activa WM, Ajinomoto) in PBS^TM) Is characterized in that. The mTG solution was kept at room temperature.

Prior to administration, 1mL of gelatin solution was added to 0.5mL of mTG solution in a 2mL leppendorf tube. The tube was inverted 2-3 times to mix the solution and then the solution was applied to the wound site with a 1mL pipette tip. This is the experimental solution.

For the control solution, the procedure was repeated without adding mTG solution, so that gelatin was administered alone.

For the application of the experiments and controls, the pipetting head was cut off approximately 1/2cm from the end to enlarge the opening and to allow the passage of the viscous gelatin-mTG solution.

Liver trauma

For experimental and control administration, the left lobe of the liver was cut using a scalpel in the mouth-to-tail (rostral-to-caudal) direction, resulting in a sagittal incision 1cm long and 1/2cm deep. After approximately 10 seconds of bleeding, accumulated blood was removed immediately prior to the application of the gelatin (control) or gelatin-mTG (experimental) solutions using cotton gauze.

First, the test solution was applied to the incision on the left side of the leaf. Gel formation occurs about two minutes after administration and bleeding is completely stopped in less than about 2.5 minutes after administration. After 5 minutes, the tissue was shaken vigorously and a pulling force was applied across the wound site using forceps, but the gel was still intact and the wound closed. Fig. 8 is a photograph showing the formation of a gel and the induction of hemostasis (fig. 8A shows the full area and fig. 8B shows a portion of the area, which is exaggerated for further details).

After that, the control solution was applied to the incision on the right side of the leaf. No gel is formed and the solution is almost flushed out of the wound site by the blood flow. Even after 6-7 minutes, no blood clot had formed and the liver continued to bleed (fig. 9A).

The control solution was removed and then the test solution was applied to the wound site without removing the accumulated blood. Although the accumulated blood significantly hindered the adhesion of the experimental solution to the liver, a gel was formed that significantly slowed blood flow after about one minute and completely stopped blood flow after 4.5 minutes (fig. 9B). This demonstrates that the compositions of the present invention slow blood flow and induce hemostasis even in the presence of accumulated blood.

Femoral artery cutting

The left femoral artery of the rat was severed using a surgical scalpel. After approximately 10 seconds of severe bleeding, accumulated blood was removed immediately prior to the application of the gelatin-mTG (test) solution using cotton gauze. When the solution was applied, the blood was mixed with the experimental gel as it underwent gelation. Under such stringent conditions, the gel still completely stopped bleeding in less than 3 minutes. After 5 minutes, the gel was manually examined using forceps. The gel is significantly less stiff and non-sticky when it is mixed with a large amount of blood, but still forms a strong blood clot on the severed arterial site. FIGS. 10A-D show photographs of an artery (10A) while the artery is being cut; a photograph (10B) of a cut artery with a large amount of bleeding; photograph (10C) of a cut artery administered with the composition of the present invention; and a photograph of hemostasis with the formation of a biomimetic clot (10D).

The right femoral artery of the rat was severed using a surgical scalpel. After bleeding for about 10 seconds, accumulated blood was removed immediately before the application of the gelatin-mTG (test) solution using cotton gauze. Severe bleeding was observed but was stopped almost immediately by the gel and was completely stopped in less than one minute. The gel remained very firm and blood trapped by the gel was easily observed. After 5 minutes, the gel was manually examined using forceps. Although there is trapped blood in the gel formed, it adheres very firmly to the tissue in the region of the artery.

Thus, it is clear that the composition according to the invention is capable of slowing the speed of bleeding and inducing haemostasis in an in vivo model even in the presence of accumulated blood and/or severe bleeding (such as, for example, from arteries and/or vascularized organs, including but not limited to, for example, liver, stomach, kidney, heart, lungs and/or skin).

Example 4

Hemostasis in pig models

This example provides an initial in vivo demonstration that gelatin-mTG compositions in accordance with the present invention achieve hemostasis in large animal models. The potential for hemostatic utility in large animal models is clearly demonstrated.

Material

The gelatin solution was characterized by 25% w/w gelatin (porcine, type a, 300 bloom, from Sigma-Aldrich (st. louis, MO)) in PBS (ph7.4) and was prepared as described herein. When the gelatin powder was gradually added, the PBS was continuously stirred at 60 ℃ using a hot plate magnetic stirrer. Occasionally, manual stirring was performed using a glass rod to increase the dissolution rate of the powder and to obtain a homogeneous solution. Throughout the experiment, the gelatin solution was stored in a thermostatic bath adjusted to-50 ℃ to maintain its liquid phase and prevent the formation of thermoreversible gel.

The mTG solution was 20% w/w microbial transglutaminase (Activa WM, Ajinomoto) in PBS (pH7.4)^TM) Is characterized in that. It was prepared as follows. The Room Temperature (RT) PBS solution was stirred using a magnetic stirrer and mTG powder was added gradually. Throughout the experiment, except for actual use, mTG solution was stored in a constant temperature bath adjusted to-30 ℃.

Adult female pigs weighing 45kg were placed under general anesthesia prior to starting the experiment. Throughout the experiment, the pigs were ventilated and monitored for vital signs.

Prior to application to a wound site as described hereinbefore, a gelatin-mTG solution according to the invention is prepared and a sealant is placed on the wound site using an applicator. Several different applicators have been examined as support materials for bandages. Unless otherwise stated, 6mL of the new surgical sealant solution was spread over the applicator and allowed to cool at RT for 1 minute immediately prior to its application to the wound site. This pad containing the sealing material is considered a "bandage prototype". A similar procedure was followed for the "control bandage" but with the control solution spread over the applicator.

New surgical sealant solution-A2: 1 mixture of gelatin: mTG was prepared. Unless otherwise stated, the mixture was prepared by adding 2mL of mTG solution to 4mL of gelatin solution in a 15mL tube and inverting the tube 5 times to mix the solutions.

Control solution-for the control solution, the procedure described for the preparation of a new surgical sealant was repeated except that PBS alone was used instead of the mTG solution. Accordingly, the gelatin was immersed in a thermostatic bath at-30 ℃ and diluted with PBS (pH7.4) at a ratio of 2: 1. Unless otherwise stated, the mixture was prepared by adding 2mL of PBS solution to 4mL of gelatin solution in a 15mL tube and inverting the tube 5 times to mix the solutions.

The applicator was used as follows:

1.4cmx4cm cotton gauze pad.

2.4cmx4cm disposable plastic backed absorbent pad. The solution is spread over the non-absorbent side of the plastic of the pad.

3. A silicon mold.

4. A 4cmx4cm disposable plastic backing absorbent pad placed in a silicon mold. The solution is spread over the non-absorbent side of the plastic of the pad.

5. A transparent, flexible plastic mold having a high margin.

6. The sealant is applied directly to the wound site using a syringe or by pouring from a 15mL tube.

The application of the new surgical sealant in this study was done by the surgeon manually placing the sealant on the wound site using a different applicator. If desired, accumulated blood is removed with cotton gauze immediately prior to application. Hemostatic pressure was applied to the opposite side of the bandage for 3 minutes. After 3 minutes, the surgeon relieves the pressure and observes the wound site for hemostasis. If complete hemostasis does not occur, the wound site is closed by accepted surgical hemostasis techniques. The control solution was applied following the same technique, using a recognized technique of hemostasis immediately if no hemostasis was observed after removal of the control bandage.

Injury of hip muscles

The animals were placed in a prone position and the skin was removed from the gluteal muscles. A total of 7 experiments were performed in which hemostasis and tissue adhesion were examined. Unless otherwise stated, in each test, a 3cmx3cm square muscle was cut into the muscle to a depth of 2cm using a #15 surgical scalpel. Excess blood is removed from the wound area as needed and a new surgical sealing solution or control solution is applied as previously described.

Tables 5 and 6 summarize and describe the experimental procedures and results for each experiment. Table 5 relates to hemostasis and table 6 relates to tissue adhesion.

First to see hemostasis, a cotton gauze pad was used to apply the control solution to the wound site (table 5, control # 1). The control solution was applied to cotton yarn and allowed to cool for 1 minute and 20 seconds immediately before its application. The wound site bleeds only slightly and complete hemostasis is observed 2 minutes after application of the control bandage. Although no biomimetic blood clot is observed at the wound site, the hemostatic pressure applied to the wound site is sufficient to promote hemostasis.

The test was repeated at different wound sites with the difference that the applicator used was a disposable plastic backed absorbent pad and the control solution was allowed to stand for 30 seconds before its application (table 5, control 2). The wound site showed very little bleeding and complete hemostasis was observed 2 minutes after application of the control bandage. As in the previous example, this may be due to the hemostatic pressure exerted on the site when the bandage is applied. No biomimetic blood clot was observed at the wound site.

Since a small amount of bleeding was observed during the first two control experiments, the experiment was repeated except that a deeper 4cm incision was made (table 5, control # 3). Thus, severe bleeding was observed. The control solution was applied to an absorbable pad and allowed to stand for 50 seconds. The surgeon removes excess blood from the wound area and applies a control bandage. After 3 minutes, bleeding was reduced but complete hemostasis was not observed.

The control solution was removed from the wound site generated in the previous experiment using a cotton gauze pad. Bleeding was still observed. New surgical sealant was applied to the wound area to achieve hemostasis (table 5, sealant # 1). The sealant solution was placed on an absorbent pad, left for 1 minute and applied to the wound site. After 3 minutes, complete hemostasis was observed. The sealing material forms a bionic blood clot at the wound site. The gel was shaken using forceps and strong adhesion to the tissue was observed. The gel is removed by applying some force and it looks like a film. These results thus demonstrate the hemostatic properties of the composition according to the invention.

TABLE 5 gluteal muscles

Experiment of

RT (℃)

Heart rate

Application technique

Description of the invention

Hemostasis time (minutes)

Results

Control 1#1

21

99

Cotton yarn pad

Little bleeding occurred at the wound site after incision. Prior to application, the control solution was placed on an applicator and left to cool for 1 minute and 20 seconds.

2

It was noted that very little bleeding in the wound area was observed after incision. Hemostasis may be achieved by applying pressure only on the wound site.

Control No. 1#2

21

98

Disposable plastic backing absorbent pad

The wound site showed little bleeding after incision. The control solution was placed on the applicator and left to cool for 30 seconds prior to application to the wound site.

2

It was noted that very little bleeding in the wound area was observed after incision. Hemostasis may be achieved by applying pressure only on the wound site.

Control 1#3

22

98

Disposable plastic-backed absorbent pad

An incision was made 4cm deep. A large amount of bleeding was observed. The control bandage was left to cool for 50 seconds. Excess blood is removed prior to its application to the wound site.

-

Due to the large amount of bleeding, the surgeon applies a hemostatic pressure. After 3 minutes, bleeding decreased but did not stop.

Sealing Material #1

22

99

Disposable plastic backing absorbent pad

Control solution was removed from the wound site that had been subjected to control # 3. Excess blood was removed. The sealant is placed over the bleeding wound site.

3

A firm biomimetic clot is formed on the wound site. Complete hemostasis and secure adhesion of the sealing material was observed.

After demonstrating the hemostatic ability of the sealant in the hip muscle model, tissue adhesion was examined (table 6). A surgical incision is made to lift a piece of tissue from the muscle bed, opening the wound site.

In the first adhesion test (table 6, seal #2), the seal was applied directly to the wound site and the surgeon immediately applied strong pressure on top of the tissue for 3 minutes, expelling all the seal from the wound site and creating no adhesion.

The test was repeated except that the surgeon applied only moderate pressure after applying the seal (table 6, seal 3). The tissue appeared to be adhered after 3 minutes. When the upper portion of the tissue is shaken, a moderate amount of resistance to its complete removal is felt.

The experiment was repeated with special care not to expel the seal material from the wound site upon application of pressure (table 6, seal # 4). At different wound sites, the sealing material was applied to two portions of tissue and left for 10 seconds. Thereafter, the upper part of the tissue is repositioned and moderate pressure is applied. After 3 minutes, strong tissue adhesion was observed. A large amount of force is required to later separate the bonded tissue.

TABLE 6 tissue adhesion

Experiment of

RT (℃)

Heart rate

Application technique

Description of the invention

Tissue adhesion

Results

Sealing Material #2

23

99

Direct administration from a tube

Excess blood was removed with a cotton gauze pad. The sealant material is placed over the wound site and immediately a discharge pressure is applied by the surgeon.

N/A

No sealant material remaining in the wound site

Sealing Material #3

23

94

Direct administration from a tube

Excess blood was removed with a cotton gauze pad. The sealant material is placed over the wound site and moderate pressure is applied by the surgeon.

+

Adhesion with slight resistance was observed

Sealing Material #4

24

95

Direct administration from a tube

The sealant solution was placed on the wound site on both parts of the tissue and allowed to stand for-10 seconds. The upper side of the tissue is then repositioned and very low pressure is applied.

+

A strong bond was observed. The tissue is removed only after a strong force is applied.

Hemostasis in the liver

The pigs were placed supine and their livers exposed by midline laparotomy. A series of incisions were made to remove the increasingly deep liver biopsies, thereby exposing larger blood vessels. A total of 5 biopsies were taken. When required, the accumulated blood is removed immediately before application of the composition according to the invention with cotton gauze.

For the initial series of biopsies, hemostatic pressure was applied to the control bandage on the reverse side of the bandage for 3 minutes. After 3 minutes, the surgeon relieves the pressure and observes the wound site for hemostasis. When complete hemostasis has not occurred, a deeper biopsy is taken, followed by the application of a new surgical sealant. Again, hemostatic pressure was applied to the sealing material on the reverse side of the bandage for 3 minutes and hemostasis was then examined. When complete hemostasis was observed, deeper liver biopsies were removed and the experiment repeated with the sealing material. This demonstrates the hemostatic ability of the sealing material to higher blood pressures. Table 7 summarizes the experimental procedures and results for each experiment.

A biopsy was removed from the left lobe of the liver at a depth of 4cm (Table 7, control # 1). The control solution was applied on an absorbable pad placed in a silicon mold and left for 1 minute. A control bandage was applied to the wound site with hemostatic pressure. After 3 minutes, the pressure was removed and no hemostasis was observed.

After hemostasis was not achieved by application of the control bandage, the biopsy 1cm deeper was removed and allowed to bleed for 30 seconds. The experiment was then repeated with a new sealing material (table 7, sealing material # 1). The new sealant was placed on the pad in a silicon mold and 1 minute later applied to the wound site with hemostatic pressure. After 3 minutes, the pressure was relieved, the prototype bandage was peeled off and checked for hemostasis. The sealing material produced a visible biomimetic film. Hemostasis is achieved but not completely because the sealing material does not cover the entire wound. It can be seen that the area covered by the sealing material stopped bleeding. When the biomimetic membrane is removed after a few minutes, bleeding resumes.

A biopsy 1cm deeper was then removed, resulting in severe bleeding. The experiment was repeated except that the silicon mold was used as an applicator and excess blood was removed prior to application (table 7, seal # 2). The surgeon then applies pressure to the wound site for 3 minutes. When the surgeon removes his hand, a biomimetic blood clot is visible on the wound site. The blood pressure pushing against the biomimetic clot is significant and after a few more minutes the blood breaks through from the edges of the biomimetic sealing material. The laceration passes through the side portion of the wound site that is not covered by the sealing material. This indicates that at this stage the haemostatic capacity of the sealant is dependent on covering the entire wound site.

To avoid breakthrough, previous experiments were repeated; except that a greater amount of seal material was applied to the wound site (table 7, seal # 3). Biopsies 0.5cm deeper were removed from liver lobes. 9mL of sealant was applied to the wound site with pressure. Unfortunately, during application, almost all of the sealant drips onto the sides of the wound site, leaving no discernible sealant on the wound site after pressure is applied by the surgeon.

The experiment was repeated (table 7, seal # 4). A further 1cm biopsy was removed and a large bleeding was observed. At this point, 15mL of sealing material was applied over the wound site using a clear plastic mold with high margins that held the sealing material in place. The seal material was placed on the applicator and cooled for 1 minute and 20 seconds. A thick layer of biomimetic blood clot was observed and complete hemostasis was achieved after 4 minutes of applying the sealing material on the wound site. After 50 minutes, the tissue was again examined and hemostasis was observed. This indicates the strong hemostatic ability of the sealant when sufficient sealant is applied to the wound site and held in place. The formed biomimetic membrane is difficult to remove because it adheres strongly to the tissue surface, and removal of the membrane results in a small amount of bleeding.

TABLE 7 hemostasis in left liver leaves

Experiment of

RT (℃)

Heart rate

Application technique

Description of the invention

Hemostasis time (minutes)

Results

Control #1

25

87

Disposable plastic backed absorbent pad in silicon mold

Remove 4cm of biopsy. The control solution was placed on the applicator, left for 1 minute and applied at the wound site with hemostatic pressure for 3 minutes.

-

Massive bleeding after administration. No hemostatic or biomimetic membranes were observed.

Sealing Material #1

24

86

Disposable plastic backed absorbent pad in silicon mold

A further 1cm of living tissue was removed for examination and left for bleeding for 30 seconds.

3

The new sealing material partially stops the massive bleeding by creating a biomimetic membrane. Hemostasis was not achieved because the sealing material did not cover the entire wound.

Sealing Material #2

24

86

Silicon die

Remove another 1cm of living tissue for examination and remove excess blood. Applying a sealing material at a hemostatic pressure

3

The sealing material does not cover the entire wound area, but hemostasis is achieved where the sealing material is present.

Sealing Material #3

24

84

Silicon die

A further 0.5cm of live tissue was removed for examination and excess blood was removed prior to application of the sealing material. 9mL of sealant material was applied.

-

All the sealing material is expelled during the application process.

Sealing Material #4

24

80

Transparent flexible plastic mold with high edge

A further 1cm biopsy was removed. 15mL of sealant was applied to the applicator, left for 1 minute and 20 seconds to cool and then placed on the wound site.

4

A thick bionic blood clot layer is formed. Complete hemostasis is realized. After 50 minutes the tissue was again examined and hemostasis was still observed. The formed film is difficult to remove and some bleeding continues after removal.

Hemostasis in femoral artery

Next, the ability of the compositions of the invention to induce hemostasis in wounds or trauma to arteries, particularly femoral arteries, was examined. The right femoral artery of the animal was exposed. A circular 2mm longitudinal cut was then made using a surgical blade. A large amount of bleeding was observed and hence hemostats were used. The excess blood was removed with a cotton gauze pad immediately prior to application of the sealing material. Approximately 9mL of the new surgical sealant was prepared and applied to the wound area using a syringe. After 4 minutes, the hemostat was gently removed and hemostasis through the sealant was tested. Biomimetic blood clots were observed and complete hemostasis was achieved. Table 9 summarizes the experimental procedures and results of this experiment.

TABLE 9 hemostasis in femoral artery

Experiment of

RT (℃)

Heart rate

Application technique

Description of the invention

Hemostasis time (minutes)

Results

Sealing Material #1

24

96

Injection device

A 2mm puncture was made and massive blood flow was stopped using hemostats. Using a syringe,. -9 mL of the sealant solution was applied to the wound site. After 4 minutes, the hemostatic forceps were gently removed.

3

After removal of the hemostat, complete hemostasis was observed. The sealing material creates a biomimetic clot at the wound site that seeks to prevent massive bleeding.

Example 5

procedure-Effect of guanidine hydrochloride on gelation and crosslinking

This example relates to the effect of an exemplary denaturant, guanidine hydrochloride (described herein as "GuCl") on compositions according to certain embodiments of the present invention. Preferred concentration ratio ranges are as follows: from about 1: 2 to about 2: 2 GuHCl: gelatin, weight/weight.

Preparation of solutions

1) 10g of GuCl (Fluka, St. Louis, MO) were dissolved in 30mL of Dulbecco's PBS (Biological Industries, Israel) at Room Temperature (RT). Separately, 10g of type a, 300 bloom porcine gelatin powder (Sigma, st. louis, MO) was weighed, after which the gelatin and GuCl solution were mixed with appropriate stirring to form a homogeneous solution having a gelatin: GuCl ratio (w: w) of 1: 1.

The Molecular Weight (MW) of GuCl was 95.53. In this solution, the final concentration of GuCl is therefore 3.489M. The final solution was 20% gelatin w/w in PBS, but on a volume basis, corresponded to a 25% w/w gelatin solution.

2) 2g of GuCl was dissolved in 30mL of PBS at RT. 10g of type A, 300 bloom of porcine gelatin powder was separately weighed, after which the gelatin and GuCl solutions were mixed with appropriate stirring to form a homogeneous solution having a gelatin: GuCl ratio (w: w) of 5: 1. The final concentration of GuCl was 698 mM. The final solution was 23.8% gelatin w/w in PBS, but on a volume basis, corresponded to a 25% w/w gelatin solution.

3) 6g of GuCl was dissolved in 30mL of PBS at RT. 10g of type A, 300 bloom of porcine gelatin powder was separately weighed, after which the gelatin and GuCl solutions were mixed with appropriate stirring to form a homogeneous solution having a gelatin: GuCl ratio (w: w) of 5: 3. The final concentration of GuCl was 2.09M. The final solution was 21.7% gelatin w/w in PBS, but on a volume basis, corresponded to a 25% w/w gelatin solution.

4) 4g of GuCl was dissolved in 30mL of PBS at RT. 10g of type A, 300 bloom of porcine gelatin powder was separately weighed, after which the gelatin and GuCl solutions were mixed with appropriate stirring to form a homogeneous solution having a gelatin: GuCl ratio (w: w) of 5: 2. The final concentration of GuCl was 1.40M. The final solution was 22.7% gelatin w/w in PBS, but on a volume basis, corresponded to a 25% w/w gelatin solution.

5) 8g of GuCl was dissolved in 30mL of PBS at RT. 10g of type A, 300 bloom of porcine gelatin powder was separately weighed, after which the gelatin and GuCl solutions were mixed with appropriate stirring to form a homogeneous solution having a gelatin: GuCl ratio (w: w) of 5: 1. The final concentration of GuCl was 2.79M. The final solution was 20.8% gelatin w/w in PBS, but on a volume basis, corresponded to a 25% w/w gelatin solution.

Addition of mTG

A20% w/w solution of 1% microbial transglutaminase powder (mTG) (Ajinomoto Activa TI-WM, Japan) in PBS was prepared

A) 2mL of each gelatin-GuCl solution was mixed with 1mL of mTG solution in a clear 4mL plastic tube. The mTG solution was injected into the gelatin-GuCl solution. Each tube was inverted several times and then allowed to stand.

B) 2mL of each gelatin-GuCl solution was mixed with 1mL of mTG solution in a plastic weighing dish. The mixture was mixed manually with a pipetting head.

C) The gelatin-GuCl solution was heated to 43 ℃ and then 2mL aliquots of the gelatin-GuCl solution were mixed with 1mL mTG solution in plastic weighing dishes. The mixture was mixed manually with a pipetting head.

D) gelatin-GuCl solution 1(10g GuCl) and 5(8g GuCl) were heated to 43 ℃ and then 2mL aliquots of the gelatin-GuCl solution were mixed with 2mL mTG solution in plastic weighing dishes. The mixture was mixed manually with a pipetting head.

Results

1) A1: 1 solution of gelatin: GuCl formed a homogeneous solution within 2 minutes at room temperature. Immediately after formation, the solution contains a number of bubbles. After 2 hours of standing at RT almost all bubbles had left the solution. Like a standard gelatin solution, the solution remains in liquid form and appears transparent with a yellow color. After 24 hours, the solution properties were unchanged and no more bubbles were seen.

2) GuCl at RT does not form a homogeneous solution with gelatin 5: 1. Gelatin granules swelled but did not dissolve as normally happens for gelatin like PBS at RT. The solution was heated to 42 ℃ until it formed a solution. When cooled to RT, it forms a gel at about 32 ℃.

3) GuCl and gelatin 5: 3 form a homogeneous solution after vigorous stirring for 5-6 minutes at room temperature. Immediately after formation, the solution contains a number of bubbles. After 2 hours of standing at RT almost all bubbles had left the solution. Like a standard gelatin solution, the solution remains in liquid form and appears transparent with a yellow color. The solution was in liquid form but was more viscous than a 1: 1 solution of gelatin: GuCl. But it can still be pipetted without difficulty. After 24 hours, the solution properties were unchanged and no more bubbles were seen.

4) GuCl, gelatin 5: 2, formed a slightly fine-grained solution after vigorous stirring for 10 minutes at room temperature. Immediately after formation, the solution contains many bubbles. Immediately after formation, the solution appeared to be very viscous but still in liquid form. However, after 2 hours of standing at RT, many bubbles were still visible in the solution and the solution was gelatinous. The solution is not easily pipetted and is too viscous to mix with other solutions. After 24 hours, many bubbles remained in the solution and the solution had formed a thermoreversible gel.

5) 5: 4 gelatin GuCl forms a homogeneous solution after stirring for 2-3 minutes at room temperature. Immediately after formation, the solution contains a number of bubbles. After 2 hours of standing at RT almost all bubbles had left the solution. Like a standard gelatin solution, the solution remains in liquid form and appears transparent with a yellow color. The solution is in liquid form, more viscous than a 1: 1 solution of gelatin to GuCl but less viscous than a 5: 3 solution of gelatin to GuCl, and it can be pipetted without difficulty. After 24 hours, the solution properties were unchanged and no more bubbles were seen.

mTG results

A) 2mL of gelatin-GuCl solution and 1mL of mTG solution in a 4mL tube at room temperature

1) For a 1: 1 gelatin: GuCl solution, a small gelatinous clot formed near the top of the tube after 4 minutes. The clot was removed and an additional 1mL of mTG solution was added. After 35 minutes, a soft-medium gel was formed. Microwave heating confirmed that the clot was not thermoreversible. It is also not as strongly cohesive as the crosslinked gel. The soft-moderate gel was confirmed to be thermally irreversible using microwave heating.

2) A5: 1 solution of gelatin: GuCl is too viscous to mix with a solution of mTG.

3) For a 5: 3 gelatin: GuCl solution, a small gelatinous clot formed near the top of the tube after 4 minutes. No clot was removed. After 20 minutes, a moderate gel was formed throughout the solution. The coagulum had a distinctly different consistency from the rest of the gel. As above, although microwave heating demonstrates that it is not thermoreversible, it is also not very cohesive and easily breaks apart upon touch. The resulting intermediate gel became slightly soft when heated in a microwave, but was thermally irreversible.

4) Since the solution became very viscous due to thermoreversible gelation within the first few minutes after mTG addition (as indicated by the control solution without mTG addition), the results for the 5: 2 gelatin: GuCl solution were very inconsistent. After 15 minutes, a moderately firm gel was formed but it was partially thermoreversible and became much softer upon heating.

5) For a 5: 4 gelatin: GuCl solution, a small gelatinous clot formed near the top of the tube after 4 minutes. No clot was removed. After 30 minutes, a moderate gel was formed throughout the solution. The coagulum had a distinctly different consistency from the rest of the gel. As above, although microwave heating demonstrates that it is not thermoreversible, it is also not very cohesive and easily breaks apart upon touch. The resulting intermediate gel became slightly soft when heated in a microwave, but was thermally irreversible.

B) 2mL of gelatin-GuCl solution and 1mL of mTG solution in a plastic dish at room temperature

The gelation time results obtained from the solution mixed in the plastic dish are almost identical to those found in the solution mixed in the 4mL plastic tube: 1: 1 gelatin: GuCl formed a soft-moderate gel after 35 minutes, 5: 3 gelatin: GuCl formed a moderate gel after 20 minutes, and 5: 4 gelatin: GuCl formed a moderate gel after 30 minutes.

But the gelatinous clot observed when mTG was injected into the gelatin-GuCl solution was not observed in these experiments.

C) 2mL of a 43 ℃ gelatin-GuCl solution, 1mL of a mTG solution in a plastic dish-the results are as follows: for a 1: 1 gelatin: GuCl solution, no gel was formed after 35 minutes; for a 5: 3 gelatin: GuCl solution, a moderate gel formed after 17 minutes; for a 5: 4 gelatin: GuCl solution, a moderate gel formed after 25 minutes.

D) 2mL of a 43 ℃ gelatin-GuCl solution, 2mL of a mTG solution in a plastic dish, the results are as follows: for a 1: 1 gelatin: GuCl solution, no gel was formed after 25 minutes; for a 5: 4 gelatin: GuCl solution, a moderate gel formed after 9 minutes.

From the above results, it was found that GuCl significantly improved the solubility of gelatin in PBS. For 25% w/w gelatin in PBS, GuCl was added at a ratio of 5: 4 and 1: 1 gelatin to GuCl so that the gelatin could be almost immediately dissolved in room temperature PBS. This effect is significantly reduced at a gelatin: GuCl ratio of 5: 3. For a concentration of 25% w/w gelatin in PBS, the gelatin-GuCl solution can be permanently maintained in liquid form by adding GuCl at gelatin: GuCl ratios of 5: 3, 5: 4, and 1: 1. At a gelatin: GuCl ratio of 5: 2, the solution underwent delayed thermoreversible gelation and formed a complete gel after 2 hours. At a ratio of 2: 1 gelatin-GuCl solution to mTG solution, no gel is formed if the gelatin to GuCl solution ratio is 1: 1. At lower GuCl concentrations, crosslinked gels were formed. Gelation time appears to be dependent on GuCl concentration.

When the gelatin: GuCl ratio is 5: 4 and 5: 3, heating the gelatin-GuCl solution to 43 deg.C before mixing with mTG accelerates the crosslinking process. This is expected because mTG activity increases with increasing reaction temperature to 55 ℃. It is possible that if the mTG activity is increased, the gelatin GuCl solution will be cross-linked more quickly by mTG.

At a 1: 1 ratio of gelatin-GuCl solution to mTG solution, a cross-linked gel is formed, even when the gelatin-GuCl solution ratio is 1: 1. The increase in the amount of mTG significantly reduced the gelation time of the gel that formed the gel at a 2: 1 ratio of gelatin-GuCl solution to mTG solution. Gelation time was observed to be dependent on GuCl concentration. This is expected because GuCl may denature a certain amount of mTG. The amount of mTG added above is arbitrary for crosslinked gelatin.

Without wishing to be bound by a single hypothesis, it is possible that if more mTG enzyme is added, the gelatin: GuCl solution will be cross-linked much more rapidly by mTG. This can be achieved, for example, alternatively by removing the carrier from the mTG powder and forming a concentrated solution of the enzyme itself.

Example 6

The process comprises the following steps: adding MgCl to gelatin ₂ Effect on gelation and crosslinking

This example relates to the effect of an exemplary reducing agent, magnesium chloride, on compositions according to certain embodiments of the present invention. The preferred concentration range for dissolving gelatin in magnesium chloride-PBS solution is preferably from about 2 to about 4M, more preferably from about 2.5 to about 3.5M.

Materials and methods

Material

Type a 300 bloom porcine gelatin and MgCl2 powder, -325 mesh was obtained from Sigma-Aldrich corporation (st. ActivataTI-WM microbial transglutaminase (mTG) was supplied by Ajinomoto (Japan). Dulbecco's PBS (pH7.4) was obtained from Biological Industries (Kibbutz BeitHaEmek, Israel).

Preparation of mTG solution:

fresh ActivatI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG) mixtures were prepared by dissolving in Dulbecco's PBS to form a 20% w/w solution. During the experiment, the solution was kept at Room Temperature (RT).

Preparation of gelatin-MgCl 2 solution:

gelatin was dissolved in MgCl2 solutions of different concentrations as follows.

Solution A-5 gr of MgCl2 were dissolved in 15mL of Dulbecco's PBS to a final concentration of 3.5M. The dissolution reaction of MgCl2 is exothermic and reaches 90 ℃, and the solution is left to cool to room temperature. 25% gelatin aliquots (w/w) were prepared by dissolving 5gr of gelatin in 15gr of 3.5M MgCl2 solution and stirring at RT.

Solution B-2.5 gr of MgCl2 were dissolved in 15mL of Dulbecco's PBS to a final concentration of 1.75M. The dissolution reaction of MgCl2 was exothermic and reached 63 ℃ and the solution was allowed to stand to cool to room temperature. A25% gelatin aliquot (w/w) was prepared by dissolving 5gr of gelatin in 15gr of a 1.75M solution of MgCl2 at 63 deg.C and stirring.

Effect of MgCl2 on gelation of gelatin:

the gelatin dissolved in different concentrations of MgCl2 solution was left to cool to RT. The temperature of each solution was monitored with a thermometer. The appearance and viscosity of the gelatin-MgCl 2 solution was evaluated by observing and touching the solution with a glass rod as the temperature was decreased.

Effect of MgCl2 on chemical crosslinking of gelatin solutions using mTG:

the gelatin-MgCl 2 "solution a" was tested for chemical crosslinking using mTG. 1 or 2ml of a 20% w/w solution of mTG was mixed with 2ml of a gelatin-MgCl 2 solution in a 4ml technicon tube. The gelatin-MgCl 2 solution was added either at room temperature or preheated to 50 ℃ using a water bath. The solution was mixed by gently stirring and inverting the tube 4 times with a pipetting head and the gelation time was measured. The appearance and viscosity of the gelatin-MgCl 2 solution after mixing with mTG was evaluated by observation and by touching the solution with a glass rod. When the gel was formed, its thermoreversibility was tested by heating the gel to 50 ℃ with a water bath.

Results

Effect of MgCl2 on gelation of gelatin:

solution A-3.5M MgCl2 solution reduced the transition point of the gelatin. The gelatin-MgCl 2 solution was viscous at room temperature. The solution appeared rather opaque and comprised small black particles. These particles may be magnesium particles that have been oxidized. Solution B-1.75M gelatin-MgCl 2 solution gelled at room temperature. The transition point is 29 ℃. The gel was opaque and included few black particles that could be formed by oxidation of Mg.

Effect of MgCl2 on chemical crosslinking of gelatin solutions using mTG:

the effect of MgCl2 on crosslinking was tested with solution a as follows. Mixing 2ml of solution A of RT with 1ml of mTG solution-90 minutes yields an irreversible gel. The gel was very viscous and somewhat soft. Mixing 2ml of 50 ℃ heated solution A with 1ml of mTG solution produced an irreversible gel-after 70 minutes. Mixing 2ml of heated solution A at 50 ℃ with 2ml of mTG solution produced an irreversible gel-after 25 minutes. The gel is rather weak. The viscosity of the gel increased after heating it in a water bath at 50 ℃.

From the above, it was shown that the addition of magnesium chloride to a gelatin solution significantly reduces the transition point of gelatin. The transition point is shown to be inversely proportional to the concentration of magnesium chloride. The transition point was reduced below RT by the addition of 3.5M magnesium chloride. In the case of 1.75M magnesium chloride, the transition point of the gelatin solution is slightly above RT. The addition of magnesium chloride to gelatin should be optimized to find the minimum concentration that will reduce the gelatin transition point below RT.

It has also been shown that cross-linking of gelatin using mTG in the presence of magnesium chloride is possible. The crosslinking ratio of the gelatin is adversely affected by magnesium chloride. The addition of larger amounts of mTG may optionally be performed to overcome this effect.

The mTG activity at 50 ℃ is much greater than that at RT. This confirms the theoretical data and demonstrates the utility of adding an exothermic element to the gelatin-mTG mixture to ensure a reaction temperature above RT.

From the data above, the exothermic dissolution of magnesium chloride was optionally used to liquefy gelatin and increase the activity of mTG.

Example 7

The process comprises the following steps: effect of Hydroquinone addition to gelatin on gelation and crosslinking

This example relates to the effect of an exemplary reducing agent, hydroquinone, on compositions according to certain embodiments of the invention. Hydroquinone is a naturally occurring water-soluble reducing agent. The reducing agent increases the solubility of the gelatin, allowing it to remain liquid at Room Temperature (RT). The preferred concentration range for dissolving gelatin in hydroquinone-PBS solution is preferably determined to be a concentration of about 0.2 to about 0.5M, more preferably about 0.3 to about 0.4M.

Materials and methods

Material

Porcine gelatin and hydroquinone form A300 bloom (RegentPlus. TM. > 99%) were obtained from Sigma-Aldrich corporation (St. Louis, Mo.). Activata TI-WM microbial transglutaminase (mTG) was supplied by Ajinomoto (Japan). Dulbecco's PBS (pH7.4) was obtained from Biological Industries (Kibbutz Bait HaEmek, Israel).

Preparation of mTG solution:

fresh ActivatI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG) mixtures were prepared by dissolving in Dulbecco's PBS to form a 20% w/w solution. During the experiment, the solution was kept at Room Temperature (RT).

Preparation of gelatin-hydroquinone solution:

gelatin was dissolved in hydroquinone solutions of different concentrations as follows. Solution A-2.75 gr of hydroquinone were dissolved in Dulbecco's PBS to a final concentration of 0.5M. Throughout the experiment, the solution was kept in a sealed beaker wrapped in aluminum foil to avoid exposure to air and light. A25% gelatin aliquot (w/w) in 0.5M hydroquinone solution was prepared by mixing 5gr of gelatin with 15gr of 0.5M hydroquinone solution and stirring.

Solution B-1.32 gr of hydroquinone were dissolved in Dulbecco's PBS to a final concentration of 0.4M. Throughout the experiment, the solution was kept in a sealed beaker wrapped in aluminum foil to avoid exposure to air and light. A25% gelatin aliquot (w/w) in 0.4M hydroquinone solution was prepared by mixing 5gr of gelatin with 15gr of 0.4M hydroquinone solution. The mixture was stirred and then heated in a water bath at 50 ℃.

Effect of hydroquinone on gelation of gelatin:

gelatin dissolved in different concentrations of MgCl2 solutions was either kept at RT or cooled to RT after heating in a water bath at 50 ℃. The temperature of each solution was monitored with a thermometer. The appearance and viscosity of the gelatin-hydroquinone solution was evaluated by observing and touching the solution with a glass rod as the temperature decreased.

Effect of hydroquinone on chemical crosslinking of gelatin solutions using mTG:

gelatin-hydroquinone "solution B" was tested for chemical crosslinking using mTG. 1ml of a 20% w/w solution of mTG was mixed with 2ml of a gelatin-hydroquinone solution in a 4ml technicon tube and heated to 50 ℃. The solution was mixed by gently stirring and inverting the tube 4 times with a pipetting head and the gelation time was measured. The appearance and viscosity of the gelatin-hydroquinone solution after mixing with mTG was evaluated by observation and by touching the solution with a glass rod. When the gel was formed, its thermoreversibility was tested by heating the gel to 50 ℃ with a water bath.

Results

Effect of hydroquinone on gelation of gelatin:

solution A-0.5M Hydroquinone solution at room temperature did not dissolve gelatin. Gelatin powder was soaked in hydroquinone solution to produce a fine-grained, brown homogeneous solution. Solution B-0.4M hydroquinone solution managed to lower the transition point of gelatin. The hydroquinone solution did not dissolve the gelatin at room temperature. After heating the mixture in a water bath at 50 ℃, the gelatin dissolved and a homogeneous solution was obtained. The solution was cooled to RT. Gelatin remains soluble at 28 ℃ but is very viscous. A gel was formed when cooled to room temperature. The gel was brown.

Effect of hydroquinone on chemical crosslinking of gelatin solutions using mTG:

the effect on crosslinking was tested with solution B.2ml of solution B heated to 50 ℃ are mixed with 1ml of mTG solution. After 4 minutes, an irreversible gel was formed. The gel was firm, similar to a crosslinked gel formed from a mixture of 25% (w/w) gelatin alone with 20% (w/w) mTG.

From the above, it was shown that the addition of hydroquinone to a gelatin solution lowers the transition point of gelatin. In the case of 0.4M hydroquinone, the transition point of the gelatin solution is slightly above RT (28 ℃).

In contrast to other substances which have been tested as a method of reducing the transition point of gelatin, hydroquinone has no significant inhibitory effect on the crosslinking of gelatin with mTG.

As demonstrated by the data above, hydroquinone can alternatively be used to lower the sol-gel transition temperature of gelatin without adversely affecting mTG crosslinking of the gelatin. As previously described, this is highly desirable for many embodiments of the present invention.

Example 8

The process comprises the following steps: adding CaCl to gelatin ₂ Effect on gelation and crosslinking

This example relates to the effect of an exemplary reducing agent, calcium chloride, on compositions according to certain embodiments of the present invention. The preferred concentration range for dissolving gelatin in calcium chloride-PBS solution is preferably in the range of from about 1 to about 2M to lower the transition point of gelatin. To produce an exothermic reaction that can help dissolve gelatin or increase mTG activity, a solution of about 0.2-0.7g of sodium chloride per ml can be selected and preferably added for each degree celsius temperature increase above ambient temperature, but the exact amount depends on several factors.

A gelatin-calcium chloride solution prepared at DPBS as follows: A4M stock solution of CaCl2 was prepared by dissolving 44.396g of CaCl2 (97%, MW 110.99, Alfa Aesar, Lancaster) in 100mL of Dulbecco's PBS (Biological Industries, Israel) with stirring. After dissolution, the solution reached a peak temperature of 80 ℃ as a result of the exothermic dissolution reaction of CaCl 2.

Solution 1 was prepared as follows. 5g of type A, 300 bloom porcine gelatin powder (Sigma, St. Louis, Mo.). A25% w/w gelatin solution in PBS-CaCl2 was formed by adding 15g of a different concentration of PBS-CaCl2 solution to 5g of gelatin. The gelatin-CaCl 2 was mixed with a stir bar and occasionally hand agitation to break up the clot. The CaCl2 concentrations tested were:

a. 2M CaCl2 solution in PBS.

b.2M CaCl2 solution in PBS.

c. 1M CaCl2 solution in PBS.

For both a & b, 2mL of gelatin-CaCl 2 solution was mixed with 1mL of 20% w/w microbial transglutaminase (mTG) powder solution in 4mL plastic tubes each. The mTG product used (Activa TI-WM, Ajinomoto, Japan) had a powder specific activity of mTG product of about 100U/g.

Solution 2 was prepared as follows. A base solution of 20% w/w of mTG was formed by dissolving 10g of mTG powder in 40mL of PBS.

a. A25% w/w gelatin solution was prepared by dissolving 5g of gelatin in 15mL of PBS by heating the gelatin-PBS mixture in a microwave for 5 seconds followed by 15 seconds. The solution was stirred immediately after each microwave heating period. The temperature after the second heating was 72 ℃. Thereafter 3.325g of CaCl2 were added to form a 2M CaCl2 solution. The temperature after the addition of CaCl2 was 74 ℃. Immediately after CaCl2 was dissolved and the temperature was measured, 2mL of gelatin-CaCl 2 solution was mixed with 1mL of 20% w/w microbial transglutaminase (mTG) powder solution in a 4mL plastic tube.

b. 5g of gelatin powder was mixed with 3.33g of CaCl2 powder. Then 30mL of PBS was stirred into this mixture to form a solution. The solution temperature reached 42 ℃ upon dissolution. After a homogeneous solution was formed, 2mL of gelatin-CaCl 2 solution was mixed with 1mL of 20% w/w microbial transglutaminase (mTG) powder solution in a 4mL plastic tube.

Solution 3 was prepared as follows. A25% gelatin solution (1a, supra) in 2M CaCl2-PBS was allowed to stand for 2 hours. Then 2mL of gelatin-CaCl 2 solution was mixed with 2mL of 20% w/w mTG solution in a 4mL plastic tube.

Solution 4 was prepared as follows. After stirring for two hours, a 25% gelatin solution in 2M CaCl2-PBS (1b, supra) was heated to 43 ℃. Then 2mL of gelatin-CaCl 2 solution was mixed with a.1mL of 20% w/w mTG solution or b.2mL of 20% w/w mTG solution; each in 4mL plastic tubes.

Results

Solutions were formed at all CaCl2 concentrations. A 2M CaCl2 solution (1a) was used to form a homogeneous solution and maintain it in liquid form. The solution is moderately viscous and contains many bubbles. The gelatin-CaCl 2 solution was allowed to sit for 30 minutes to allow the bubbles to dissipate before mixing with the mTG solution.

The second 2M solution (1b) was the same as the first except that it required more manual agitation to disperse the formed gelatin clot.

Using a 1M solution of CaCl2(1c), a homogeneous solution formed but gelled after a few minutes. After 2 hours a fully thermoreversible gel had formed. But this gel is much softer than thermoreversible gelatin normally formed from gelatin at room temperature. After half an hour the solution was too viscous to mix with the mTG solution.

After addition of mTG solution to 2M gelatin-CaCl 2 solution, the solution gradually became more viscous over a 20 minute period but no cohesive gel formed.

Heating the gelatin-CaCl 2 solution increased the gelling effect of mTG. The solution heated by the microwave gradually became more viscous. After 20 minutes, a very soft, irreversible (as evidenced by heating to 50 ℃) gel had formed. The heated solution of CaCl2 became progressively more viscous. After 20 minutes, a very soft, irreversible (as evidenced by heating to 50 ℃) gel had formed.

For a gelatin-CaCl 2 solution (2M) mixed with 2mL of a 20% w/w solution of mTG, a soft gel formed after 10 minutes. After 20 minutes, a medium strength gelatin was formed, and after 35 minutes a medium-firm strength gel was formed.

For gelatin-CaCl 2 solution (2M) heated to 43 deg.C, after mixing with 1mL of 20% w/w mTG solution, a soft gel formed after 10 minutes and a soft-medium strength gel formed after 20 minutes. However, after mixing with 2mL of a 20% w/w solution of mTG, a moderate strength gel was formed after 10 minutes and a moderate-firm strength gel was formed after 20 minutes

Example 9

The process comprises the following steps: microwave drying of gelatin-effect on gelation and crosslinking

This example examines the effect of drying gelatin in microwaves on solubility. Increased solubility was observed. The selectable and preferred microwave radiation energy range is preferably characterized by an overall Specific Absorption Rate (SAR) of from about 1 to 100 mW/cubic centimeter, more preferably from about 30 to about 60 mW/cubic centimeter. The process is carried out as follows.

Gelatin preparation and drying

10gr portions of type A, 300 bloom, porcine gelatin powder (Sigma, St. Louis, Mo.) were weighed into 50mL or 250mL beakers. The gelatin was then heated in 700W and 2,450MHz microwaves (Kennedy type KN-949, China) for the following amounts of time:

sample a: 30 second, 50mL beaker

Sample B: 60 second, 250mL beaker

Sample C: 120 second, 250mL beaker

Sample D: 180 second, 250mL beaker

Comparison: gelatin without microwave heating

After heating, 30mL of 37 ℃ Dulbecco's PBS (Biological Industries, Israel) was added to the gelatin and the mixture was stirred at 37 ℃. For sample a, PBS was added immediately after the gelatin was removed from the microwave. For the remaining samples, the gelatin was cooled to Room Temperature (RT) before adding PBS.

For sample C, after mixing gelatin with PBS, portions of the mixture were separated, heated to 50 ℃ and then mixed according to a 2: 1 gelatin solution: the mTG solution ratio was mixed with a 20% w/w solution of microbial transglutaminase (mTG) (Ajinomoto Activa TI-WM, Japan).

Microwave heating of gelatin/PBS mixtures

In sample F, gelatin was not heated in powder form. Instead, gelatin was poured directly into 30mL of RT PBS in a 50mL beaker and the mixture was then heated twice in a microwave for 15 seconds in succession with a 5 second pause between heating periods. After heating, it was stirred manually. The gelatin solution was then mixed with a 20% w/w solution of mTG in a 2: 1 ratio of gelatin solution to mTG solution.

Heating of mTG

A 20% w/w mTG solution was heated twice in the microwave for 15 seconds in succession with a 5 second pause between heating periods. Then mTG was added to a 25% w/w gelatin solution at a 1: 2 ratio of mTG to gelatin solution.

Results

Sample a: gelatin readily dissolved in PBS to form a viscous solution. Upon cooling to RT, the gelatin solution formed a firm thermoreversible gel that was comparable to the thermoreversible gel formed at RT from a standard gelatin solution.

Comparison: gelatin was not completely soluble in PBS. Despite its complete immersion in PBS, gelatin remains very fine particulate.

Sample B: gelatin was not completely soluble in PBS. Despite its complete immersion in PBS, gelatin remains very fine particulate.

Sample C: gelatin was not completely soluble in PBS. It was completely soaked in PBS, but the gelatin remained very fine particulate. The finely divided gelatin-PBS mixture was then heated in a microwave for 30 seconds. The temperature when removed from the microwave was 76 ℃. The mixture was then stirred manually to form a homogeneous solution. The solution was allowed to cool to room temperature and the solution formed a thermoreversible gel comparable to that formed at RT from a standard gelatin solution.

When the solution was heated to 50 ℃ and mixed with mTG, firm and sticky gelatin formed after 3 minutes. This gel was heated in a microwave for 10 seconds to confirm its irreversibility. Upon exiting from the microwave, the gel was more viscous but stronger. It appears to be somewhat dry.

Sample D: during heating, the gelatin forms carbonized bubbles. Bubbles are formed inside the gelatin powder by burning the gelatin. There was a strong scorched smell associated with this event.

Heated gelatin/PBS mixture: the gelatin/PBS mixture was heated in a microwave to form a liquid solution. Addition of mTG to the solution produced a firm, irreversible gel.

Heated mTG: mTG that has been heated in the microwave does not crosslink the gelatin.

From the above, it was shown that heating gelatin powder in a microwave reduced the moisture content of the gelatin as indicated by a significant reduction in gelatin weight (data not shown). Heating the gelatin powder in a microwave immediately followed by 37 ℃ PBS reduces the dissolution time of the gelatin. However, if the gelatin powder is cooled to RT, no improvement in the dissolution time of the gelatin occurs.

If the microwave heated gelatin is heated after it has been mixed with PBS, the resulting solution can be cross-linked by mTG. It is possible to dissolve gelatin in PBS at RT and then heat it in a microwave by heating for 15 seconds followed by another 15 seconds. Dissolving it in this way does not adversely affect the crosslinking of the mTG.

If the dried gelatin is heated in the microwave for more than 2 minutes, it will burn. Heating mTG in microwaves significantly reduces its activity.

Example 10

Effect of Urea on gelation and Cross-linking of gelatin

This example relates to the effect of urea as part of an exemplary composition according to the present invention. Urea was found to lower the transition point of the gelatin solution. The data below demonstrates that it lowers the transition point of even a highly concentrated gelatin solution sufficiently below room temperature that the solution remains in liquid form at room temperature. Transglutaminase was found to be capable of cross-linking gelatin even in the presence of urea.

Preparing a gelatin solution:

type a, 300 bloom porcine gelatin (Sigma, st. 25% and 15% w/w gelatin solutions were prepared by dissolving 50gr and 30gr of gelatin in 150mL and 170mL of Dulbecco's PBS (Biological Industries, Israel), respectively, while stirring on a hot plate at 50 ℃. Gelatin was gradually added to PBS and manually stirred with a glass rod. The gelatin solution was placed in a water bath at 50 ℃ during the experiment.

Preparation of transglutaminase solution:

activa TI-WM (Ajinomoto, Japan) microbial transglutaminase (mTG) mixture was dissolved in Dulbecco's PBS to form a 20% w/w solution. The solution was kept at Room Temperature (RT) during the experiment.

Preparation of gelatin-urea solution

A40 g aliquot of 25% or 15% w/w gelatin solution was transferred to a 100mL beaker and stirred at 50 ℃. As detailed in the table below, urea (98%, Alfa Aesar, Lancaster, UK) was added to the beakers at different ratios:

after urea addition, the gelatin-urea solution was stirred at 50 ℃ for 5 minutes to ensure homogeneity and then transferred to a 37 ℃ water bath.

Influence of urea on thermoreversible gelation of gelatin:

each gelatin-urea solution was removed from the 37 ℃ water bath sequentially and left to cool to RT. The temperature decrease of each solution was monitored with a thermometer. The appearance and viscosity of the gelatin-urea solution was evaluated by observing and touching the solution with a glass rod as the temperature decreased. The results of this experiment are described in the following table:

crosslinking of gelatin-urea solution using mTG:

the gelatin-urea solution was cross-linked using mTG. 1 or 2ml of a 20% w/w solution of mTG was manually mixed with 2ml of a gelatin-urea solution in a plastic dish. The gelatin-urea solution is added at room temperature or after preheating to 50 ℃. In some tests, it was heated to 50 ℃ to facilitate comparison with gelatin alone, which needed to be heated to mix with mTG. The solution was mixed by gentle stirring with a pipetting head and allowed to crosslink for a few minutes. As in the test of thermoreversible gel formation, the appearance and viscosity of the gelatin-urea solution after mixing with mTG was evaluated by observing and touching the solution with a glass rod. The results are summarized in the following table:

the above studies show that the addition of urea to a gelatin solution significantly reduces the transition point of gelatin. For 15% and 25% w/w gelatin solutions, the transition point was reduced below RT by adding urea in the ratio 1: 1 urea to gelatin and above. In the case of a urea to gelatin ratio of 0.5: 1, the transition point of the gelatin solution is slightly above RT. It is likely that a urea to gelatin ratio between 0.5: 1 and 1: 1 will be sufficient to reduce the transition point of the gelatin below RT.

It has also been shown that cross-linking of gelatin using mTG in the presence of urea is possible. However, urea has an adverse effect on mTG activity. It appears that this effect is related to the concentration of urea, such that mTG activity in the presence of urea is inversely proportional to urea concentration.

As expected, the transglutaminase activity at 50 ℃ is much greater than the mTG activity at RT. The addition of urea to gelatin can be optimized to find the minimum concentration that will reduce the gelatin transition point below RT. mTG, if added in sufficient quantities, will be able to overcome the adverse effects of urea.

Example 11

Effect of pH on gelatin transition Point

This example demonstrates the effect of varying the pH of gelatin solutions on the transition point of those gelatin solutions.

Preparation of solutions

58.82gr of 99% sodium citrate dihydrate (Alfa Aesar, Lancaster, UK) was dissolved in 100mL of double distilled water to produce a 2M stock of sodium citrate. A basic solution of type A, 300 bloom, porcine gelatin (Sigma, St. Louis, Mo.) in Dulbecco PBS (biologicals industries, Israel) at 25% w/w gelatin solution was prepared. The gelatin solution was continuously stirred and maintained at 50 ℃. 19.21gr of anhydrous citric acid (Frutarom, Israel) was dissolved in 50mL of double distilled water to produce a 2M citric acid stock.

pH measurement

The pH of the solution was measured using a pH meter (Eutech pH510, Singapore) with a glass electrode. The pH meter was calibrated prior to the experiment using calibration solutions with pH values of 4.01, 7 and 10.01. The accuracy of the pH measurements was determined periodically during the experiment. The pH of the 2M sodium citrate solution was 8.54. The pH of the 2M citric acid solution was 1.4.

Adding sodium citrate

A20 mL aliquot of 25% w/w gelatin solution was separated into a 100mL beaker, which was maintained at 50 ℃ with moderate agitation. The initial pH of the gelatin solution was measured to be 4.89. Various amounts of 2M sodium citrate solution were added to 20mL of gelatin solution to form the following solutions:

solution 1: pH of 5.87-2 mL sodium citrate solution

Solution 2: pH of 6.55-4 mL sodium citrate solution

Solution 3: pH of 6.7-6 mL sodium citrate solution

Each solution was then cooled to RT.

Adding citric acid

A100 mL aliquot of 25% w/w gelatin solution was separated into a 250mL beaker, which was maintained at 50 ℃ with moderate agitation. The initial pH of the gelatin solution was measured to be 5.19.

Various amounts of 2M citric acid solution were added to the gelatin solution to form the following solutions:

solution 1: pH of 3.99

Solution 2: pH of 3.54

Solution 3: pH of 2.72

Solution 4: pH of 2.35

Solution 5: pH of 2.17

Solution 6: pH of 2.04

Solution 7: pH of 1.7

Each solution was then cooled to RT.

Sodium citrate results

When sodium citrate was added, the addition formed a cloudy white clot in the gelatin solution. Vigorous stirring first dispersed the coagulum into smaller coagulums, followed by a homogeneous solution. The homogeneous solution formed was turbid and opaque.

A pH of 5.87 allows the cooled gelatin solution aliquot to form a thermoreversible gel in about the same amount of time as gelatin alone forms thermoreversible gelatin.

A pH of 6.55 allows the cooled gelatin solution aliquot to form a thermoreversible gel very rapidly in less than one minute. This is much faster than gelatin alone.

At a pH of 6.70, the thermoreversible gel formed almost immediately in small portions. After holding the gelatin-sodium citrate solution at 50 ℃ for several minutes, the whole solution formed a gel. The transition point has increased to a point above 50 ℃.

At all pH values, the gel proved to be thermoreversible since it returned to a liquid form when immersed in water at 60 ℃.

Citric acid results

No significant difference in transition point was observed at pH values above 3.54. At a pH of 3.54, from 50 ℃ up to about 32 ℃ (point where a very viscous gel is formed) the gelatin solution remains in liquid form.

At a pH of 2.72, the transition point is about 31 ℃ and the formed gelatin is loose: fine grained, with many bubbles.

At a pH of 2.04, the transition point drops to 29 ℃. The gel formed was more porous than the gel formed at a pH of 2.72.

At a pH of 1.7, the transition point was lowered to 27-28 ℃. The gel formed was rather loose.

At all pH values, the gel proved to be thermoreversible since it returned to a liquid form when immersed in water at 50 ℃.

However, after the gel was held in water at 50 ℃ for 30 minutes, a gel was formed that did not revert back to liquid form. This may have shown that citric acid at 50 ℃ causes the gelatin to crosslink after 30 minutes.

The above shows that lowering the pH of a gelatin solution by adding citric acid can significantly lower the gelatin transition point. The addition of citric acid, which reduces the pH of the gelatin solution to 2, did not result in cross-linking of the gelatin. Further addition of citric acid at 50 ℃ to lower the pH below 2 can result in crosslinking of the gelatin after 30 minutes.

Example 12

Effect of polyols on gelation and crosslinking of gelatin

This example relates to the effect of polyols such as sorbitol on gelatin crosslinking.

Materials and methods

Material

300 bloom type A porcine gelatin and 97% D-sorbitol were obtained from Sigma-Aldrich corporation (St. Louis, Mo.). Glycerol was purchased from Frutarom (Israel) at 99%. ActivataTI-WM microbial transglutaminase (mTG) is supplied by Ajinomoto (Japan). Dulbecco' S PB S (pH7.4) was obtained from biologicalcal industries (Kibbutz Bait HaEmek, Israel).

Preparation of mTG solution:

a fresh mixture of Activa TI-WM Ajinomoto (Japan) microbial transglutaminase (mTG) was prepared by dissolving in Dulbecco's PBS to form a 20% w/w solution. The solution was kept at Room Temperature (RT) during the experiment.

Preparation of gelatin-polyol solution:

solution A-10 gr of glycerol was dissolved in 30ml of PBS. 10gr of gelatin were then immersed in the glycerol solution at RT for 1.5 hours. After 1.5 hours of soaking, 10ml of PBS was added and the mixture was heated to 50 ℃. The mixture was stirred by hand until a homogeneous liquid solution (1: 1 glycerol: gelatin ratio) was formed.

Solution B-with stirring, 27.5ml of a 20% w/w gelatin solution were heated to 50 ℃. To the gelatin solution was added 11gr of glycerol (2: 1 glycerol: gelatin ratio). The glycerol-gelatin solution was then cooled. A2: 1 solution of glycerol and gelatin was allowed to soak at 50 deg.C for 1.5 h. The solution was then removed and allowed to cool at RT.

Solution C-with stirring, a 20% gelatin solution containing glycerol in a 2: 1 ratio of glycerol to gelatin was heated to 50 ℃. Sorbitol 11gr was added to form a homogeneous solution (glycerol: sorbitol: gelatin ratio 2: 1)

Solution D-to 20ml of a 20% w/w gelatin solution at 50 ℃ 12gr of sorbitol was added to form a homogeneous solution with a sorbitol to gelatin ratio of 3: 1. The solution was then allowed to cool.

Solution E-A5: 1 solution of glycerol to gelatin was prepared by adding 25gr of RT glycerol to 20ml of a 20% w/w gelatin solution at 50 ℃. The mixture was mixed by stirring at 50 ℃.

Solution F-20 gr of sorbitol was added to 20ml of a 20% gelatin solution, resulting in a sorbitol to gelatin ratio of 5: 1. The mixture was mixed at 50 ℃. The mixture was then cooled at RT.

Effect of polyols on gelation of gelatin:

2ml of gelatin solution containing polyol prepared as described above (see solutions A-F above) were removed and cooled to RT. The temperature of each solution was measured with a thermometer. The appearance and viscosity of the gelatin-polyol solution was evaluated by observation and touching the solution with a glass rod as the temperature decreased.

Effect of polyols on crosslinking of gelatin solutions using mTG:

gelatin-polyol solutions were tested for chemical crosslinking using mTG. 1ml of a 20% w/w solution of mTG was mixed with 2ml of a gelatin-polyol solution in a small plastic dish. The gelatin-polyol solution was added after preheating to 50 ℃ at RT or using a water bath. The solution was mixed manually by gentle stirring with a pipetting head and the time to gelation was measured. The appearance and viscosity of the gelatin-polyol solution after mixing with mTG was evaluated by observing and touching the solution with a glass rod. When the gel was formed, its thermoreversibility was tested by heating the gel to 50 ℃ using a water bath.

Results

Effect of polyols on gelation of gelatin:

solution a-after immersion in glycerol, the gelatin particles cluster together to form a very friable solid, fine particulate mass. The presence of glycerol does not appear to slow down thermoreversible gelatin gelation at all. Thermoreversible gels were formed within 2-3 minutes as occurred with a 20% w/w gelatin solution without glycerol. At 35 ℃, the gelatin-glycerol solution was very viscous, approaching gelation. As with cooling to room temperature, the phase transition of gelatin-glycerol is nearly identical to that of gelatin alone.

Solution B-like the 1: 1 glycerol to gelatin solution, glycerol at a 2: 1 glycerol to gelatin ratio did not lower the gelatin transition temperature. The solution was extremely viscous at 35 ℃ and formed a cohesive gel at 33-34 ℃. Soaking in a 2: 1 solution of glycerol and gelatin for 1.5 hours had no effect on the gelatin transition point. The thermoreversible gel begins to form at 34 ℃.

At solution C-50 ℃, the gelatin-glycerol-sorbitol solution is more viscous and opaque than the gelatin-glycerol solution alone. When this mixture was cooled, a gel formed at 35 ℃. As a result of sorbitol and glycerol, the transition point is not reduced at all.

The gelatin-sorbitol solution started to gel at solution D-40 ℃, indicating that a high concentration of sorbitol did raise the gelatin transition point. At RT, the thermoreversible gel formed was much more elastic than the gelatin gel alone.

Solution E-the transition temperature of gelatin is not lowered even at very high concentrations of glycerol to gelatin ratio of 5: 1. Thermoreversible gelatin is formed as formed with gelatin alone.

The solution F forms thermoreversible gel at 40 ℃. Very little difference was observed between the properties of the solutions of sorbitol: gelatin 3: 1 and 5: 1. Both solutions raise the transition point of gelatin slightly but produce a thermoreversible gel that is extremely viscous and elastic.

Effect of polyols on crosslinking of gelatin solutions using mTG:

solution a-became a hard gel within 3 minutes via mTG gelatin-glycerol solution. The gel formed after 3 minutes was more cohesive than the gel formed by gelatin and mTG in the same time period without glycerol. Using a water bath to reheat the gel to 50 ℃ while the gel remains solid, it was confirmed that mTG cross-linking is the mechanism of gelation.

After 3 minutes for solution B, an irreversible gel was formed. Using a water bath to reheat the gel to 50 ℃ while the gel remains solid, it was confirmed that mTG cross-linking is the mechanism of gelation. The presence of glycerol produced a firmer gel after 3 minutes as noted in the 1: 1 glycerol to gelatin ratio solution. In a 2: 1 glycerol to gelatin ratio solution, it was also observed that the gel formed was significantly more brittle than the gel formed with gelatin alone and also significantly more brittle than the gel formed from a solution having a 1: 1 ratio of glycerol to gelatin.

After 3 minutes of solution C, a solid, viscous, very elastic gel was formed. This gel is not brittle at all and is not easily separated. This is considered to be a very important result since the cross-linked gel with glycerol alone is very brittle. Sorbitol greatly increases the elasticity of an otherwise brittle material. The gel was reheated to 50 ℃ with a water bath while the gel remained solid, confirming that mTG crosslinking is the mechanism of gelation.

Solution E-results are very similar to those of glycerol at a ratio of 2: 1 glycerol to gelatin: the presence of glycerol results in the formation of a gel that is much more brittle than a gel formed from gelatin alone. However, in the presence of mTG, after 3 minutes the gel formed was stronger than the gel formed with mTG from gelatin alone after 3 minutes.

After solution F-3 minutes, a solid but extremely elastic and sticky gel was formed by crosslinking the mTG of the gelatin-sorbitol solution. In addition to greatly increasing the elasticity and viscosity of mTG crosslinked gelatin gels, sorbitol appears to have no effect on mTG crosslinking of gelatin.

From the above it was found that the addition of glycerol to gelatin did not seem to reduce the transition point of gelatin at all. Soaking gelatin in glycerol does not appear to significantly alter its tendency to form thermoreversible gels. The presence of glycerol appears to result in a harder gel after the gelatin solution is mixed with mTG for 3 minutes. This may indicate an acceleration of mTG crosslinking of gelatin when glycerol is present in the gelatin.

The presence of high concentrations of glycerol during mTG crosslinking of gelatin appears to make the resulting crosslinked gel more brittle than a gel formed by crosslinking of gelatin alone. Glycerol appears to accelerate mTG crosslinking of gelatin.

The addition of sorbitol in combination with glycerol did not lower the turning point of gelatin. Sorbitol, however, greatly increases the elasticity and viscosity of the gelatin gel. Sorbitol may be able to be used to increase the elasticity of gelatin gels that appear more brittle with the addition of other substances. Sorbitol does not appear to inhibit mTG crosslinking of gelatin. Although sorbitol appears to slightly increase the transition point of gelatin, it greatly increases the elasticity and viscosity of the gelatin gel.

Increasing the glycerol to gelatin ratio to 5: 1 makes the cross-linked gelatin gels more brittle than those made with a 2: 1 glycerol to gelatin solution but has no further effect on the gelatin transition point. The slight crosslinking acceleration effect at this higher glycerol to gelatin ratio still occurs but is not more pronounced than at 1: 1 and 2: 1 glycerol to gelatin ratios.

A solution having a sorbitol to gelatin ratio of 5: 1 increased the gelatin transition point but not more than a solution having a sorbitol to gelatin ratio of 3: 1. However, the crosslinked gelatin gel formed from the 5: 1 solution was even more elastic and viscous. This further indicates that the amount of sorbitol can be varied to alter the elasticity of the crosslinked gelatin gel.

Example 13

Effect of spray drying on gelation and crosslinking of gelatin

This example relates to the effect of spray drying on gelation and crosslinking of gelatin. Preferred ranges of particle size formed via use of spray drying are preferably as follows: from about 20 to about 140 μm, more preferably from about 60 to about 100 μm (diameter).

Various strategies for particle formation are optionally contemplated. One possible strategy is to form granules of gelatin and mTG that are easily reconstituted using a special drying technique for this purpose or by including additives and then drying the gelatin and mTG including additives into granules, respectively. Another possible strategy is to form easily reconstituted particles that include gelatin and mTG together. These particles may be formed solely by using specialized drying techniques or by including additives that improve the reconstitutability of these particles. Furthermore, these particles may be produced when the gelatin and mTG have not undergone any cross-linking or after they have undergone partial cross-linking.

Materials and methods

Material

Porcine gelatin form a 300 bloom was obtained from Sigma-Aldrich corporation (st. louis, MO). Activata TI-WM microbial transglutaminase (mTG) is supplied by Ajinomoto (Japan). Dulbecco's PBS (pH7.4) was obtained from Biological Industries (Kibbutz Bait HaEmek, Israel). Urea was obtained from Alfa Aesar (Lancaster, UK), 98%.

Preparation of mTG solution:

a20% (w/w) solution of microbial transglutaminase (mTG) was prepared by dissolving 4gr of mTG in 16gr of Dulbecco's PBS. The solution was kept at Room Temperature (RT) during the experiment.

A4% (w/w) solution of microbial transglutaminase (mTG) was prepared by dissolving 2.04gr of mTG in 50gr of Dulbecco's PBS. The solution was kept at Room Temperature (RT) during the experiment.

Preparing a gelatin solution:

a5% (w/w) gelatin solution was prepared by dissolving 10.52gr of gelatin powder in 200gr of Dulbecco's PBS. The mixture was heated to 50 ℃ and stirred until a homogeneous solution was formed. Solution 1 is characterized by 50ml of a 5% gelatin solution. Solution 2-A1: 1(w/w) solution of gelatin-mannitol was prepared by adding 2.63gr of mannitol to 50ml of a 5% gelatin solution. The solution was stirred and placed in a water bath at 50 ℃. Throughout the experiment, the solution was placed in a-50 ℃ water pan to prevent thermoreversible gelation.

Solution 3-A1: 1(w/w) gelatin-trehalose solution was prepared by dissolving 2.63gr trehalose in 50ml of a 5% gelatin solution while heating to 50 ℃ with stirring. Throughout the experiment, the solution was maintained at 50 ℃.

Solution 4-A1: 1(w/w) gelatin-urea solution was prepared by dissolving 2.63gr of urea in 50ml of a 5% gelatin solution while stirring and heating to 50 ℃. Throughout the experiment, the solution was maintained at 50 ℃.

Solution 5-40 gr of 5% gelatin solution was mixed with 20gr of 4% mTG solution. Throughout the experiment, the solution was maintained at 50 ℃.

Spray drying of gelatin solutions

To prepare spray-dried gelatin particles, different 5% gelatin solutions (solutions 1-5) in Dulbecco's PBS were prepared. Use ofThe micro spray dryer spray-dries the gelatin solution. The flow type is co-current and the air and liquid mix at the spray head. The aspiration rate (asparator rate) and the inlet temperature were kept constant at 100% and 100 ℃ respectively. The liquid feed rate is varied according to the process conditions, affecting the outlet temperature as given below.

Solution 1-50 ml of a 5% (w/w) gelatin solution was heated continuously and spray dried at 15% feed rate, exit temperature 57 ℃.

Solution 2-50 ml of a 1: 1(w/w) solution of gelatin-mannitol were kept soluble in a water bath at 50 ℃. The liquid feed rate was 15% and the exit temperature was 62 ℃.

Solution 3-50 ml of a 1: 1(w/w) gelatin-trehalose solution was kept soluble in a water bath at 50 ℃. The liquid feed rate was 15% and the exit temperature was 54 ℃.

Solution 4-50 ml of a 1: 1(w/w) gelatin-urea solution was kept soluble in a water bath at 50 ℃. The liquid feed rate was set at 15% and the exit temperature was 57 ℃. Throughout the experiment, the liquid feed rate was varied to 20% and the exit temperature 54 ℃ to enable powder formation.

Solution 5-40 ml of a 5% gelatin solution mixed with a 4% mTG solution was spray dried at a liquid feed rate of 20% and an exit temperature of 56 ℃. The solution was placed in a 37 ℃ water dish throughout the experiment.

Effect of spray drying on gelation of gelatin solutions:

spray dried gelatin powder was dissolved with Dulbecco's PBS in a 4ml vial and mixed by inverting the tube 4 times. The precipitated solution was heated in a water bath at 50 ℃ and mixed by hand until dissolved. The solutions were then cooled at RT and the appearance and viscosity of each solution was evaluated by observing and touching the solution with a glass rod as the temperature decreased.

Solution 1-0.33 gr of a 5% solution of spray dried gelatin was dissolved in 1ml of RT of Dullbeco's PBS to a final 25% (w/w) gelatin. Then another 1ml of Dullbeco's PBS was added to reduce the gelatin content to 12.5% (w/w).

Solution 2-0.33 gr of spray dried 1: 1 gelatin-mannitol solution was dissolved in 1ml of RT Dullbeco's PBS.

Solution 3-0.33 gr of spray dried 1: 1 gelatin-trehalose solution was dissolved in 1ml of RT in Dullbeco's PBS.

Spray drying of the solution 4-gelatin-urea failed to produce a powder.

Solution 5-0.25 gr of spray dried gelatin-mTG solution was dissolved in 0.75ml of 37 ℃ Dullbeco's PBS.

Effect of spray drying on chemical crosslinking of gelatin solutions using mTG:

the spray-dried gelatin powder was dissolved and placed in a water bath at 50 ℃ as described in the previous section. A20% solution of RT in mTG was added at a 2: 1 ratio of gelatin to mTG and the pipetting head was used and gently mixed 4 times through the inverted tube. The gelation time was measured and the appearance and viscosity of the solution after mixing with mTG was evaluated by observing and touching the solution with a glass rod. When the gel was formed, its thermoreversibility was tested by heating the gel to 50 ℃ with a water bath.

As a result:

spray drying of gelatin solutions

Spray drying of the gelatin solution produced varying amounts of fine white powder. Solution 1-50 ml of 5% gelatin solution yields 0.78 gr. Solution 2-40 ml of a 5% gelatin solution mixed with mannitol in a 1: 1 ratio (w/w) yields 0.73 gr. Solution 3-50 ml of a 5% gelatin solution mixed with trehalose in a 1: 1 ratio (w/w) yields 1.135 gr. Solution 4-no powder was produced. The experiment was concluded because gelatin mixed with urea produced a very viscous paste that could not be collected. Solution 5-40 ml of a 5% gelatin solution mixed with a 4% mTG solution yields 1.27 gr.

Effect of spray drying on gelation of gelatin solutions:

solution 1-gelatin powder was partially dissolved in PBS at RT to a final 25% (w/w) gelatin, forming a white insoluble precipitate. After heating in a water bath at 50 ℃, the powder dissolved to give a homogeneous solution. On cooling to RT, the solution gelled and an additional 1ml of PBS was added to reduce the gelatin to 12.5 (w/w). A12.5% gelatin solution gelled at 26-27 ℃.

Solution 2-partially dissolve gelatin-mannitol powder in PBS at RT to a final-12.5% (w/w) gelatin (gelatin is expected to be 1/2 amount of powder produced, but the exact percentage of gelatin is unknown). A white insoluble precipitate formed. After heating in a water bath at 50 ℃, the powder dissolved to give a homogeneous solution. Upon cooling to RT, the solution gelled at 28-29 ℃.

Solution 3-partially dissolve gelatin-trehalose powder in PBS at RT to a final-12.5% (w/w) gelatin (gelatin is expected to be 1/2 amount of powder produced, but the exact percentage of gelatin is unknown). A white insoluble precipitate formed. After heating in a water bath at 50 ℃, the powder dissolved to give a homogeneous solution. Upon cooling to RT, the solution gelled at 28-29 ℃.

Solution 4-solution contains a cross-linking agent so cross-linking of the solution is examined rather than gelation.

Effect of spray drying on chemical crosslinking of gelatin solutions using mTG:

solution 1-to 2ml of 12.5% gelatin solution was added 500ul of 20% mTG solution. After 4 minutes a firm white gel formed. The gel is irreversible.

Solution 2-to 1ml of a 12.5% gelatin solution containing mannitol was added 250ul of a 20% mTG solution. After 2.5 minutes a firm white gel formed. The gel is irreversible.

Solution 3-to 1ml of a 12.5% gelatin solution containing trehalose, 250ul of a 20% mTG solution was added. After 3 minutes a firm white gel formed. The gel is irreversible.

Solution 4-mTG-gelatin solution was dissolved in 1ml of Dulbecco's PBS heated to 37 ℃. A white, brittle gelatin immediately formed, preventing complete dissolution in PBS. The gel formed is irreversible.

From the above, it was shown that spray drying of gelatin solutions is possible. For example, a 5% gelatin solution may be spray dried to produce a fine white powder. Gelatin comprising mannitol or trehalose in a 1: 1(w/w) gelatin to mannitol or trehalose ratio may be spray dried. The solution of spray-dried gelatin-mannitol has a higher transition point than the spray-dried gelatin alone. Spray dried gelatin-trehalose has a lower transition point than spray dried gelatin alone.

Cross-linking of a solution of spray-dried gelatin is possible. Spray drying of a 1: 1(w/w) gelatin-mannitol solution improves crosslinking. Spray drying of a 1: 1(w/w) gelatin-trehalose solution did not improve crosslinking.

Spray drying of the gelatin solution mixed with mTG is possible. The formed particles may form a gel immediately upon reconstitution.

Example 14

Applicator for applying sealing material

This example relates to an exemplary illustrative applicator for applying a hemostatic sealing material according to certain embodiments of the present invention. Fig. 11A shows an example of a dual syringe applicator 1700 that features two syringes 1702 and 1704 for containing each component of a two-component hemostatic sealing material. Syringe 1702 may optionally contain gelatin or a substitute as described herein, while syringe 1704 may optionally include transglutaminase or a substitute as described herein. The difference in vial volume will reflect the relative ratio of mixing between the two components. The two components may be mixed in the nozzle 1705. To improve mixing, the nozzle 1705 may include an element 1706 (shown in more detail in fig. 11B) that creates a vortex. The injector applicator 1700 is optionally connected to a pressurized air system 1708 at the nozzle 1705 to create a spray effect. Pressurized air may optionally enter the proximal or distal end of the nozzle 1705 depending on the desired application.

Example 15

Catheter and method of use thereof

This example relates to an exemplary, illustrative catheter and method of use thereof according to certain embodiments of the present invention. Fig. 12A shows an example of vascular insertion point closure, where a catheter 1200 is preferably characterized by a coating 1202 comprising a sealing material composition described herein. The coating 1202 may be selected and preferably characterized by at least one gelatin layer 1204, two of which gelatin layers 1204 are shown for illustrative purposes only and not for any limiting purpose. The gelatin layer 1204 may optionally be replaced with another protein substrate as described herein. The coating 1202 may also optionally and preferably feature at least one transglutaminase layer 1206, which may also optionally be substituted with another crosslinking agent as described herein. The coating 1202 preferably encases a vascular introducer 1208, also known as a trocar.

The sleeve 1208 is optionally covered by another jacket 1209, the jacket 1209 creating a mechanical barrier between the dried sealant and bodily fluids, as shown in fig. 12B. Once the sheath 1209 is removed, the sealant composition can be activated by body fluids to create a closed plug of perivascular access points.

The present invention has been described with reference to the foregoing detailed description and preferred embodiments thereof, but the foregoing description is intended to illustrate and not limit the invention, which is defined by the scope of the included claims. Other aspects, advantages, and modifications are within the scope of those claims.

Claims (19)

1. A composition comprising a combination of gelatin and transglutaminase, wherein the transglutaminase is added as part of a transglutaminase composition, wherein the activity of the transglutaminase in a gelatin-transglutaminase composition is from 40U to 200U/g gelatin, and wherein the weight ratio of gelatin to transglutaminase composition is from 1: 1 to 300: 1 in the range of;

wherein the composition comprising a combination of gelatin and transglutaminase promotes in situ cross-linking between gelatin chains and endogenous collagen of the tissue extracellular matrix when applied to a wound site to create a barrier to fluid exudation.

2. The composition of claim 1, wherein the fluid exudation is bleeding.

3. The composition according to claim 1 or 2, wherein the transglutaminase comprises a plant, fish, mammal, or microorganism-derived transglutaminase other than blood-derived factor XIII.

4. The composition of claim 3, wherein the microbial-derived transglutaminase is extracted from one or more of streptoverticillium bardahliae, streptomyces hygroscopicus, or escherichia coli.

5. The composition of claim 1 or 2, having a pH in the range of from 5 to 8.

6. The composition according to claim 1 or 2, wherein the gelatin is acid-treated or base-treated.

7. The composition of claim 1 or 2, further comprising a stabilizer or filler.

8. The composition according to claim 1 or 2, wherein the gelatin is purified to remove salt.

9. The composition of claim 1 or 2, wherein the fluid is selected from the group consisting of blood, cerebrospinal fluid, intestinal fluid, gas, bile and urine.

10. The composition of claim 1 or 2, which induces the formation of a biomimetic blood clot at the site of an injured blood vessel.

11. The composition of any one of claims 1-2 and 4, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

12. The composition of claim 3, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

13. The composition of claim 5, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

14. The composition of claim 6, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

15. The composition of claim 7, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

16. The composition of claim 8, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

17. The composition of claim 9, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

18. The composition of claim 10, wherein the wound site is selected from the group consisting of surgically cut tissue, surgically repaired tissue, and traumatized tissue.

19. A hemostatic bandage comprising the composition of any one of claims 11-18.