HK1228807A1

HK1228807A1 - Rapidly acting dry sealant and methods for use and manufacture

Info

Publication number: HK1228807A1
Application number: HK17102398.5A
Authority: HK
Inventors: Woonza M. Rhee; Cary J. Reich; A. Edward Osawa; Felix Vega
Original assignee: Baxter International Inc.; Baxter Healthcare S.A.
Priority date: 2006-08-02
Filing date: 2017-03-08
Publication date: 2017-11-10

Description

Quick-acting dry sealant and methods of use and preparation

This application is a divisional application with application number 200780036641.0. The filing date of the parent case is 8/1/2007; the invention provides a quick-drying sealant and methods of use and preparation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. patent application No. 60/821,190 filed on 8/2/2006. This application is also related to U.S. patent nos. 5,874,500, 6,063,061, 6,066,325, 6,166,130 and 6,458,889. The contents of each of these submissions are hereby incorporated by reference.

Statement regarding rights to inventions resulting from research and development with federal government assistance

Not applicable to

Appendix to "sequence Listing", forms or computer program lists submitted on compact disks

Not applicable to

Background

In U.S. patent No. 5,162,430 issued to Rhee et al on 11/10 1992, there is a discussion of collagen-synthetic polymer conjugates prepared by covalently linking collagen to synthetic hydrophilic polymers such as various derivatives of polyethylene glycol. In U.S. patent No. 5,324,775 issued to Rhee et al at 28.6.1994, there is a discussion of various embedded natural biocompatible polymers (e.g., polysaccharides) covalently linked to synthetic, non-immunogenic hydrophilic polyethylene glycol polymers. In U.S. patent No. 5,328,955 issued to Rhee et al, 7/12, 1994, there is a discussion of various activated forms of polyethylene glycol and various linkages that can be used to produce collagen-synthetic polymer conjugates having a range of physical and chemical properties.

In sequence number 08/403,358 filed 3/14 in 1995, there is a discussion of crosslinked biomaterial compositions prepared with hydrophobic crosslinkers or mixtures of hydrophilic and hydrophobic crosslinkers. The hydrophobic crosslinking agent can include any hydrophobic polymer that contains or can be chemically derivatized to contain two or more succinimide groups.

In U.S. patent No. 5,580,923 issued to Yeung et al, 12/3/1996, there is a discussion of a composition useful for preventing surgical adhesions, the composition comprising a base material and an anti-adhesive, wherein the base material preferably comprises collagen, and preferably the adhesive comprises at least one tissue-reactive functional group and at least one matrix-reactive functional group.

In U.S. patent No. 5,614,587 issued to Rhee et al, 3/25 1997, there is a discussion of bioadhesive compositions comprising collagen crosslinked with a multifunctional activated synthetic hydrophilic polymer, and methods of using such compositions to accomplish adhesion between a first surface and a second surface, wherein at least one of the first and second surfaces may be a native tissue surface.

In Japanese patent publication No. 07090241, there is a discussion about a composition for lens material for temporary adhesion to support and mount the material in a mechanical device, which contains a mixture of polyethylene glycol having an average molecular weight of 1000-5000 and poly-N-vinylpyrrolidone having an average molecular weight of 30,000-200,000.

West and Hubbell in Biomaterials (1995) 16: 1153-1156 discusses the use of photopolymerizable polyethylene glycol-lactic acid copolymer diacrylate hydrogels and physically cross-linked polyethylene glycol-polypropylene glycol copolymer hydrogel poloxamersPreventing postoperative adhesion.

In U.S. patent nos. 5,672,336 and 5,196,185 there is a discussion of wound dressings containing particulate fibrous collagen of 0.5 to 2.0 μm particle size. The composition comprises predominantly an aqueous phase, but does not form a hydrogel as described in the present invention. A discussion of crosslinked aliphatic polyester hydrogels that can be used as absorbable surgical devices and drug delivery vehicles is provided in U.S. patent No. 5,698,213. Acrylate or methacrylate based hydrogel adhesives are discussed in U.S. Pat. No. 5,674,275. A discussion of polyoxyalkylene based thermoreversible hydrogels useful as drug delivery vehicles is presented in U.S. patent No. 5,306,501.

Hydrogels containing protein components crosslinked with polysaccharides or mucopolysaccharides useful as drug delivery vehicles are discussed in U.S. patent nos. 4,925,677 and 5,041,292.

In U.S. patent No. 5,384,333 and Jeong et al (1997) "Nature," 388: 860-862 are discussed with respect to biodegradable injectable delivery polymers. Biodegradable hydrogels for controlled drug delivery are discussed in U.S. patent No. 4,925,677. A discussion of resorbable collagen-based delivery systems is provided in U.S. patent nos. 4,347,234 and 4,291,013. In U.S. patent nos. 5,300,494 and 4,946,870 there is a discussion of aminopolysaccharide-based biocompatible membranes for drug delivery. U.S. patent No. 5,648,506 is directed to a water-soluble carrier for delivery of paclitaxel.

Polymers have been used as carriers for therapeutic drugs to achieve local and sustained release (Langer, et al, Rev.Macro.Chem.Phys., C23(1), 61, 1983; Controlled Drug Delivery, Vol.I and II, Bruck, S.D., (ed.), CRC Press, Boca Raton, Fla., 1983; Leong et al, adv.drug Delivery review, 1: 199, 1987). These therapeutic drug delivery systems are similar to infusion, offering the potential to enhance therapeutic efficacy and reduce systemic toxicity.

Other classes of synthetic polymers proposed for Controlled drug Release include polyesters (Pitt, et al, Controlled Release of Bioactive Materials, R.Baker, Ed., Academic Press, New York, 1980); polyamides (Sidman, et al, Journal of Membrane Science, 7: 227, 1979); polyurethanes (Maser, et al, Journal of Polymer Science, Polymer Symposium, 66: 259, 1979); polyorthoesters (Heller, et al, Polymer Engineering science, 21: 727, 1981); and polyanhydrides (Leong, et al, Biomaterials, 7: 364, 1986).

In U.S. Pat. nos. 5,428,024; 5,352,715; and 5,204,382 for a collagen-containing composition that undergoes a change in physical property by mechanical disruption. These patents are primarily concerned with fiber and insoluble collagen. There is a discussion of injecting a collagen composition in U.S. Pat. No. 4,803,075. There is a discussion of bone/cartilage injectable compositions in U.S. patent No. 5,516,532. In WO 96/39159 there is a discussion of collagen-based release matrices comprising dry particles of 5 μm to 850 μm size, which can be suspended in water and have a specific surface charge density. In us patent No. 5,196,185 there is described a collagen preparation having a particle size of 1 μm to 50 μm, which can be used as an aerosol spray for forming a wound dressing. Other patents that discuss collagen compositions include U.S. patent nos. 5,672,336 and 5,356,614. In WO 96/06883 there is a discussion of polymer non-erodible hydrogels which are cross-linkable and injectable by means of a syringe.

The following co-pending applications, assigned to the assignee of the present invention, contain relevant subject matter: U.S. patent application serial No. 08/903,674 filed on 31/7/1997; U.S. patent application serial No. 60/050,437 filed on 18/6/1997; U.S. patent application serial No. 08/704,852 filed on day 8, 27 of 1996; U.S. patent application serial No. 08/673,710 filed on 19/6/1996; U.S. patent application serial No. 60/011,898 filed 2/20 1996; U.S. patent application serial No. 60/006,321 filed 11/7 1996; U.S. patent application serial No. 60/006,322 filed 11/7 1996; U.S. patent application serial No. 60/006,324 filed 11/7 1996; and U.S. patent application serial No. 08/481,712 filed on 7.6.1995. Each of these applications is incorporated by reference herein in its entirety. Each of the above documents and publications cited therein is incorporated herein by reference. There are a variety of materials suitable for use in bioadhesives for tissue augmentation, prevention of surgical adhesions, coating of synthetic implant surfaces, as drug delivery matrices, ophthalmic applications, and the like. In addition, in many cases, the setting time of these materials may be less than optimal, but for surgical and other medical applications, fast-acting materials are generally preferred. In other cases, currently available materials may exhibit swelling properties that are undesirable for certain surgical applications. Thus, there is a need for fast acting materials for use as tissue sealants, e.g., for hemostasis and/or wound sealing. It is also desirable to provide materials that exhibit minimal swelling properties.

Disclosure of Invention

The present invention provides compositions that achieve hemostasis or other fluid containment in an in vivo environment. The compositions of the present invention comprise first and second cross-linkable components and at least one hydrogel-forming component, and such compositions are suitable for application to a vertebrate to promote fluid containment. The compositions include fast-acting materials for use as tissue sealants, e.g., for hemostatic and/or wound sealing applications. Such compositions exhibit minimal swelling properties.

In a first aspect, embodiments of the present invention provide a composition comprising: a first crosslinkable component, a second crosslinkable component which crosslinks with the first crosslinkable component under conditions capable of reaction, and a hydrogel-forming component. The first cross-linkable component is cross-linked with the second cross-linkable component to form a porous matrix having voids, and the hydrogel-forming component is capable of hydrating to form a hydrogel to fill at least some of the voids. In certain aspects, the pH of the hydrogel-forming component can affect the reaction time to form the sealant matrix barrier. For example, in certain embodiments, a composition comprising a hydrogel-forming component at pH 6.75 is provided that has a slower reaction time than a composition comprising a hydrogel-forming component at pH 9.5.

The first cross-linkable component can comprise a plurality of nucleophilic groups and the second cross-linkable component can comprise a plurality of electrophilic groups. In certain aspects, the first crosslinkable component comprises a multi-nucleophilic polyalkylene oxide having m nucleophilic groups and the second crosslinkable component comprises a multi-electrophilic polyalkylene oxide having n electrophilic groups, wherein m and n are each greater than or equal to 2, and wherein m + n is greater than or equal to 5. In certain aspects, n is 2 and m is greater than or equal to 3. The multi-nucleophilic polyalkylene oxide may be tetrafunctionally activated. In certain aspects, m is 2 and n is greater than or equal to 3. The multi-electrophilic polyalkylene oxide can be tetrafunctionally activated. In some cases, both the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are tetrafunctionally activated. The multi-nucleophilic polyalkylene oxide may contain 2 or more nucleophilic groups such as NH₂、-SH、-H、-PH₂and/or-CO-NH₂. In some cases, the multi-nucleophilic polyalkylene oxide includes 2 or more primary amino groups. In some cases, the multi-nucleophilic polyalkylene oxide comprises 2 or more thiol groups. The multi-nucleophilic polyalkylene oxide may be polyethylene glycol or a derivative thereof. In thatIn some cases, the polyethylene glycol contains 2 or more nucleophilic groups, which may include primary amino groups and/or thiol groups. The multi-electrophilic polyalkylene oxide may comprise 2 or more electrophilic groups such as-CO₂N(COCH₂)₂、-CO₂H、-CHO、-CHOCH₂、-N＝C＝O、-SO₂CH＝CH₂、-N(COCH)₂and/or-S-S- (C)₅H₄N). The multi-electrophilic polyalkylene oxide can include 2 or more succinimide groups. The multi-electrophilic polyalkylene oxide can include 2 or more maleimide groups. In some cases, the multi-electrophilic polyalkylene oxide can be polyethylene glycol or a derivative thereof.

In certain aspects, the composition comprises a polysaccharide or a protein. The polysaccharide may be a glycosaminoglycan, such as hyaluronic acid, chitin, chondroitin sulfate a, chondroitin sulfate B, chondroitin sulfate C, keratin sulfate, heparin, or derivatives thereof. The protein may be collagen or a derivative thereof. The multi-nucleophilic polyalkylene oxide or the multi-electrophilic polyalkylene oxide, or both the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide may contain a linking group. In some cases, the multi-nucleophilic polyalkylene oxide can be given by the formula: Polymer-Q¹-X_m. The multi-electrophilic polyalkylene oxide can be given by the formula: Polymer-Q²-Y_n. X can be an electrophilic group, Y can be a nucleophilic group, m and n can each be from 2 to 4, m + n can be ≦ 5, Q¹And Q²Each may be a linking group such as-O- (CH)₂)_n′-、-S-、-(CH₂)_n′-、-NH-(CH₂)_n′-、-O₂C-NH-(CH₂)_n′-、-O₂C-(CH₂)_n′-、-O₂C-CR¹H and/or-O-R²-CO-NH. In some cases, n' may be 1 to 10, R¹Can be-H, -CH₃or-C₂H₅，R²Can be-CH₂-or-CO-NH-CH₂CH₂-，Q¹And Q²May be the same or different, or may be absent. In certain aspects, Y can be-CO₂N(COCH₂)₂or-CO₂N(COCH₂)₂In certain aspects, the hydrogel-forming component is capable of hydration to form a biocompatible hydrogel segment comprising gelatin and absorbs water when released to a target site in moist tissue, the hydrogel may comprise equilibrium swollen subunits having a size of from about 0.01mm to about 5mm and from about 400% to about 5000% when fully hydrated, in certain aspects, the hydrogel has an in vivo degradation time of less than 1 year.

In another aspect, embodiments of the present invention provide methods of delivering an active agent to a patient. The method can include administering to the patient a target site an amount of a composition described herein. In certain aspects, embodiments include methods of releasing a sealant to a patient. The method can comprise administering a composition described herein to the bleeding target site in an amount sufficient to inhibit bleeding. In certain aspects, embodiments include methods of releasing thrombin into a patient. The method can comprise administering a composition described herein to the bleeding target site in an amount sufficient to inhibit bleeding.

In yet another aspect, embodiments of the present invention include compositions comprising a multi-nucleophilic polyalkylene oxide, a multi-electrophilic polyalkylene oxide, and a hydrogel-forming component. The multi-nucleophilic polyalkylene oxide can further comprise at least one primary amino group and at least one thiol group. Under conditions capable of reaction, the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are capable of substantially immediate crosslinking. Embodiments include compositions wherein the multi-nucleophilic polyalkylene oxide comprises two or more thiol groups and the multi-electrophilic polyalkylene oxide comprises two or more electrophilic groups, such as succinimide groups and/or maleimide groups. Embodiments also include compositions wherein the multi-nucleophilic polyalkylene oxide comprises two or more nucleophilic groups, such as primary amino groups and/or thiol groups. The multi-electrophilic polyalkylene oxide can include two or more succinimide groups. In certain instances, embodiments include compositions comprising: a first polyethylene glycol having two or more thiol groups, a second polyethylene glycol having two or more succinimide or maleimide groups, and a hydrogel-forming component. The sum of thiol groups and succinimide or maleimide groups may be at least 5, and the first polyethylene glycol and the second polyethylene glycol may be capable of substantially immediate crosslinking under conditions capable of reaction. In some cases, the first polyethylene glycol contains 4 thiol groups and the second polyethylene glycol contains 4 succinimide groups. In some cases, the composition contains a protein or a polysaccharide. The polysaccharide may be a glycosaminoglycan such as hyaluronic acid, chitin, chondroitin sulfate a, chondroitin sulfate B, chondroitin sulfate C, keratin sulfate, heparin, or derivatives thereof. The protein may be collagen or a derivative thereof.

In another aspect, embodiments of the invention include a method of sealing a tissue tract. The method may include at least partially filling a tissue tract with a composition comprising a first crosslinkable component, a second crosslinkable component that crosslinks with the first crosslinkable component under conditions capable of reacting, and a hydrogel-forming component. The first and second cross-linkable components can cross-link to form a porous matrix having voids, and the hydrogel-forming component can be capable of being hydrated to form a hydrogel to fill at least some of the voids. In some cases, the hydrogel contains subunits having a size of about 0.05mm to about 5mm when fully hydrated and an equilibrium swelling of about 400% to about 1300%, which degrade after about 1 to about 120 days in the tissue tract. In some cases, the first crosslinkable component comprises a plurality of nucleophilic groups and the second polymer comprises a plurality of electrophilic groups.

In yet another aspect, embodiments of the invention include a method of inhibiting bleeding at a target site in a patient's body. The method can include delivering to the target site an amount of a composition sufficient to inhibit bleeding, wherein the composition comprises a first crosslinkable component, a second crosslinkable component that crosslinks with the first crosslinkable component under conditions that enable reaction, and a hydrogel-forming component. The first and second cross-linkable components can cross-link to form a porous matrix having voids, and the hydrogel-forming component can be hydrated to form a hydrogel to fill at least some of the voids. When fully hydrated, hydrogels may contain subunits of about 0.05mm to about 5mm in size, with equilibrium swelling of about 400% to about 1300%, which degrade after about 1 to about 120 days in the tissue tract. The first crosslinkable component can comprise a plurality of nucleophilic groups and the second crosslinkable component can comprise a plurality of electrophilic groups. In another aspect, embodiments of the invention include methods of delivering a biologically active substance to a target site in a patient's body. The method can include delivering a composition and a bioactive agent to the target site in combination, wherein the composition comprises a first cross-linkable component, a second cross-linkable component that cross-links with the first cross-linkable component under conditions that enable reaction, and a hydrogel-forming component. The first and second cross-linkable components can cross-link to form a porous matrix having voids, and the hydrogel-forming component can be hydrated to form a hydrogel to fill at least some of the voids. When fully hydrated, hydrogels may contain subunits of about 0.05mm to about 5mm in size, with equilibrium swelling of about 400% to about 1300%, which may degrade after about 1 to about 120 days in the tissue tract. In some cases, the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups. The biologically active substance may be a haemostatic agent, such as thrombin.

In another aspect, embodiments of the invention include methods of delivering a swellable composition to a target site in a tissue. The method can include applying a composition to a target site, wherein the composition comprises a first crosslinkable component, a second crosslinkable component that crosslinks with the first crosslinkable component under conditions that enable reaction, and a hydrogel-forming component. The first and second cross-linkable components can cross-link to form a porous matrix having voids, and the hydrogel-forming component can hydrate to form a hydrogel that fills at least some of the voids. When fully hydrated, hydrogels may contain subunits of about 0.05mm to about 5mm in size, which swell at equilibrium from about 400% to about 1300%, and which degrade after about 1 to about 120 days in the tissue tract. When applied to a target site where it swells to its equilibrium swelling value, the composition can be hydrated at less than its equilibrium swelling. In certain aspects, the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups. In certain aspects, the target site may be in the following tissues: muscle, skin, epithelial tissue, smooth muscle, skeletal or cardiac muscle, connective or supporting tissue, neural tissue, ocular and other sensory organ tissue, vascular and cardiac tissue, gastrointestinal organs and tissue, pleural and other lung tissue, kidney, endocrine glands, male and female reproductive organs, adipose tissue, liver, pancreas, lymphatic glands, cartilage, bone, oral tissue and mucosal tissue, and spleen and other abdominal organs. In certain aspects, the target site includes a void region in selected tissue, such as a tissue cortex (divot), a tissue tract, a space within the spine, or a body cavity. In some cases, the hydrogel has a degree of hydration of 50% to 95% upon equilibrium swelling. In some cases, the hydrogel contains a plasticizer such as polyethylene glycol, sorbitol, or glycerin. The plasticizer may be present at 0.1% to 30% by weight of the composition of the hydrogel component. In some cases, the hydrogel comprises a crosslinked protein hydrogel. The protein may include gelatin, soluble collagen, albumin, hemoglobin, fibrinogen (fibrinogen), fibrin, casein, fibronectin, elastin, keratin, laminin and derivatives and combinations thereof. In some cases, the hydrogel contains a cross-linked polysaccharide. The polysaccharide may comprise glucosamine polysaccharides, starch derivatives, cellulose derivatives, hemicellulose derivatives, xylan, agarose, alginate esters, and chitosan and combinations thereof. In some cases, the hydrogel contains a crosslinked non-biological polymer. The crosslinked non-biological polymer may comprise polyacrylates, polymethacrylates, polyacrylamides, polyvinyl resins, polylactide-glycolides, polycaprolactones, polyoxyethylenes, and combinations thereof. In some cases, the hydrogel comprises at least two components selected from the group consisting of cross-linked proteins, cross-linked polysaccharides, and cross-linked non-biological polymers. The hydrogel may include a hydrogel polymer and a hydrogel cross-linking agent. The hydrogel polymer and the hydrogel crosslinking agent may be reacted under conditions that result in crosslinking of the hydrogel polymer molecules. In some cases, the hydrogel comprises a molecularly crosslinked hydrogel polymer that is prepared by irradiating the hydrogel under conditions that produce crosslinking of the hydrogel polymer molecules. In some cases, the hydrogel comprises a molecularly crosslinked hydrogel prepared by reacting monounsaturated and polyunsaturated hydrogel monomers under conditions that produce crosslinking of hydrogel polymer molecules.

In yet another aspect, embodiments of the present invention include a method of forming a three-dimensional synthetic polymer matrix. The method includes providing a first crosslinkable component comprising m nucleophilic groups and a second crosslinkable component comprising n electrophilic groups. Reacting the electrophilic groups with the nucleophilic groups to form covalent bonds between the groups, each of m and n being greater than or equal to 2, and m + n being greater than or equal to 5. The method further includes combining the first cross-linkable component with the second cross-linkable component, adding the hydrogel-forming component to the first cross-linkable component and the second cross-linkable component, and cross-linking the first cross-linkable component and the second cross-linkable component with each other to form the three-dimensional matrix. The method can also include contacting the first tissue surface and the second surface with a first crosslinkable component, a second crosslinkable component, and a hydrogel-forming component. In some cases, the second surface is a native tissue surface. In some cases, the second surface is a non-native tissue surface, such as a synthetic implant. The synthetic implant may be a donor cornea, an artificial blood vessel, a heart valve, an artificial organ, an adhesive prosthesis, an implantable lenticule, a vascular graft, a stent or a stent/graft combination. In some cases, the first crosslinkable component, the second crosslinkable component, and the hydrogel-forming component are each coated in powder form on the first tissue surface. In some cases, the first crosslinkable component, the second crosslinkable component, and the hydrogel-forming component are each coated on the first tissue surface as a powder of a single combined mixed powder formulation. The mixed powder formulation may contain proteins and/or polysaccharides. The first tissue surface may be on or in hard or soft tissue. The first tissue surface may comprise the vicinity or vicinity of the surgical site. The method may also include sealing the surgical site. In some cases, the mixed powder formulation contains collagen. In some cases, the mixed powder formulation contains a bioactive agent. In certain aspects, embodiments of the invention include mixing a powder composition comprising a first crosslinkable component having a plurality of nucleophilic groups in powder form, a second crosslinkable component having a plurality of electrophilic groups in powder form, and a hydrogel-forming component in powder form. The first and second crosslinkable components are capable of substantially immediate crosslinking under conditions capable of reaction.

In a related aspect, the first cross-linkable component added to the second cross-linkable component provides a combined cross-linkable component composition. The first cross-linkable component may be present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition. In some cases, the second cross-linkable component can be present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition. The weight ratio of the first cross-linkable component to the second cross-linkable component can be about 45% to about 55%. In a related aspect, the weight ratio of the first cross-linkable component to the second cross-linkable component can be about 50%. In some instances, the weight ratio of the first and second cross-linkable components and the hydrogel-forming component may be from about 28% to about 42% w/w. In a related aspect, the weight ratio between the first and second cross-linkable components and the hydrogel-forming component can be about 20% to about 30% w/w. In certain aspects, the first cross-linkable component can be present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition. Relatedly, the second cross-linkable component can be present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition. The weight ratio of the first cross-linkable component to the second cross-linkable component can be about 45% to about 55%. Similarly, the weight ratio of the first cross-linkable component to the second cross-linkable component can be about 50%.

In another aspect, embodiments of the present invention provide sealant base composition kits. The kit may comprise, for example, a container and a mixed powder composition disposed within the container. The composition can include a first crosslinkable component having a plurality of nucleophilic groups and a second crosslinkable component having a plurality of electrophilic groups. The first crosslinkable component, the second crosslinkable component, or both may be in powder form. The kit may also contain a hydrogel-forming component in powder form. The first and second crosslinkable components are capable of substantially immediate crosslinking under conditions capable of reaction. In some cases, the container contains a syringe barrel and a syringe plunger. The kit may also contain written instructions for applying the mixed powder composition to a target site of bleeding in a patient. In some cases, the mixed powder contains an active agent. The active agent may include thrombin. In another aspect, the kit may comprise a collagen sponge or other suitable support, and a mixed powder composition immobilized on the surface of the sponge or support. The composition can include a first crosslinkable component having a plurality of nucleophilic groups and a second crosslinkable component having a plurality of electrophilic groups. The first crosslinkable component, the second crosslinkable component, or both can be in powder form. The kit may also contain a hydrogel-forming component in powder form. The first and second crosslinkable components are capable of substantially immediate crosslinking under conditions capable of reaction.

For a fuller understanding of the nature and advantages of the present invention, reference should be made to the detailed description taken together with the accompanying figures.

The present invention includes, but is not limited to:

31. a composition comprising:

a first crosslinkable component;

a second crosslinkable component which crosslinks with the first crosslinkable component under conditions capable of reaction; and

a hydrogel-forming component;

wherein the first and second cross-linkable components cross-link to form a porous matrix having voids, and wherein the hydrogel-forming component is capable of being hydrated to form a hydrogel to fill at least some of the voids.

32. The composition of paragraph 31 wherein the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups.

33. The composition of paragraph 31 wherein the first crosslinkable component comprises a multi-nucleophilic polyalkylene oxide having m nucleophilic groups and the second crosslinkable component comprises a multi-electrophilic polyalkylene oxide having n electrophilic groups, wherein m and n are each greater than or equal to 2, and wherein m + n is greater than or equal to 5.

34. The composition of paragraph 33, wherein n is 2, and wherein m is greater than or equal to 3.

35. The composition of paragraph 34, wherein the multi-nucleophilic polyalkylene oxide is tetrafunctionally activated.

36. The composition of paragraph 33, wherein m is 2, and wherein n is greater than or equal to 3.

37. The composition of paragraph 36 wherein the multi-electrophilic polyalkylene oxide is tetrafunctionally activated.

38. The composition of paragraph 33 wherein both the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are tetrafunctionally activated.

39. The composition of paragraph 33 wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more nucleophilic groups selected from the group consisting of: NH (NH)₂、-SH、-H、-PH₂and-CO-NH₂。

40. The composition of paragraph 33, wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more primary amino groups.

41. The composition of paragraph 33, wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more thiol groups.

42. The composition of paragraph 33, wherein the multi-nucleophilic polyalkylene oxide is polyethylene glycol or a derivative thereof.

43. The composition of paragraph 42, wherein the polyethylene glycol further comprises 2 or more nucleophilic groups selected from the group consisting of primary amino groups and thiol groups.

44. The composition of paragraph 33 wherein the multi-electrophilic polyalkylene oxide further comprises 2 or more electrophilic groups selected from the group consisting of: CO 2₂N(COCH₂)₂、-CO₂H、-CHO、-CHOCH₂、-N＝C＝O、-SO₂CH＝CH₂、N(COCH)₂and-S-S- (C)₅H₄N)。

45. The composition of paragraph 33 wherein the multi-electrophilic polyalkylene oxide further comprises 2 or more succinimide groups.

46. The composition of paragraph 33 wherein the multi-electrophilic polyalkylene oxide further comprises 2 or more maleimide groups.

47. The composition of paragraph 33 wherein the multi-electrophilic polyalkylene oxide is polyethylene glycol or a derivative thereof.

48. The composition of paragraph 33, further comprising a polysaccharide or a protein.

49. The composition of paragraph 33, further comprising a polysaccharide, wherein the polysaccharide is a glycosaminoglycan.

50. The composition of paragraph 49, wherein the glycosaminoglycan is selected from the group consisting of hyaluronic acid, chitin, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratin sulfate, keratosulfate, heparin, and derivatives thereof.

51. The composition of paragraph 33, further comprising a protein, wherein the protein is collagen or a derivative thereof.

52. The composition of paragraph 33 wherein the multi-nucleophilic polyalkylene oxide or the multi-electrophilic polyalkylene oxide, or both the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide further comprise a linking group.

53. The composition of paragraph 33 wherein the multi-nucleophilic polyalkylene oxide is given by the formula:

Polymer-Q¹-X_m

And wherein the multi-electrophilic polyalkylene oxide is given by the formula:

Polymer-Q²-Y_n

Wherein X is an electrophilic group and Y is a nucleophilic group;

wherein m and n are each 2 to 4;

wherein m + n is less than or equal to 5;

wherein Q¹And Q²Each is a linking group selected from the group consisting of-O- (CH)₂)_n′-、-S-、-(CH₂)_n′-、-NH-(CH₂)_n′-、-O₂C-NH-(CH₂)_n′-、-O₂C-(CH₂)_n′-、-O₂C-CR¹H and-O-R²-CO-NH；

Wherein n' is 1-10;

wherein R is¹＝-H、-CH₃or-C₂H₅；

Wherein R is²＝-CH₂-or-CO-NH-CH₂CH₂-; and

wherein Q¹And Q²May be the same or different, or may be absent.

54. The composition of paragraph 53, wherein Y is given by:

-CO₂N(COCH₂)₂。

55. the composition of paragraph 53, wherein Y is given by:

-N(COCH)₂。

56. the composition of paragraph 33 wherein the multi-nucleophilic polyalkylene oxide or the multi-electrophilic polyalkylene oxide, or both the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide further contain a biodegradable group.

57. The composition of paragraph 56, wherein the biodegradable group is selected from the group consisting of lactide, glycolide, -caprolactone, poly (alpha-hydroxy acids), poly (amino acids), and poly (anhydrides).

58. The composition of paragraph 31, wherein the hydrogel-forming component is capable of being hydrated to form biocompatible hydrogel segments comprising gelatin that will absorb water when released to the wet tissue target site, wherein the hydrogel comprises subunits having a size of about 0.01mm to about 5mm when fully hydrated and an equilibrium swelling of about 400% to about 5000%.

59. The composition of paragraph 58, wherein the hydrogel has a degradation time in vivo of less than 1 year.

60. The composition of any of paragraphs 58 and 59, wherein at least a portion of the hydrogel is hydrated with an aqueous medium containing an active agent.

61. The composition of paragraph 60, wherein the active agent is a coagulant.

62. The composition of paragraph 61, wherein the clotting agent is thrombin.

63. A method of delivering an active agent to a patient, the method comprising administering to the patient an amount of the composition of paragraph 60 at a target site.

64. A method of delivering a sealant to a patient, the method comprising administering the composition of paragraph 31 to a bleeding target site in an amount sufficient to inhibit bleeding.

65. A method of delivering thrombin to a patient, the method comprising administering the composition of paragraph 62 to a bleeding target site in an amount sufficient to inhibit bleeding.

66. A composition comprising a multi-nucleophilic polyalkylene oxide, a multi-electrophilic polyalkylene oxide, and a hydrogel-forming component, wherein the multi-nucleophilic polyalkylene oxide further comprises at least one primary amino group and at least one thiol group, and wherein the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are capable of substantially immediate crosslinking under conditions capable of reaction.

67. A composition comprising a multi-nucleophilic polyalkylene oxide, a multi-electrophilic polyalkylene oxide, and a hydrogel-forming component, wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more thiol groups, the multi-electrophilic polyalkylene oxide further comprises 2 or more electrophilic groups selected from the group consisting of succinimidyl groups and maleimidyl groups, and wherein the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are capable of substantially immediate crosslinking under reaction-enabling conditions.

68. A composition comprising a multi-nucleophilic polyalkylene oxide, a multi-electrophilic polyalkylene oxide, and a hydrogel-forming component, wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more nucleophilic groups selected from the group consisting of primary amino groups and thiol groups, and the multi-electrophilic polyalkylene oxide further comprises 2 or more succinimidyl groups, wherein the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are capable of substantially immediate crosslinking under reaction-enabling conditions.

69. A composition comprising a first polyethylene glycol having 2 or more thiol groups, a second polyethylene glycol having 2 or more succinimide groups or maleimide groups, and a hydrogel-forming component, wherein the total number of thiol groups and succinimide or maleimide groups is at least 5, and wherein the first polyethylene glycol and the second polyethylene glycol are capable of substantially immediate crosslinking under conditions capable of reaction.

70. The composition of paragraph 69, wherein the first polyethylene glycol further comprises 4 thiol groups and the second polyethylene glycol further comprises 4 succinimide groups.

71. The composition of paragraph 69, further comprising a protein or a polysaccharide.

72. The composition of paragraph 69, further comprising a polysaccharide, wherein the polysaccharide is a glycosaminoglycan.

73. The composition of paragraph 72, wherein the glycosaminoglycan is selected from the group consisting of hyaluronic acid, chitin, chondroitin sulfate A, chondroitin sulfate B, chondroitin sulfate C, keratin sulfate, keratosulfate, heparin, and derivatives thereof.

74. The composition of paragraph 69, further comprising a protein, wherein the protein is collagen or a derivative thereof.

75. A method of sealing a tissue tract, the method comprising at least partially filling the tissue tract with a composition comprising:

a first crosslinkable component;

a hydrogel-forming component;

wherein the first and second cross-linkable components are cross-linked to form a porous matrix having voids, the hydrogel-forming component is capable of being hydrated to form a hydrogel to fill at least some of the voids, the hydrogel comprises subunits having a size of from about 0.05mm to about 5mm when fully hydrated and an equilibrium swelling of from about 400% to about 1300%, such hydrogel degrading after from about 1 to about 120 days in the tissue tract.

76. The method of paragraph 75 wherein the first crosslinkable component comprises a plurality of nucleophilic groups and the second polymer comprises a plurality of electrophilic groups.

77. A method of inhibiting bleeding at a target site in a patient's body, the method comprising delivering to the target site an amount of a composition sufficient to inhibit bleeding, the composition comprising:

a first crosslinkable component;

a hydrogel-forming component;

wherein the first and second cross-linkable components are cross-linked to form a porous matrix having voids, the hydrogel-forming component is capable of being hydrated to form a hydrogel to fill at least some of the voids, the hydrogel comprises subunits having a size of from about 0.05mm to about 5mm when fully hydrated and an equilibrium swelling of from about 400% to about 1300%, and such hydrogel degrades after from about 1 to about 120 days in the tissue tract.

78. The method of paragraph 77, wherein the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups.

79. A method for delivering a biologically active substance to a target site in a patient's body, the method comprising delivering to the target site in combination a composition and the biologically active substance, the composition comprising:

a first crosslinkable component;

a hydrogel-forming component;

80. The method of paragraph 79, wherein the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups.

81. The method of paragraph 79, wherein the biologically active substance is a hemostatic agent.

82. The method of paragraph 79, wherein the biologically active substance is thrombin.

83. A method of delivering a swellable composition to a target site in a tissue, said method comprising applying to the target site a composition comprising:

a first crosslinkable component;

a hydrogel-forming component;

wherein the first and second cross-linkable components are cross-linked to form a porous matrix having voids, the hydrogel-forming component being capable of being hydrated to form a hydrogel to fill at least some of the voids, the hydrogel comprising subunits having a size of from about 0.05mm to about 5mm when fully hydrated and an equilibrium swelling of from about 400% to about 1300%, such hydrogel degrading after from about 1 to about 120 days in the tissue tract and the composition hydrating less than its equilibrium swelling when coated at a target site wherein it has been expanded to its equilibrium swelling value.

84. The method of paragraph 83, wherein the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups.

85. The method of paragraph 83, wherein the target site is located in a tissue selected from the group consisting of: muscle, skin, epithelial tissue, connective or support tissue, neural tissue, ocular and other sensory organ tissue, vascular and cardiac tissue, gastrointestinal organs and tissue, pleural and other lung tissue, kidney, endocrine glands, male and female reproductive organs, adipose tissue, liver, pancreas, lymph glands, cartilage, bone, oral tissue and mucosal tissue, and spleen and other abdominal organs.

86. The method of paragraph 85, wherein the target site is a void region in the selected tissue.

87. The method of paragraph 86, wherein the void region is selected from the group consisting of a tissue cortex, a tissue tract, an intraspinal space, and a body cavity.

88. The method of paragraph 83, wherein the hydrogel has a degree of hydration of 50% to 95% upon equilibrium swelling.

89. The method of paragraph 83, wherein the hydrogel contains a plasticizer.

90. The method of paragraph 89, wherein the plasticizer is selected from the group consisting of polyethylene glycol, sorbitol, and glycerol.

91. The method of paragraph 89, wherein the plasticizer is present at 0.1% to 30% by weight of the composition of the hydrogel component.

92. The method of any one of paragraphs 75-91, wherein the hydrogel comprises a crosslinked protein hydrogel.

93. The method of paragraph 92, wherein the protein is selected from the group consisting of gelatin, soluble collagen, albumin, hemoglobin, fibrinogen, fibrin, casein, fibronectin, elastin, keratin, laminin and derivatives and combinations thereof.

94. The method of any one of paragraphs 75-91, wherein the hydrogel comprises a crosslinked polysaccharide.

95. The method of paragraph 94, wherein the polysaccharide is selected from the group consisting of glycosaminoglycans, starch derivatives, cellulose derivatives, hemicellulose derivatives, xylans, agaroses, alginates, and chitosans, and combinations thereof.

96. The method of any one of paragraphs 75-91, wherein the hydrogel comprises a crosslinked non-biological polymer.

97. The method of paragraph 96, wherein the cross-linked non-biological polymer is selected from the group consisting of polyacrylates, polymethacrylates, polyacrylamides, polyvinyl resins, polylactide-glycolides, polycaprolactones, polyoxyethylenes, and combinations thereof.

98. The method of any of paragraphs 75-91, wherein the hydrogel comprises at least two components selected from the group consisting of cross-linked proteins, cross-linked polysaccharides, and cross-linked non-biological polymers.

99. The method of any one of paragraphs 75-91, wherein the hydrogel comprises a hydrogel polymer and a hydrogel cross-linking agent, wherein the hydrogel polymer and the hydrogel cross-linking agent are reacted under conditions that result in cross-linking of hydrogel polymer molecules.

100. The method of any one of paragraphs 75-91, wherein the hydrogel comprises a molecularly crosslinked hydrogel polymer prepared by irradiating the hydrogel under conditions that produce crosslinking of hydrogel polymer molecules.

101. The method of any of paragraphs 75-91, wherein the hydrogel comprises a molecularly crosslinked hydrogel prepared by reacting monounsaturated and polyunsaturated hydrogel monomers under conditions that produce crosslinking of hydrogel polymer molecules.

102. A method of forming a three-dimensional synthetic polymer matrix, the method comprising the steps of:

providing a first crosslinkable component comprising m nucleophilic groups and a second crosslinkable component comprising n electrophilic groups, wherein the electrophilic groups react with the nucleophilic groups to form a covalent bond therebetween, wherein m and n are each greater than or equal to 2, and wherein m + n is greater than or equal to 5;

combining a first crosslinkable component with a second crosslinkable component;

adding a hydrogel-forming component to the first crosslinkable component and the second crosslinkable component;

the first crosslinkable component and the second crosslinkable component are crosslinked with each other to form a three-dimensional matrix.

103. The method of paragraph 102, further comprising contacting the first tissue surface and the second surface with a first crosslinkable component, a second crosslinkable component, and a hydrogel-forming component.

104. The method of paragraph 103, wherein the second surface is a native tissue surface.

105. The method of paragraph 103, wherein the second surface is a non-native tissue surface.

106. The method of paragraph 105, wherein the non-native tissue surface is a synthetic implant.

107. The method of paragraph 106, wherein the synthetic implant is selected from the group consisting of a donor cornea, an artificial blood vessel, a heart valve, an artificial organ, an adhesive prosthesis, an implantable microlens, a vascular graft, a stent, and a stent/graft combination.

108. The method of paragraph 102, wherein the first cross-linkable component, the second cross-linkable component and the hydrogel forming component are each coated onto the first tissue surface in powder form.

109. The method of paragraph 102, wherein the first crosslinkable component, the second crosslinkable component, and the hydrogel-forming component are each coated onto the first tissue surface as a powder of a single combined mixed powder formulation.

110. The method of paragraph 109, wherein the mixed powder formulation further comprises a protein or polysaccharide.

111. The method of paragraph 102, wherein the first tissue surface is on or in a hard or soft tissue.

112. The method of paragraph 102, wherein the first tissue surface comprises, surrounds, or is adjacent to the surgical site, and wherein the method further comprises the step of sealing the surgical site.

113. The method of paragraph 102, wherein the mixed powder formulation further comprises collagen.

114. The method of paragraph 102, wherein the mixed powder formulation further comprises a bioactive agent.

115. A mixed powder composition comprising:

a first crosslinkable component comprising a plurality of nucleophilic groups, the first crosslinkable component being in powder form;

a second crosslinkable component comprising a plurality of electrophilic groups, the second crosslinkable component being in powder form; and

a hydrogel-forming component in powder form;

wherein the first and second cross-linkable components are capable of substantially immediate cross-linking under conditions capable of reaction.

116. The mixed powder composition of paragraph 115, wherein the first cross-linkable component is added to the second cross-linkable component to provide a combined cross-linkable component composition, and the first cross-linkable component is present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition.

117. The mixed powder composition of paragraph 115, wherein the first cross-linkable component is added to the second cross-linkable component to provide a combined cross-linkable component composition and the second cross-linkable component is present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition.

118. The mixed powder composition of paragraph 115, wherein the weight ratio of the first cross-linkable component to the second cross-linkable component is about 45% to about 55%.

119. The mixed powder composition of paragraph 115, wherein the weight ratio of the first cross-linkable component to the second cross-linkable component is about 50%.

120. The mixed powder composition of paragraph 115, wherein the weight ratio between the first and second cross-linkable components and the hydrogel-forming component is about 28% to about 42% w/w.

121. The mixed powder composition of paragraph 115, wherein the weight ratio between the first and second cross-linkable components and the hydrogel-forming component is about 20% to about 30% w/w.

122. The mixed powder composition of paragraph 121, wherein the first cross-linkable component is added to the second cross-linkable component to provide a combined cross-linkable component composition, and the first cross-linkable component is present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition.

123. The mixed powder composition of paragraph 121, wherein the first cross-linkable component is added to the second cross-linkable component to provide a combined cross-linkable component composition, and the second cross-linkable component is present at a concentration of about 0.5% to about 20% by weight of the combined cross-linkable component composition.

124. The mixed powder composition of paragraph 121, wherein the weight ratio of the first cross-linkable component to the second cross-linkable component is between about 45% and about 55%.

125. The mixed powder composition of paragraph 121, wherein the weight ratio of the first cross-linkable component to the second cross-linkable component is about 50%.

126. A kit, comprising:

a container; and

a mixed powder composition disposed in the container, the composition comprising:

a hydrogel-forming component in powder form;

127. The kit of paragraph 126, wherein the container contains a syringe barrel and a syringe plunger.

128. The kit of paragraph 126, further comprising written instructions for applying the mixed powder composition to a target site of bleeding in a patient.

129. The kit of paragraph 126, wherein the mixed powder further comprises an active agent.

130. The kit of paragraph 129, wherein the active agent comprises thrombin.

131. A kit, comprising:

a collagen sponge; and

a mixed powder composition fixed to the surface of the sponge, the mixed powder composition comprising:

a hydrogel-forming component in powder form;

132. A composition for use in the preparation of a medicament, the composition comprising:

a first crosslinkable component;

a hydrogel-forming component;

wherein the first and second cross-linkable components are cross-linked to form a porous matrix having voids, and wherein the hydrogel-forming component is capable of being hydrated to form a hydrogel to fill at least some of the voids.

133. The composition of paragraph 132 wherein the first crosslinkable component comprises a plurality of nucleophilic groups and the second crosslinkable component comprises a plurality of electrophilic groups.

134. The composition of paragraph 133 wherein the first crosslinkable component comprises a multi-nucleophilic polyalkylene oxide having m nucleophilic groups and the second crosslinkable component comprises a multi-electrophilic polyalkylene oxide having n electrophilic groups, wherein m and n are each greater than or equal to 2, and wherein m + n is greater than or equal to 5.

135. The composition of paragraph 134, wherein n is 2, and wherein m is greater than or equal to 3.

136. The composition of paragraph 135, wherein the multi-nucleophilic polyalkylene oxide is tetrafunctionally activated.

137. The composition of paragraph 134, wherein m is 2, and wherein n is greater than or equal to 3.

138. The composition of paragraph 137 wherein the multi-electrophilic polyalkylene oxide is tetrafunctionally activated.

139. The composition of paragraph 134 wherein both the multi-nucleophilic polyalkylene oxide and the multi-electrophilic polyalkylene oxide are activated with tetrafunctionality.

140. The composition of paragraph 134 wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more nucleophilic groups selected from NH₂、-SH、-H、-PH₂and-CO-NH₂。

141. The composition of paragraph 134, wherein the multi-nucleophilic polyalkylene oxide further comprises 2 or more primary amino groups.

142. A composition, comprising:

a collagen sponge comprising native collagen fibers; and

a mixed powder composition for fixing to the surface of a sponge, the mixed powder composition comprising:

a first cross-linkable component comprising a plurality of nucleophilic groups, the first cross-linkable component being in the form of a powder and comprising about 10% of the mixed powder;

a second cross-linkable component comprising a plurality of electrophilic groups, the second cross-linkable component being in the form of a powder and comprising about 10% of the mixed powder; and

a hydrogel-forming component in powder form comprising about 80% of the mixed powder;

wherein the first and second cross-linkable components are capable of substantially immediate cross-linking under reaction-enabling conditions to form a porous matrix having voids, and the hydrogel-forming component is capable of hydrating to form a hydrogel to fill at least some of the voids.

Drawings

FIG. 1 illustrates a first crosslinkable component of certain embodiments of the present invention.

FIG. 2 illustrates a second crosslinkable component of certain embodiments of the present invention.

Fig. 3 shows the formation of a cross-linked matrix composition from a hydrophilic polymer according to certain embodiments of the present invention.

Figure 4 shows the formation of a cross-linked matrix composition from a hydrophobic polymer according to certain embodiments of the present invention.

FIG. 5 illustrates hydrogel-forming component subunits of certain embodiments of the present invention.

FIG. 6 illustrates the correlation between percent swelling and percent solids of crosslinked polymer gel segments that can be used as hydrogel-forming components in sealant compositions according to certain embodiments of the invention.

Figures 7A-E illustrate administration of a sealant matrix composition to treat a splenic artery puncture, according to embodiments of the present invention.

Figures 8A-E illustrate administration of a sealant matrix composition to treat liver resection, according to embodiments of the present invention.

Fig. 9 illustrates the handling and packaging of a sealant matrix composition according to an embodiment of the present invention.

Fig. 10 illustrates the handling and packaging of a sealant matrix composition according to an embodiment of the present invention.

Figure 11 illustrates the effect of PEG concentration on gel strength according to embodiments of the invention.

Figure 12 illustrates the effect of PEG concentration on swelling ratio according to embodiments of the invention.

Figure 13 illustrates the effect of PEG concentration on swelling ratio according to embodiments of the invention.

Figure 14 illustrates the effect of PEG concentration on swelling ratio according to embodiments of the invention.

Detailed Description

According to certain embodiments, the present invention provides a dry sealant matrix composition that achieves hemostasis or other fluid inhibition in an in vivo environment by sealing a tissue breach or defect. The compositions of certain embodiments of the present invention include a dry composition suitable for coating vertebrate tissue to promote fluid containment, the composition comprising first and second cross-linkable components and at least one hydrogel-forming component. The first and second components of the composition of the present invention react in an in vivo environment to form a cross-linked matrix, and the hydrogel-forming component rapidly absorbs biological fluids that reach the tissue breach and strengthens the resulting physical sealant matrix barrier formed by the cross-linking of the first and second components. As used herein, the term "sealant matrix composition" can refer to the composition of the present invention prior to application to a tissue site in the body, and the term "sealant matrix barrier" can refer to the matrix barrier created upon contact of the composition of the present invention with biological fluid, and the porous cross-linked matrix of the first and second components cross-linked to form an aqueous gel. The sealant matrix composition can be prepared in a variety of forms including powder, cake, pad, and the like. The cake embodiment includes a powder sample of the sealant matrix composition that is formed into an aggregate by heating or oven drying. Pad embodiments include a sample of the sealant matrix composition powder placed on a sponge, such as a collagen sponge or other support, which is then dried to form a solidified powder fused to the sponge or support.

Although the present invention may be used to contain non-blood biological fluids (e.g., lymph or spinal fluid), sealant matrices formed from compositions in certain embodiments of the invention may also be referred to as "hemostatic matrices" as this is the primary use described herein.

In addition to providing rapid hemostasis and a barrier with high adhesion to surrounding tissue, the sealant matrix of certain embodiments of the present invention has several advantages over the disclosed materials for hemostasis. First, the sealant matrix of certain embodiments of the present invention can be used in a relatively moist environment of a breach in tissue (e.g., rapid bleeding or jet of arterial bleeding, such as internal organ friction or severe trauma). In contrast, many compositions currently on the market for hemostasis require a relatively dry site to properly adhere the composition and maintain hemostasis. For example, in some cases, certain PEG mixtures may be placed at sites of rapid bleeding, but it is possible that they may be washed away. Also, in some cases, certain gelatin compositions may hydrate at the site of rapid bleeding, but it may be difficult to retain them at the site. Advantageously, it has been found that a formulation comprising a first cross-linkable component, a second cross-linkable component and a hydrogel-forming component can provide a material that remains immobilized even under conditions that enable reaction, and which forms a mechanically stable clot-like substance with excessive bleeding, stopping the flow of blood. Second, the sealant matrix of certain embodiments of the present invention functions by physically sealing a tissue breach without relying on any endogenous clotting capabilities of the vertebrate. Thus, the sealant matrix can be used for vertebrates with low fibrinogen concentration in their blood, or even for fibrinogen-free blood substitutes. For example, when the first and second cross-linkable components are combined with the hydrogel-forming component and coated on the bleeding surface, the cross-linkable component and the hydrogel-forming component may interact synergistically. According to certain embodiments, the first and second cross-linkable components may react and cross-link at the bleeding target site in the presence of the hydrogel-forming component to form a relatively rigid structure. In connection therewith, the hydrogel-forming component may fill the relatively rigid structure, mediating the formation of the physical seal.

According to certain embodiments of the present invention, the sealant matrix composition can be prepared by mixing the first cross-linkable component with the second cross-linkable component and the hydrogel-forming component under conditions in which the first and second cross-linkable components are not cross-linked (i.e., in the absence of moisture, at an appropriate pH, temperature, etc.). Upon contact with biological fluid, or under other conditions capable of reaction, the cross-linked first and second components cross-link to form a porous matrix having voids, and the hydrogel-forming component hydrates to form a hydrogel, filling at least some of the voids. Optionally, the crosslinkable component may also crosslink with the hydrogel-forming component and/or surrounding tissue.

I. Sealant matrix cross-linking component

Typically, the first crosslinkable component comprises two or more nucleophilic groups and the second crosslinkable component comprises two or more electrophilic groups capable of covalently bonding to the nucleophilic groups on the first crosslinkable component. The first and second components can be crosslinked to form a porous matrix. In U.S. patent nos. 5,874,500; 6,166,130, respectively; 6,312,725, respectively; 6,328,229; and 6,458,889, the contents of which are incorporated herein by reference, are described with respect to exemplary first and second components and porous matrices.

The first and second components are generally selected to be non-immunogenic, and therefore, may not require a "skin test" prior to initiation of treatment. In addition, these components and the hydrogel-forming component can be selected to prevent enzymatic cleavage by matrix metalloproteinases (e.g., collagenase), providing a longer in vivo duration than existing collagen compositions. Alternatively, the first and second components and the hydrogel-forming component may be selected to be eliminated or resorbed during wound healing to avoid the formation of fibrous tissue around the sealant matrix in the body.

In one embodiment, the first component may be a synthetic polymer containing a plurality of nucleophilic groups (hereinafter "X") that is reactive with a second component containing a plurality of electrophilic groups (hereinafter "Y") to form a covalently bonded polymer network as follows:

Polymer-X_m+ Polymer-Y_n→ Polymer-Z-Polymer

Wherein m is more than or equal to 2, n is more than or equal to 2, and m + n is more than or equal to 5;

X＝-NH₂、-SH、-OH、-PH₂、-CO-NH-NH₂etc., and may be the same or different;

Y＝-CO₂N(COCH₂)₂、-CO₂H、-CHO、-CHOCH₂、-N＝C＝O、SO₂CH＝CH₂、-N(COCH)₂)、-S-S-(C₅H₄n), etc., and may be the same or different; and

z is a functional group formed by bonding a nucleophilic group (X) and an electrophilic group (Y)

As mentioned above, X and Y may be the same or different, i.e. the first component may have two different nucleophilic groups and/or the second component may have two different electrophilic groups. FIG. 1 illustrates an exemplary first component polymer or first crosslinkable component. FIG. 2 illustrates an exemplary second component polymer or second cross-linkable component.

The backbone of the first and second component polymers can be an alkylene oxide, especially ethylene oxide, propylene oxide, and mixtures thereof. Examples of difunctional alkylene oxides can be represented by the formula:

X-Polymer-X Y-Polymer-Y

Wherein X and Y are as defined above, the term "polymer" denotes- (CH)₂CH₂O)_n-or- (CH)₃)CH₂O)_n-or- (CH)₂CH₂O)_n-(CH(CH₃)CH₂O)_n-。

The functional groups X or Y are typically coupled to the polymer backbone through a linking group (hereinafter represented by "Q"), a variety of which are known or may be present. Although the components of the present invention have two or more functional groups, for the sake of simplicity, the following examples show only one functional group and the resulting crosslinking. There are a variety of methods for preparing various functionalized polymers, some of which are listed below:

Polymer-Q¹-X + Polymer-Q²-Y → Polymer-Q¹-Z-Q²-polymers

Wherein

In each case, n is 1-10;

R¹＝H、CH₃、C₂H₅，...C_pH_2p+1；

R²＝CH₂、CO-NH-CH₂CH₂；

Q¹and Q²May be the same or different.

For example, when Q²＝OCH₂CH₂(in this case Q)₁Absent); y ═ CO₂N(COCH₂)₂(ii) a And X ═ NH₂-SH or-OH, the resulting reactants and the Z group are as follows:

Polymer-NH₂+ Polymer-OCH₂CH₂CO₂-N(COCH₂)₂→-NH-OCH₂CH₂CO-polymers (amides)

Polymer-SH + Polymer-OCH₂CH₂CO₂-N(COCH₂)₂→-S-OCH₂CH₂CO-polymers (thioesters)

Polymer-OH + Polymer-OCH₂CH₂CO₂-N(COCH₂)₂→-O-OCH₂CH₂CO-polymers (esters)

Another group, represented by the following "D", can be inserted between the polymer and the linking group to provide increased degradation of the crosslinked polymer composition in vivo, for example for drug delivery applications:

Polymer-D-Q-X + Polymer-D-Q-Y → Polymer-D-Q-Z-Q-D-Polymer-

Some useful biodegradable groups "D" include lactide, glycolide, -caprolactone, poly (. alpha. -hydroxy acids), poly (amino acids), poly (anhydrides), and various di-or tripeptides.

A. First and second components having a polymer backbone

As noted above, to prepare the compositions of the present invention, it is useful to provide a first component polymer comprising two or more nucleophilic groups, such as primary amino groups or thiol groups, and a second component polymer comprising two or more electrophilic groups capable of covalently bonding to the nucleophilic groups on the first component polymer. The first and second component polymers may be composites.

The term "polymer" used in connection with the first and second component polymers refers in particular to the polyalkyl group; di-, tri-, oligo-and polyamino acids; and tri-, oligo-, or polysaccharides.

The term "synthetic polymer" as used in connection with the first and second component polymers includes polymers that are not natural and are prepared by chemical synthesis. Thus, native proteins such as collagen and native polysaccharides such as hyaluronic acid may not be included. Including synthetic collagen and synthetic hyaluronic acid and derivatives thereof. Synthetic polymers containing nucleophilic or electrophilic groups include "multifunctional activated synthetic polymers". The term "multifunctional activated" (or simply "activated") can refer to a synthetic polymer having, or modified chemically to have, two or more nucleophilic or electrophilic groups capable of reacting with each other (i.e., a nucleophilic group reacts with an electrophilic group) to form a covalent bond. Types of multifunctional activated synthetic polymers include difunctional activated, tetrafunctional activated and star-branched polymers.

The multifunctional activated synthetic polymers useful in the present invention typically contain at least 2 or at least 3 functional groups to form a 3-dimensional crosslinked network with a synthetic polymer containing multiple nucleophilic groups (i.e., a "multi-nucleophilic polymer"). In other words, they are generally at least difunctional, trifunctional or tetrafunctional activated. If the first synthetic polymer is a difunctional activated synthetic polymer, the second synthetic polymer will typically contain 3 or more functional groups to give a 3-dimensional crosslinked network. Both the first and second component polymers may contain at least 3 functional groups.

B. A first component polymer

The first component polymer comprising a plurality of nucleophilic groups is also referred to herein collectively as a "multi-nucleophilic polymer". For use in the present invention, the polynucleophilic polymers will generally contain at least 2 or at least 3 nucleophilic groups. If synthetic polymers containing only two nucleophilic groups are used, synthetic polymers containing 3 or more electrophilic groups are generally used to give a 3-dimensional crosslinked network.

The multi-nucleophilic polymers useful in the compositions and methods of the present invention include synthetic polymers that contain, or are modified to contain, a plurality of nucleophilic groups, such as primary amino groups and thiol groups. Such multi-nucleophilic polymers may include: (i) synthesizing a synthetic polypeptide comprising two or more primary amino groups or thiol groups; and (ii) a polyethylene glycol modified to contain two or more primary amino groups or thiol groups. Generally, thiol groups tend to react slower with electrophilic groups than primary amino groups.

The multi-nucleophilic polypeptide can be a synthetic polypeptide that is synthesized to incorporate amino acids containing primary amino groups (e.g., lysine) and/or amino acids containing thiol groups (e.g., cysteine). For example, the first component polymer may be a di-polylysine, a tri-polylysine, a tetra-polylysine, a penta-polylysine, or a di-cysteine, tri-cysteine, tetra-cysteine, penta-cysteine, or an oligopeptide or polypeptide containing two or more lysines or cysteines and other amino acids (e.g., glycine, alanine), preferably containing non-hydrophobic amino acids. Poly (lysine), a polymer of synthetically prepared lysine, is commonly used (145 MW). Poly (lysine) s having 6 to about 4,000 primary amino groups at any position, corresponding molecular weights of about 870 to about 580,000, are prepared. Poly (lysine) is available in various molecular weights from Peninsula Laboratories, Inc (Belmont, Calif.).

May be prepared, for example, according to the methods described in Poly (ethylene glycol) Chemistry: polyethylene glycol is chemically modified to contain multiple primary amino or thiol groups by methods set forth in Biotechnical and biomedical Applications, chapter 22, j.milton Harris, ed., Plenum Press, n.y. (1992). Modified polyethylene glycols containing two or more primary amino groups are referred to herein as "polyamino PEGs". Modified polyethylene glycols containing two or more thiol groups are referred to herein as "polythiol-based PEGs". The term "polyethylene glycol" as used herein includes modified and derivatized polyethylene glycols.

Various forms of polyaminoPEGs known as "Jeffamines" are commercially available from Shearwater Polymers (Huntsville, Ala.) and Texaco chemical company (Houston, Tex.). Polyamino PEGs useful in the present invention include Texaco's Jeffamine diamines ("D" series) and triamines ("T" series) containing 2 and 3 primary amino groups per molecule, respectively.

Polyamines, e.g. ethylenediamine (H)₂N-CH₂CH₂-NH₂)1, 4-butanediamine (H)₂N-(CH₂)₄-NH₂)1, 5-Pentanediamine (cadaverine) (H)₂N-(CH₂)₅-NH₂)1, 6-hexanediamine (H)₂N-(CH₂)₆-NH₂) Bis (2-hydroxyethyl) amine (HN- (CH)₂CH₂OH)₂) Bis (2) aminoethyl amine (HN- (CH)₂CH₂NH₂)₂) And tris (2-aminoethyl) amine (N- (CH)₂CH₂NH₂)₃) Also useful as a first component synthetic polymer containing a plurality of nucleophilic groups.

C. A second component polymer

The second component polymer comprising a plurality of electrophilic groups is also referred to herein as a "multi-electrophilic polymer". For use in the present invention, the multi-electrophilic polymer typically contains at least 2 or at least 3 electrophilic groups to form a 3-dimensional cross-linked network with the multi-nucleophilic polymer.

The multi-electrophilic polymer used in the compositions of the present invention can be a polymer containing two or more succinimide groups capable of forming covalent bonds with nucleophilic groups on other molecules. Succinimidyl and substituted esters of carboxylic acids containing primary amino groups (-NH)₂) Such as polyaminopeg, poly (lysine) or collagen are very reactive. Succinimidyl groups are somewhat unreactive with thiol group (-SH) containing materials such as polythiol PEG or synthetic polypeptides containing multiple cysteine residues.

The term "containing two or more succinimide groups" as used herein is intended to include both commercially available polymers containing two or more succinimide groups and those polymers which have been chemically derivatized to contain two or more succinimide groups. The term "succinimidyl" as used herein is intended to include sulfosuccinimidyl and other such variations of the "superordinate" succinimidyl. The presence of a sodium sulfite moiety on the sulfosuccinimidyl group serves to increase the solubility of the polymer.

D. Hydrophilic polymers as backbones for the first or second component

According to certain embodiments of the present invention, hydrophilic polymers and, in particular, various polyethylene glycols may be used in the first and second component polymer backbones. The term "PEG" as used herein includes a polymer having a repeating structure (OCH)₂CH₂)_nThe polymer of (1).

The structure of the tetrafunctionally activated form of PEG is shown in FIG. 3, which is a common reaction product obtained by reacting tetrafunctionally activated PEG with polyaminoPEG. As shown in the figure, the succinimidyl group is composed of-N (COCH)₂)₂A 5-membered ring structure. In FIG. 3, the symbols^ΛΛΛRepresenting an open key.

Embodiments include reacting tetrafunctionally activated PEG succinimidyl glutarate, referred to herein as SG-PEG, with polyaminopeg to obtain a reaction product. Another activated form of PEG is known as PEG succinimidyl propionate (SE-PEG). Embodiments include tetrafunctionally activated SE-PEG and products obtained by reacting it with polyaminopeg. In certain embodiments, there are 3 repeating CH2 groups on either end of the PEG. Further embodiments include conjugates comprising non-hydrolyzable "ether" linkages. This conjugate is distinct from the conjugate shown in figure 3 in which the ester linkage is provided. The ester bond is hydrolyzed under physiological environment. Other functionally activated forms of polyethylene glycol are contemplated by embodiments of the present invention, such as conjugates formed by reacting tetrafunctionally activated PEGs with polyaminopegs. In certain embodiments, the conjugate contains both an ether and an amide bond. These bonds are stable in physiological environments.

Another functionally activated form of PEG is known as PEG succinimidyl succinamide (SSA-PEG). Embodiments include tetrafunctionally activated forms of the compounds and reaction products obtained by reacting them with polyaminopegs. These and related compounds may also be used in compositions in embodiments of the invention. Embodiments also include conjugates that contain an "amide" linkage, as with the aforementioned ether linkages, which is less susceptible to hydrolysis and therefore more stable than the ester linkage. In the compound embodiment referred to as PEG succinimidyl carbonate (SC-PEG), yet another activated form of PEG is provided. Embodiments include tetrafunctionally activated SC-PEG and conjugates formed by reacting it with polyaminopeg.

As described above, the activated polyethylene glycol derivatives used in embodiments of the present invention may contain a succinimide group as an active group. However, different activating groups can be attached at positions along the length of the PEG molecule. For example, PEG can be derivatized to form a functionally activated PEG propionaldehyde (a-PEG). Embodiments include tetrafunctional activated forms and conjugates formed by reacting A-PEG with polyaminoPEGs. A bond may be referred to as- (CH)₂)_m-NH-bond, wherein m is 1-10.

Yet another form of activated polyethylene glycol is functionally activated PEG glycidyl ether (E-PEG). Embodiments include tetrafunctionally activated compounds and conjugates formed by reacting the compounds with polyaminopegs. Another activated derivative of polyethylene glycol is a functionally activated PEG-isocyanate (I-PEG). Embodiments include conjugates formed by reacting such compounds with polyaminoPEGs. Another activated derivative of polyethylene glycol is functionally activated PEG-vinylsulfone (V-PEG). Embodiments include conjugates formed by reacting such compounds with polyaminoPEGs.

The multifunctional activated polyethylene glycols useful in the compositions and other embodiments of the present invention may include succinimidyl-containing polyethylene glycols such as SG-PEG and SE-PEG, which may be in trifunctional or tetrafunctional activated forms. Polyethylene glycols in various activated forms as described above are now commercially available from Shearwater Polymers, Huntsville, ala. and Union Carbide, South Charleston, w.va.

E. Derivatizing the first and second component polymers to contain functional groups

Certain polymers, such as polyacids, can be derivatized to contain 2 or more functional groups, such as succinimidyl groups. The polybasic acids useful in the present invention include, but are not limited to, trimethylolpropane-tricarboxylic acid, ditrimethylolpropane-tetracarboxylic acid, pimelic acid, suberic acid (suberic acid), and hexadecanedioic acid (taparic acid). A variety of such polyacids are commercially available from DuPont Chemical Company.

According to the general method, polyacids can be chemically derivatized to contain 2 or more succinimidyl groups by reaction with an appropriate molar amount of N-hydroxysuccinimide (NHS) in the presence of N, N' -Dicyclohexylcarbodiimide (DCC).

Polyols such as trimethylolpropane and di (trimethylolpropane) can be converted to carboxylic acids by various methods described in U.S. patent application serial No. 08/403,358, and then further derivatized by reaction with NHS in the presence of DCC to yield trifunctional and tetrafunctional activated polymers, respectively. Polybasic acids such as pimelic acid (HOOC- (CH) s) by the addition of a succinimidyl group₂)₂-COOH), suberic acid (HOOC- (CH)₂)₂-COOH) and hexadecanedioic acid (HOOC- (CH)₂)₁₄-COOH) derivatization to give a difunctional activated polymer.

Polyamines such as ethylenediamine (H)₂N-CH₂CH₂-NH₂)1, 4-butanediamine (H)₂N-(CH₂)₄-NH₂)1, 5-Pentanediamine (cadaverine) (H)₂N-(CH₂)₅-NH₂)1, 6-hexanediamine (H)₂N-(CH₂)₆-NH₂) Bis (2-hydroxyethyl) amine (HN- (CH)₂CH₂OH)₂) Bis (2) aminoethyl amine (HN- (CH)₂CH₂NH₂)₂) And tris (2-aminoethyl) amine (N- (CH)₂CH₂NH₂)₃) The chemical derivatization to a polyacid, which can then be derivatized to contain 2 or more succinimide groups by reaction with an appropriate molar amount of N-hydroxysuccinimide in the presence of DCC, as described in U.S. patent application Ser. No. 08/403,358. A variety of such polyamines are commercially available from DuPont chemical Company.

In certain embodiments, the first cross-linkable component (e.g., polyaminopeg) is present at a concentration of about 0.5 to about 20 weight percent based on the total cross-linkable component composition and the second cross-linkable component is present at a concentration of about 0.5 to about 20 weight percent based on the total cross-linkable component composition. For example, a final cross-linkable component composition having a total weight of 1 gram (1000 milligrams) may contain about 5 to about 200 milligrams of a first cross-linkable component (e.g., polyaminoPEG) and about 5 to about 200 milligrams of a second cross-linkable component.

In certain embodiments, the weight ratio of the first crosslinkable component to the second crosslinkable component is about 20% to about 80%. In a related embodiment, the ratio is about 45% to about 55%. In some cases, this ratio is about 50%. The weight ratio was determined according to the gel strength test. The first crosslinkable component and the second crosslinkable component may have the same molecular weight.

Hydrogel-forming component for sealant base composition

Hydrogel-forming components useful in the present invention may include those described in U.S. patent nos. 4,640,834; 5,209,776, respectively; 5,292,362, respectively; 5,714,370, respectively; 6,063,061, respectively; and 6,066,325, which are incorporated herein by reference. Materials prepared by the techniques described in these patents are available from Baxter Healthcare Corporation under the trade name FLOSEAL, and these materials and thrombin solutions are mixed together and placed in a kit for use as a hemostatic drug. Alternatively, any hydratable crosslinked polymer may be used as the hydrogel-forming component in the present invention. For example, alginate, agarose, gelatin (e.g., SURGIFOM) may be used^TMPowder) or other synthetic carbohydrate or protein baseA cross-linked polymer capable of hydration. The main properties of useful hydrogel-forming components are biocompatibility, rapid absorption and retention of fluids. Thus, although polyacrylamide may be used as the hydrogel-forming component in the present invention, it is less preferred because it is poorly biocompatible in many internal applications. Typically, the hydratable, crosslinked polymers for use as the hydrogel-forming component have a particle size of about 70 to about 300 microns and a pH of about 6.8 to about 9.5. The hydrogel-forming component may provide mechanical stability to the first and second cross-linkable components, particularly when a force, pressure or dilution is applied to the sealant matrix.

In certain embodiments, the weight ratio between the first and second cross-linkable components and the hydrogel-forming component is about 28% to about 42% w/w. In some instances, the composition may contain the first and second cross-linkable components in combination at a concentration of about 5% to about 75% by weight of the total composition, and the hydrogel-forming component at a concentration of about 95% to about 25% by weight of the total composition. In a related aspect, the composition can comprise the first and second cross-linkable components in admixture at a concentration of about 5% to about 40% by weight of the total composition, and the hydrogel-forming component at a concentration of about 95% to about 60% by weight of the total composition. Similarly, the composition may contain the first and second cross-linkable components in admixture at a concentration of about 10% to about 30% by weight of the total composition and the hydrogel-forming component at a concentration of about 90% to about 70% by weight of the total composition. For example, the composition may contain about 20% of the first and second cross-linkable components combined and about 80% of the hydrogel-forming component. In certain embodiments, the combined first and second cross-linkable component compositions may have a fixed weight ratio of 50: 50% and the w/w ratio of the combined first and second cross-linkable component compositions to the hydrogel forming components may be from about 20% to about 30%. The w/w ratio of the combined first and second cross-linkable component compositions to the hydrogel forming components may be selected based on the gel strength/tack test. The hydrogel-forming component may act as an absorbent to provide a semi-dry surface for the first and second cross-linkable components to polymerize. Embodiments of the invention include a dry sealant matrix composition kit containing crosslinking components and hydrogel-forming components in these proportional amounts.

According to certain embodiments, the term "biocompatible" refers to a material that conforms to the standard # ISO 10993-1 published by the international organization for standardization (NAMSA, Northwood, Ohio). According to certain embodiments, the term "resorbable" refers to a composition that degrades or dissolves when placed directly at a target site in a patient (and not protected by a grafting device such as a breast graft) over a period of less than 1 year, typically 1-120 days. Methods for measuring resorption and degradation are known. According to certain embodiments, the term "molecularly cross-linked" refers to a material comprising polymeric molecules (i.e., single chains) connected by bridges composed of elements, groups, or compounds, wherein the backbone atoms of the polymeric molecules are connected by primary chemical bonds. As described in more detail below, crosslinking can be achieved in a variety of ways.

According to certain embodiments, the term "hydrogel" includes compositions comprising a single phase aqueous colloid in which a biological or non-biological polymer, as defined in more detail below, absorbs water or an aqueous buffer. The hydrogel may contain multiple sub-networks, wherein each sub-network is a molecularly crosslinked hydrogel whose size depends on the degree of hydration and is within the ranges described above. Typically, these hydrogels contain little or no free water, i.e., the water in the hydrogel cannot be removed by simple filtration.

"percent swell" may be defined as the wet weight minus the dry weight divided by the dry weight and multiplied by 100, wherein the wet weight is measured after the humectant on the surface of the material has been removed as completely as possible, for example by filtration, and wherein the dry weight is measured after exposure to an elevated temperature for a time sufficient to evaporate the humectant, for example after 2 hours at 120 ℃.

"equilibrium swell" may be defined as the percentage of swell that reaches equilibrium after the hydratable cross-linked polymeric material has been soaked in the humectant for a time sufficient to allow the water content to become constant, typically 18 to 24 hours.

The "target site" is generally the intended site for release of the sealant matrix composition, typically a tissue breach or defect. Typically, the target site is a target tissue site, but in some cases, such as when the material swells in situ to cover the target site, the sealant matrix composition can be administered or dispensed to a location near the target site.

Hydratable cross-linked polymers useful as hydrogel-forming components in at least certain embodiments of the invention can be formed from biological and non-biological polymers. Suitable biopolymers include proteins such as gelatin, soluble collagen, albumin, hemoglobin, casein, fibrinogen, fibrin, fibronectin, elastin, keratin, laminin and derivatives and combinations thereof. Soluble non-fibrillar collagen is also suitable. Exemplary gelatin formulations are listed below. Other suitable biopolymers include polysaccharides such as glycosaminoglycans (e.g., hyaluronic acid and chondroitin sulfate), starch derivatives, xylans, cellulose derivatives, hemicellulose derivatives, agarose, alginates, chitosan, and derivatives and combinations thereof. Suitable non-biological polymers that are degradable can be selected by either of two mechanisms: i.e., (1) polymer backbone cleavage, or (2) degradation of the side chains that results in water solubility. Exemplary non-biological polymers include composites such as polyacrylates, polymethacrylates, polyacrylamides, polyvinyl resins, polylactide-glycolides, polycaprolactones, polyoxyethylenes, and derivatives and combinations thereof.

The hydratable, cross-linkable polymer molecules used as the hydrogel-forming component can be cross-linked in any manner suitable for forming an aqueous hydrogel. For example, these polymer molecules may be crosslinked with a bi-or multifunctional crosslinking agent that is covalently linked to two or more polymer molecular chains. Exemplary bifunctional crosslinking agents include aldehydes, epoxides, succinimides, carbodiimides, maleimides, azides, carbonates, isocyanates, divinyl sulfones, alcohols, amines, imidoesters (imides), anhydrides, halides, silanes, diazoacetates, aziridines, and the like. Alternatively, crosslinking can be accomplished by using an oxidizing agent that activates the side chains or moieties on the polymer so that they can react with other side chains or moieties to form crosslinks and other agents such as periodate. Another method of crosslinking involves exposing the polymer to radiation, such as gamma radiation, to activate the side chain polymer and allow the crosslinking reaction to proceed. Dehydrothermal crosslinking processes are also suitable. Gelatin may be dehydrothermally crosslinked by holding the gelatin at elevated temperature, typically 120 c, for at least 8 hours. Depending on the decreasing percentage of equilibrium swell, it may be shown that the degree of crosslinking can be increased by increasing the control temperature, by extending the control time, or by a combination of both. The operation under reduced pressure accelerates the crosslinking reaction. The preferred method of crosslinking gelatin molecules is discussed below.

The hydrogel may contain a plasticizer to increase the malleability, elasticity and rate of degradation of the hydrogel. The plasticizer may be an alcohol, such as polyethylene glycol, sorbitol, or glycerol. Typically, the plasticizer will be a polyethylene glycol having a molecular weight of about 200-1000D or about 400D. The plasticizer present in the hydrogel may comprise from about 0.1% to about 30%, preferably from 1% to 5%, by weight of the polymer composition. The use of plasticizers can be particularly advantageous in hydrogels having a high solids content, typically greater than 10% by weight of the composition (without plasticizer).

An exemplary method of preparing the molecularly cross-linked gelatin is as follows. Gelatin is taken and placed in an aqueous buffer to form a non-crosslinked gel, typically having a solids content of from about 1% to about 70% by weight, or from about 3% to about 10% by weight. Gelatin is typically crosslinked by exposure to glutaraldehyde (e.g., 0.01% -0.05% w/w, at 0 ℃ -15 ℃, overnight in an aqueous buffer), sodium periodate (e.g., 0.05M, held at 0 ℃ -8 ℃ for 48 hours), or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide ("EDC") (e.g., 0.5% -1.5% w/w, overnight at room temperature), or by exposure to about 0.3-3 mrad gamma or electron beam radiation. Alternatively, gelatin particles may be suspended in an alcohol such as methanol or ethanol to a solids content of about 1% to about 70% by weight, or about 3% to about 10% by weight, and then crosslinked by exposure to a crosslinking agent, typically glutaraldehyde (e.g., 0.01% to 0.1% w/w, overnight at room temperature). In the case of aldehydes, the pH is typically maintained from about 6 to about 11, or from about 7 to about 10. When crosslinked with glutaraldehyde, the crosslinks formed are formed by a schiff base and the crosslinks formed are stabilized by subsequent reduction, for example by treatment with sodium borohydride. After crosslinking, the resulting particles may be washed with water, optionally rinsed with alcohol, dried, and resuspended in an aqueous medium having the desired buffer and pH to achieve the desired degree of hydration. The resulting hydrogel may then be loaded into an applicator of the present invention as described in more detail below. Alternatively, the hydrogel may be mechanically disrupted, either before or after crosslinking, as described in more detail below.

An exemplary method for preparing a molecularly cross-linked gelatin composition having an equilibrium swell percentage of about 400% to about 1300%, or about 600% to about 950%, is as follows. Gelatin is taken and placed in an aqueous buffer (usually at a pH of about 6 to about 17, or at a pH of about 7 to about 10) containing a solution of a cross-linking agent (usually glutaraldehyde, usually at a concentration of 0.01% to 0.1% w/w) to form a gel, typically having a solids content of 1% to 70% by weight, usually 3% to 10% by weight. As crosslinking occurred, the gel was stirred well and then held at 0-15 ℃ overnight. Then rinsed 3 times with deionized water, rinsed 2 times with an alcohol (preferably methanol, ethanol or isopropanol), and dried at room temperature. The gel may optionally be treated with sodium borohydride to further stabilize the crosslinking. In some cases, the hydrogel-forming component may comprise gelatin having, for example, a plurality of glycine residues (e.g., 1 out of 3 arranged every 3 rd residue), proline residues, and 4-hydroxyproline residues. An exemplary gelatin subunit is shown in fig. 5. Gelatin embodiments include molecules having the following amino acid composition: glycine 21%, proline 12%, hydroxyproline 12%, glutamic acid 10%, alanine 9%, arginine 8%, aspartic acid 6%, lysine 4%, serine 4%, leucine 3%, valine 2%, phenylalanine 2%, threonine 2%, isoleucine 1%, hydroxylysine 1%, methionine and histidine < 1% and tyrosine < 0.5%. FIG. 6 illustrates the correlation between percent swelling and percent solids for embodiments of crosslinked polymer gel segments that can be used as hydrogel-forming components of sealant compositions.

The molecular crosslinked hydrogel is mechanically broken, preferably in a batch process, prior to use as a hydrogel-forming component. The main purpose of this mechanical disruption step is to obtain subunits of the hydrogel that are of a size that enhances the ability to fill and plug the spaces in which they are released. If mechanical breakage does not occur, the molecularly crosslinked hydrogel will have difficulty matching and filling the treated irregularly formed target spaces. By breaking the hydrogel into smaller sized subunits, such spaces can be filled very efficiently while retaining the mechanical integrity and durability of the crosslinked hydrogel.

The molecular crosslinking of the polymer chains of the hydrogel can be carried out before or after its mechanical cleavage. As long as the hydrogel composition is broken into subunits having a size within the above-mentioned range of 0.01mm to 5.0mm, the hydrogel can be mechanically broken by an intermittent operation such as stirring. Other batch mechanical disruption methods include pumping through a homogenizer or stirrer or through a pump that compresses, stretches, or shears the hydrogel to a level greater than the yield stress at break of the hydrogel. In some cases, extrusion of the polymer composition causes the hydrogel to transition from a substantially continuous network, i.e., a network spanning the size of the original hydrogel mass, to a collection of sub-networks or subunits having a size in the above-described range.

In a presently preferred embodiment, the hydratable cross-linked polymer may be prepared (e.g., by spray drying) and/or mechanically disrupted prior to cross-linking, typically to form a hydrogel prior to hydration. The hydratable cross-linked polymers can be provided as a finely divided solid or as a dry solid in powder form, which can be disintegrated by further comminution to give particles having the desired particle size which is usually well controlled within a small range. Other particle size selection and modification steps such as sieving, cyclone classification methods, etc. may also be performed. For the exemplary gelatin materials described hereinafter, it is preferred that the dry particle size range be from about 0.01mm to about 1.5mm, more preferably from about 0.05mm to about 1.0 mm. An exemplary particle size distribution would be greater than about 95% by weight of the particles having a particle size of from about 0.05mm to about 0.7 mm. Methods of comminuting the polymer feedstock include homogenizing, grinding, agglomerating, milling, jet milling, and the like. The polymer feedstock powder may also be formed by spray drying. The particle size distribution can be further controlled and refined by conventional techniques such as sieving, agglomeration, regrinding, and the like.

The dried solid powder can then be suspended in an aqueous buffer as described elsewhere herein and crosslinked. In other cases, the hydratable cross-linked polymer can be suspended in an aqueous buffer, cross-linked, and then dried. The crosslinked dried polymer can then be cleaved and the cleaved material then resuspended in an aqueous buffer. In all cases, the resulting material contained a crosslinked hydrogel with discrete subnetworks having the dimensions described above.

After mechanical disruption, the hydratable cross-linked polymers useful as hydrogel-forming components are generally resorbable, i.e. they will biodegrade on the body of the patient less than 1 year after the first application, usually 1 to 120 days, preferably 1 to 90 days, more preferably 2 to 30 days. Techniques are known to measure the length of time required for resorption.

Preparation and use of a set of sealant base composition embodiments: combination of porous matrix and hydratable cross-linked polymer

The compositions of the present invention comprise a first cross-linkable component in combination with a second cross-linkable component, which are capable of cross-linking to form a porous matrix having voids, in combination with a hydratable cross-linked polymer, which is capable of hydrating to form a hydrogel to fill at least some of the voids. It will be appreciated that the compositions of the present invention may be used in a variety of biomedical applications, including those described above with respect to varying: (1) a first and a second component (i.e., a porous matrix); and (2) a hydratable cross-linkable polymer. For example, such compositions can act as a mechanical sealant, stopping or inhibiting bleeding by forming a rapid physical barrier to blood. Thus, certain embodiments of the invention may provide results without direct "hemostatic" effects (e.g., biochemical effects on the coagulation cascade; involving coagulation initiation factors).

The hydrogel-forming component may be used as an absorbent (e.g., for blood and other fluids and tissues). By absorbing blood, the hydrogel-forming component ensures that a relatively high concentration of the first and second cross-linkable components is maintained at the treatment site and that a semi-dry surface is provided for cross-linking the first and second cross-linkable components with each other and with the surrounding tissue. In certain embodiments, the first and second components may be crosslinked while the hydrogel-forming component absorbs blood. This absorption and crosslinking can occur in seconds and the resulting sealant matrix barrier can reach full strength in 30 minutes to 1 hour.

Typically, the sealant matrix composition is "dry" although very little moisture may be present, for example, in the hydrogel-forming component. In some cases, the hydratable, cross-linkable polymer can be partially pre-hydrated prior to application, although this method may have to be carried out at a higher than physiological pH, or under other conditions that prevent cross-linking of the first and second components prior to application to the target site. Typically, the sealant matrix composition is in the form of a powder or a molten cake.

The concentrations of the first and second components used to prepare the sealant matrix composition can vary depending on a number of factors, including the type and molecular weight of the particular crosslinkable component used, and the desired end use. In certain embodiments, the weight ratio of the first and second components to the hydrogel-forming component is from 10-50% w/w, 15-45% w/w, 20-42% w/w, 30-40% w/w, and 28-about 42% w/w. In certain embodiments, the particle size of the first and second polymers may be from about 50 to about 90 microns. In certain embodiments, the particle size of the hydratable, crosslinked polymer can be from about 250 to about 400 microns.

In certain embodiments, the first and second components may be provided in dry particulate or powder form. The first and second components in this form may be mixed together and may be further mixed with the hydrogel-forming component, which may also be in the form of dry particles or a powder. The mixture of these components can be obtained by any mechanical mixing means, such as mixing with grinding blades. The resulting dried powder of the sealant base composition can then be filled into various containers such as cartons, envelopes, jars, and the like. The sealant base composition can be mixed and filled under aseptic conditions, or sterilized after packaging, for example, by gamma irradiation. The dry powder embodiment of the present invention is then ready for use. The first and second cross-linkable polymers are reactive to cross-link in a physiological environment (e.g., blood pH), so that a 3-component sealant matrix composition of the composition can be applied directly to the dry desired site to seal the tissue defect, provided that sufficient hydrated body fluid is present. Thus, the sealant matrix composition powder can simply be sprinkled over and poured into the tissue defect target site, held in place (e.g., with a gauze pad or surgical glove) until a sealant matrix barrier is formed. This is particularly effective and convenient in trauma situations where disposable products are required that can be used for tissue defects of various sizes, such as in emergency departments or battlefields.

In other embodiments, the first and second components and the hydrogel-forming component may be immobilized on a support or backer to form a "sealant matrix mat". In these embodiments, a support, such as a collagen sponge, is provided and the sealant matrix composition is then immobilized on the support for use. Because the sealant matrix composition is easily combined with tissue, organic materials, and synthetic materials, these embodiments may be preferred because the sealant matrix composition may be coated with a support that is easy to handle. In view of the fact that a relatively small amount of sealant matrix composition is required to establish an effective sealant matrix barrier, a relatively thin layer of sealant matrix composition can be immobilized on a support. For example, in the following examples, only about 0.5-1.0g of the sealant matrix composition immobilized on a surface forms a 3cm by 3cm pad with excellent hemostatic properties. As the surgical technician recognizes, these embodiments are needed in situations where the size of the tissue defect can be expected and where improved handling characteristics are needed as compared to powders. As with the dry powder embodiment, in the sealant matrix pad embodiment of the sealant matrix composition, the sealant matrix pad can be applied directly to the tissue defect without further preparation by pressing the sealant matrix composition side of the pad against the tissue defect until crosslinking of the crosslinkable component occurs.

The support in the sealant matrix pad embodiments of the present invention can be any biocompatible material. Although collagen supports are described in detail herein, other materials useful as supports may be used. For example, other protein or polysaccharide support materials that are biocompatible may be used. These support materials degrade in vivo at about the same rate as the sealant matrix barrier, or may degrade at a different rate than the sealant matrix barrier. Collagen sponges and methods for their preparation, collagen production and manipulation are well known in the surgical field and are described in detail below. Also, sponges made from fibrin may be used. Carbohydrate-based materials such as cellulose (topical) or chitosan may also be used. In addition, biocompatible and biodegradable synthetic polymers may also be used. One skilled in the art of surgery will recognize that a non-sponge form may be used for the support in the sealant matrix pad embodiments of the present invention. For example, sheets or films of collagen or other materials may be used. Alternatively, the support may take any useful shape, such as conical, hemispherical, rod-like, wedge-like, etc., to provide a pad that more closely approximates the shape of the tissue defect. For example, a sealant matrix pad using a conical collagen sponge as a support can be used to treat gunshot wounds.

Typically, such supports function as structural or mechanical components. The support may be porous to the extent that blood or other liquids may penetrate the support to increase contact with the composition. Such structures may have a swelling factor of about 1.3X to about 1.5X and are therefore desirable for surgical applications. For example, sponge-supported compositions can be used in neurosurgery to seal the dura, where excessive swelling can produce harmful pressure on the brain. Generally, the support should be flexible enough to accommodate typical tissue defects, yet have good handling properties in a surgical environment.

The sealant matrix composition can be immobilized on the support by a variety of methods. In certain embodiments described below, moderate heat is sufficient to powder immobilize the sealant matrix composition comprising the 4-arm PEG first and second components and the crosslinked gelatin hydrogel-forming component. In these embodiments, the sealant matrix composition powder is placed on a collagen sponge and then heated to 60-70 ℃ for about 1-2 minutes. The dry powder matrix melts slightly under this heat, causing it to become immobilized on the surface of the collagen sponge. Alternatively, the sealant matrix composition can be immobilized to the support with an adhesive or other excipient known in the pharmaceutical arts. In general, the technique used to fix the sealant base composition to the support will depend on the first and second components of the sealant base composition and the hydrogel-forming component. The method used to immobilize the sealant matrix composition on the support should not significantly reduce the ability of the first and second components to crosslink upon exposure to a physiological environment, or the ability of the hydrogel-forming component to absorb biological fluids.

In other embodiments, the sealant matrix composition can be formed into a unsupported sheet or film. For the sealant matrix pad embodiments, this formation of the sealant matrix composition can be accomplished using the methods described above for immobilizing the sealant matrix composition to a support.

Adding other components to the sealant base composition

In other embodiments of the present invention, other components besides the first and second cross-linkable components and the hydrogel-forming component may be added to the sealant matrix composition of the present invention. Generally, these other components can be mixed with the first and second hydrogel-forming components in dry form. Other components may add further mechanical strength or improved performance to the sealant matrix composition of the invention for a particular use. For example, fibrillar collagen is sometimes not preferred for use in bioadhesive compositions because it is opaque and less viscous than non-fibrillar collagen. Fibrous collagen or a mixture of non-fibrous collagen and fibrous collagen may be preferred for use in adhesive compositions intended to be effective for long periods of time in vivo, as described in U.S. patent No. 5,614,587. Various deacetylated and/or desulfonated glycosaminoglycan derivatives may be incorporated into the compositions in a manner similar to that described above for collagen.

Natural proteins such as collagen and various natural polysaccharide derivatives such as glycosaminoglycans may be incorporated into the sealant matrix barrier formed by reactive crosslinking of the first and second components of the present invention under physiological conditions. When these other components also contain functional groups that react with functional groups on the synthetic polymer, their presence results in the formation of a crosslinked synthetic polymer-natural polymer matrix during crosslinking of the first and second components at the target site. In particular, when the natural polymer (protein or polysaccharide) also contains nucleophilic groups, such as primary amino groups, the electrophilic groups on the second cross-linkable component will react with the primary amino groups on these components and the nucleophilic groups on the first cross-linkable component, causing these other components to become part of the sealant matrix barrier.

Generally, the glycosaminoglycan is typically chemically derivatized by deacetylation, desulfonation, or both to contain primary amino groups that can react with electrophilic groups on the second crosslinkable component. Glucosamine polysaccharides that can be derivatized by any one of the foregoing methods or both include the following: hyaluronic acid, chondroitin sulfate a, chondroitin sulfate B (dermatan sulfate), chondroitin sulfate C, chitin (which may be derivatized to chitosan), keratin sulfate, and heparin. Derivatization of glucosamine polysaccharides by deacetylation and/or desulfation and covalent attachment of the resulting glucosamine polysaccharide derivative to a synthetic hydrophilic polymer is discussed in more detail in commonly assigned granted U.S. patent No. 5,510,418, the contents of which are incorporated herein by reference.

Likewise, the electrophilic groups on the second cross-linkable component may react with primary amino groups on lysine residues or thiol groups on cysteine residues of certain native proteins. Lysine-rich proteins such as collagen and its derivatives are particularly active for reacting with electrophilic groups on synthetic polymers. The term "collagen" as used herein is intended to include any type of collagen from any source, including, but not limited to, collagen extracted from tissue or recombinantly produced, collagen analogs, collagen derivatives, modified collagens, and denatured collagens such as gelatin. Covalently bonding collagen to synthetic hydrophilic polymers is discussed in commonly assigned U.S. patent No. 5,162,430 issued to Rhee et al on 10.11.1992.

In general, collagen from any source may be used in the compositions of the present invention, e.g., collagen may be extracted and purified from human or other mammalian sources, such as bovine or porcine dermis and human placenta, or may be prepared by recombinant or other methods. Methods for preparing pure, substantially non-antigenic collagen solutions from bovine skin are well known in the art. A method for extracting and purifying collagen from human placenta is disclosed in U.S. patent No. 5,428,022 issued to Palefsky et al at 27.6.1995. U.S. patent No. 5,667,839 discloses a method for preparing recombinant human collagen from transgenic animal milk including transgenic cows. The term "collagen" or "collagen material" as used herein refers to all forms of collagen, including those collagen that have been treated or modified.

Although type I is generally preferred, any type of collagen may be used in the compositions of the present invention, including, but not limited to, type I, II, III, IV, or any combination thereof. Non-telopeptide or telopeptide-containing collagen; however, when using collagen derived from a xenogeneic source, such as bovine collagen, non-telopeptide collagen is generally preferred because it is less immunogenic than telopeptide-containing collagen.

Collagen that has not been pre-crosslinked by methods such as heat, radiation, or chemical crosslinking agents may be used in the compositions of the invention, as may pre-crosslinked collagen. Collagen Corporation (Palo Alto, Calif.) sells non-crosslinked non-telopeptide fibrous collagens with Collagen concentrations of 35mg/ml and 65mg/ml, under the respective trade namesCollagen I and Zyderm II. The Collagen Corporation markets glutaraldehyde cross-linked non-telopeptide fibrous Collagen with a Collagen concentration of 35mg/ml, under the trade nameCollagen eggWhite. The collagen used in the present invention is generally in the form of a lyophilized powder.

Due to its consistency, non-fibrillar collagen is typically used in the compositions of the present invention intended for use as a bioadhesive. The term "non-fibrillar collagen" includes any modified or unmodified collagen material that is substantially in a non-fibrillar form at pH 7, as evidenced by the optical clarity of an aqueous suspension of collagen.

Collagen, already in non-fibrillar form, may be used in the compositions of the present invention. The term "non-fibrillar collagen" as used herein is intended to include native forms of the non-fibrillar collagen type and chemically modified collagen that is non-fibrillar at or near neutral pH. The native form of the non-fibrous (or microfibrous) collagen types includes types IV, VI and VII.

Chemically modified collagens that are in a non-fibrillar form at neutral pH include succinylated collagen and methylated collagen, both of which can be prepared as described in U.S. patent No. 4,164,559 issued to Miyata et al, 8/14 1979, which is incorporated herein by reference in its entirety. Methylated collagen is commonly used in bioadhesive compositions due to its inherent viscosity, as disclosed in U.S. Pat. No. 5,614,587.

The collagen used in the sealant matrix composition of the present invention may be initially in a fibrous form and then rendered non-fibrous by the addition of one or more fibrolytic agents. The fibrinolytic agent is typically present in an amount sufficient to render the collagen substantially non-fibrillar at the pH 7 described above. The fibrinolytic agents used in the present invention include, but are not limited to, various biocompatible alcohols, amino acids, inorganic salts and carbohydrates, with biocompatible alcohols being particularly preferred. Preferred biocompatible alcohols include glycerol and propylene glycol. In some cases, non-biocompatible alcohols such as ethanol, methanol, and isopropanol may not be suitable for use in the first and second polymers of the present invention because of their potentially deleterious effects on the body of the patient receiving them. Examples of amino acids include arginine. Examples of the inorganic salt include sodium chloride and potassium chloride. Although carbohydrates such as various sugars including sucrose may be used in the practice of the present invention, they are not preferred as are other types of cellulolytic agents, as they may have a cytotoxic effect in vivo.

For use in tissue adhesion, in addition to sealing, it may be desirable to add proteins such as albumin, fibrin or fibrinogen to the sealant matrix composition to promote cell attachment. In addition, the introduction of hydrocolloids such as carboxymethylcellulose may also promote tissue adhesion and/or swelling.

The sealant matrix compositions of the present invention may also contain one or more other bioactive agents or compounds. In one embodiment, a bioactive agent, such as a paclitaxel derivative, may be added to the sealant matrix composition to prevent adhesion at the site of the tissue defect. In other embodiments, bioactive agents such as antibiotics or antimicrobial agents may be added to the sealant matrix for use in, for example, wound conditions (e.g., knife or bullet wounds) caused by trauma in which pathogenic organisms may enter the tissue defect site or wound. In other embodiments, bioactive agents, such as growth factors, in the composition may be released to the local tissue site to promote tissue healing and regeneration. In further embodiments, clotting agents, such as thrombin, may be added to further improve sealing and tissue regeneration by activating the clotting cascade. Exemplary bioactive components include, but are not limited to, proteins, carbohydrates, nucleic acids, inorganic and organic bioactive molecules such as enzymes, antibiotics, antineoplastics, bacteriostats, anti-adhesion forming agents (e.g., paclitaxel derivatives), antiseptics, antivirals, hemostatics, local anesthetics, anti-inflammatory agents, hormones, antiangiogenic agents, antibodies, neurotransmitters, psychotropic agents, agents that act on reproductive organs, and oligonucleotides, such as antisense oligonucleotides. The term "bioactive agent" or "active agent" as used herein includes organic or inorganic molecules that play a biological role in the body. Examples of active agents include, but are not limited to, enzymes, receptor antagonists or agonists, hormones, growth factors, autologous bone marrow, antibiotics, anti-adhesion forming agents, antimicrobial agents, other drugs, and antibodies. The term "active agent" will also include combinations or mixtures of two or more active agents as defined above.

Such bioactive components are generally present at relatively low concentrations, typically less than 10% by weight, typically less than 5% by weight, and typically less than 1% by weight of the composition. Two or more such active agents may be combined in a single composition and/or two or more compositions may be used to release different active components, wherein the components may interact at the site of delivery. Exemplary hemostatic agents include thrombin, fibrinogen, and blood coagulation factors. A hemostatic agent such as thrombin can be added at a concentration of, for example, from about 50 to about 10,000 units thrombin/ml hydrogel, or from about 100 to about 1000 units thrombin/ml hydrogel.

Crosslinked first and second polymer compositions containing various imaging agents, such as iodine or barium sulfate or fluorine, can also be prepared to aid in post-administration by X-ray or by¹⁹F-MRI visualises the composition.

Preferred active agents for use in the compositions of the present invention include growth factors such as Transforming Growth Factors (TGFs), Fibroblast Growth Factors (FGFs), Platelet Derived Growth Factors (PDGFs), Epidermal Growth Factors (EGFs), Connective Tissue Activating Peptides (CTAPs), osteogenic factors and biologically active analogs, fragments and derivatives of such growth factors. Especially preferred are members of the Transforming Growth Factor (TGF) supergene family, which are multifunctional regulatory proteins. TGF supergene family members include beta transforming growth factors (e.g., TGF-. beta.1, TGF-. beta.2, TGF-. beta.3); bone morphogenic proteins (e.g., BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9); heparin-binding growth factors (e.g., Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF)); statins (e.g., statin a, statin B); growth differentiation factors (e.g., GDF-1); and activins (e.g., activin a, activin B, activin AB).

The growth factors may be isolated from a native or natural source, such as mammalian cells, or may be synthetically prepared, such as by recombinant DNA techniques or various chemical methods. In addition, analogs, fragments, or derivatives of these factors may also be used, provided they exhibit at least some of the biological activity of the native molecule. For example, analogs can be prepared by altering gene expression by site-specific mutagenesis or other genetic engineering techniques.

The bioactive agent can be incorporated into the sealant matrix composition by mixing. In one embodiment, the active agent may be mixed with the sealant matrix composition powder in a dried or lyophilized form. In another embodiment, the mixture can be immobilized on a solid support, such as collagen as described above for the sealant matrix composition. In other embodiments, active agents may be incorporated into the above-described sealant matrix compositions by combining these active agents with functional groups on the first or second component forming polymers. In commonly assigned U.S. patent No. 5,162,430 issued to Rhee et al on 10.11.1992, there is a discussion of a method for covalently binding a bioactive agent, such as a growth factor, with a functionally activated polyethylene glycol. Preferably, such compositions contain linkages that are readily biodegradable, e.g., due to enzymatic degradation resulting in the release of the active agent to the target tissue, where it can exert its desired therapeutic effect.

A simple method of incorporating a nucleophilic group-containing bioactive agent into a crosslinked polymer composition involves mixing the active agent with the first component, the second component, and the hydrogel-forming component in a dry state prior to administration. After the sealant matrix composition is applied to the tissue defect and contacted with the biological fluid, the bioactive agent will react with the second component, cross-link the porous cross-linked matrix of the first and second components, and the hydrogel-forming component absorbs the biological fluid. The method covalently bonds the active agent to the cross-linkable component polymer matrix portion of the sealant matrix barrier to yield a highly effective sustained release composition.

The type and amount of active agent used will depend on, among other factors, the particular site and condition being treated and the biological activity and pharmacokinetics of the active agent selected.

Use of sealant base compositions as bioadhesives

The sealant matrix compositions of the present invention are generally viscous and therefore strongly bound to tissue because the electrophilic groups of the second cross-linking component react with the nucleophilic groups of the collagen in the target site of the tissue. Certain porous matrix compositions of the present invention can have unusually high viscosities. Thus, in addition to being used as a barrier matrix to stop bleeding, the sealant matrix compositions of the present invention can also be used as a bioadhesive to bond with wet or moist tissue in a physiological environment. As used herein, the terms "bioadhesive", "bioadhesive" and "surgical adhesive" are used interchangeably and include biocompatible compositions that enable the transient or permanent attachment of two native tissue surfaces, or the transient or permanent attachment of a native tissue surface to a non-native tissue surface or to the surface of a synthetic implant.

The sealant matrix composition (e.g., in dry powder or tablet form) is applied to the first surface in accordance with the usual method of joining the first surface to the second surface. The first surface is then brought into contact with the second surface, preferably immediately after bonding the two surfaces. At least one of the first and second surfaces is preferably a native tissue surface. When a mechanically stable hydrogel-forming component is used in a composition, such as the cross-linked gelatin used in the examples, the resulting porous matrix exhibits increased mechanical strength as opposed to a composition containing only the first and second cross-linkable components. Thus, the adhesive strength between two tissue surfaces is also increased, as the porous matrix layer between the tissues is less likely to separate internally under physiological mechanical pressure.

The two surfaces may be brought together by hand or by other suitable means, while allowing the crosslinking reaction to proceed to completion. Crosslinking is generally completed within 5 to 60 minutes after application of the sealant matrix composition. However, the time required for complete crosslinking to occur depends on a number of factors, including the type and molecular weight of the first and second crosslinkable components, and most particularly the effective concentration of both components in the target site (i.e., the higher the concentration, the faster the crosslinking time).

Preferably at least one of the first and second surfaces is a native tissue surface. The term "native tissue" as used herein includes the native biological tissue of the body itself which is the particular patient being treated. The term "native tissue" as used herein includes biological tissue (e.g., bone autograft, flap autograft, etc.) that is lifted from or removed from one part of a patient's body for transplantation to another part of the same patient's body. For example, in the case of a burn patient, a skin patch from one part of the patient's body may be adhered to another part of the body with the composition of certain embodiments of the present invention.

The other surface may be a native tissue surface, a non-native tissue surface or a surface of a synthetic implant. The term "non-native tissue" as used herein includes biological tissue (e.g., tissue and organ grafts) excised from the body of a donor patient (who may be of the same ethnicity or a different ethnicity as the recipient patient) for transplantation to the body of the recipient patient. For example, a patient's xenograft heart valve can be secured in the patient's heart and sealed around the heart valve against leakage using the crosslinked polymer compositions of the invention.

The term "synthetic implant" as used herein includes any biocompatible substance intended for implantation into the body of a patient, which is not included in the above definitions for native and non-native tissue. Synthetic implants include, for example, vascular prostheses, heart valves, artificial organs, bone prostheses, implantable microlenses, vascular grafts, stents, and stent/graft combinations, among others.

Use of a sealant base composition to prevent blocking

Another use of the sealant composition of the present invention is to coat tissue to prevent adhesion formation following surgery or internal tissue or organ injury. According to the general method of coating tissue to prevent post-surgical adhesion formation, first and second synthetic polymers are mixed or pre-mixed with a hydratable, cross-linkable polymer, and then a thin layer of the reaction mixture is coated onto the tissue that comprises, surrounds, and/or is adjacent to the surgical site before the nucleophilic groups on the first synthetic polymer significantly cross-link with the electrophilic groups on the second synthetic polymer. By extrusion, spraying, painting, spraying of powders of the composition (as described above); the reaction mixture is applied to the tissue site by placing a film or sheet of the sealant matrix composition on the tissue or by any other conventional method.

After the reaction mixture is applied to the surgical site, the cross-linking is allowed to proceed in situ before the surgical incision is closed. Once the cross-linking reaches equilibrium, the tissue in contact with the coated tissue will not adhere to the coated tissue. At this time, the surgical site can be closed by a conventional method (suturing or the like).

In general, compositions that preferably complete crosslinking within a relatively short time (i.e., 5-15 minutes after mixing the first synthetic polymer and the second synthetic polymer) can be used to prevent surgical adhesions, allowing for faster closure of the surgical site after the procedure is completed. In addition, hydratable cross-linked polymers with higher mechanical strength are preferred for the composition, such as the cross-linked gelatin used in the examples, to increase the mechanical stability of the coating.

In the following examples, the preparation and characterization of the first cross-linkable component with the second cross-linkable component and the hydrogel-forming component to form the sealant matrix composition are described and are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to prepare the preferred conjugates, compositions and device embodiments and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, molecular weight, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees centigrade, and pressure is at or near atmospheric.

VII. examples

Example 1: first and second composition for sealant matrix: preparation of Cross-Linked PolyaminoPEG compositions

The following stock solutions were prepared for various diamino PEGs: 10 grams of Jeffamine ED-2001 (available from Texaco chemical Company, Houston, Tex.) was dissolved in 9ml of water. 10 grams of Jeffamine ED-4000 (also available from Texaco chemical Company) was dissolved in 9ml of water. 0.1g of diamino PEG (3400MW from Shearwater Polymers, Huntsville, Ala.) was dissolved in 300. mu.l of water. Each of the 3 diamino PEG solutions prepared above was mixed with a trifunctional activated SC-PEG aqueous solution (TSC-PEG, 5000MW, also from Shearwater Polymers) as shown in Table 1 below.

TABLE 1

Solutions of diamino PEG and TSC-PEG were mixed using a syringe-syringe mixing method. The materials were pushed out of the syringe and left at 37 ℃ for 1 hour. Each material formed a gel. Generally, gels soften as water content increases; the gel containing the least amount of aqueous solvent (water or PBS) was the hardest.

Example 2: first and second composition for sealant matrix: preparation of crosslinked poly (lysine) compositions

10mg of poly-L-lysine hydrobromide (8,000MW, from Penninsula Laboratories, Belmont, Calif.) in 0.1ml of phosphate buffer (0.2M, pH 6.6) were mixed with 10mg of tetrafunctionally activated SE-PEG (10,000MW, from Shearwater Polymers, Huntsville, Ala.) in 0.1ml PBS. The composition formed a soft gel almost immediately.

Example 3: first and second composition for sealant matrix: effect of pH on gel formation of Tetraamino PEG/TetraSE-PEG formulations

Gels containing tetraamino-PEG and tetrasE-PEG at various concentrations of pH 6, 7 and 8 were prepared in Petri dishes. Mixing tetra-amino PEG and tetra-SE-PEG, and repeatedly inclining each vessel; the point at which the formulation stops flowing is defined as the gel time. The effect of pH on the gel time of various tetraamino PEG/tetrasE-PEG formulations at room temperature is shown in Table 2 below.

TABLE 2

The time required for gel formation increased with increasing pH and decreased with increasing concentrations of tetraamino PEG and tetrase-PEG.

Example 4: evaluation of hydrogel-forming component materials and percent Cross-linking and swelling measurements

The gelatin particles are swollen in an aqueous buffer (e.g., 0.2M sodium phosphate, pH 9.2) containing a crosslinking agent (e.g., 0.005-0.5% by weight glutaraldehyde). The reaction mixture was refrigerated overnight, then washed 3 times with deionized water, 2 times with ethanol, and dried at ambient temperature. The dried crosslinked gelatin is resuspended in aqueous buffer at low solids concentration (2-3%) for a period of time at ambient temperature. The concentration of buffer is substantially greater than that required for equilibrium swelling, and two phases (hydrogel phase and buffer) are present. The suspension containing the wet hydrogel was then filtered by applying a vacuum on a 0.8 μm nominal cut-off filter (Millipore, Bedford, Mass.). After removal of the extraneous buffer, the total weight of trapped wet hydrogel and wet filter was recorded. The hydrogel and membrane were then dried at about 120 ℃ for at least 2 hours, and the total weight of the dried hydrogel residue and the dried filter membrane was recorded. Several measurements of samples of the wet filter containing no hydrogel residue and the dry filter containing no hydrogel were also taken and these measurements were used to derive the net weights of the wet hydrogel and the dry hydrogel. The "percent swelling" is then calculated according to the following equation:

percent swelling of 100 × [ (wet weight of hydrogel-dry weight of hydrogel)/dry weight of hydrogel ]

For a given gelatin sample, swelling was measured at least in triplicate, and the measurements were then averaged. The percent swell value for the sample resuspended in buffer for 18-24 hours before measuring wet weight is defined as "equilibrium swell".

The resulting crosslinked gelatin material exhibited an equilibrium swell value in the range of 400% to 1300%. The extent of equilibrium swelling depends on the method and extent of crosslinking.

Example 5: hydrogel-forming component for sealant matrix: hydratable cross-linked polymer fragments consisting of gelatin cross-linked with EDC

Gelatin (Atlantic Gelatin, General Foods Corp., Woburn, Mass.) was dissolved in distilled water at 70 ℃ at a solid content of 1-10% (w/w) (more preferably 8%). Then 0.2% -3.5% (or 0.2% -0.3%) 1-ethyl-3- (3 dimethylaminopropyl) carbodiimide (EDC) (Sigma, st. louis, Mo.) was added. The resulting hydrogel product was stirred at room temperature for 1 hour. The hydrogel was dried using a Freezone 12 lyophilization system (Labconco, Mo.) and ground using a Waring blender model 31BC91(VWR, Willad, Ohio). The dried polymer composition was then added to a syringe and equilibrated with buffer. Equilibrium swelling was measured to be at least 1000%. The results are shown in Table 3.

TABLE 3

Example 6: hydrogel-forming component for sealant matrix: hydratable cross-linked polymer fragments consisting of gelatin and poly (L) glutamic acid cross-linked with EDC

Gelatin (Atlantic Gelatin, General Foods Corp., Woburn, Mass.) was dissolved in distilled water at 70 ℃ at a solid content of 1-10% (w/w) (more preferably 6-8%). Then 0-10% (w/w) (more preferably 2-5%) poly (L) glutamic acid (PLGA) (Sigma, St. Louis, Mo.) and 0.2-3.5% (or 0.2-0.4%) 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) (Sigma) were added. The resulting hydrogel product was stirred at room temperature for 1 hour. The hydrogel is swollen in excess saline for a period of time (e.g., 20 hours). The hydrogel was then filtered by applying a vacuum on the filter membrane (Millipore, Bedford, Mass.). Equilibrium swelling was measured to be at least 1500%. The results are shown in Table 4.

TABLE 4

Example 7: hydrogel-forming component for sealant matrix: preparation of hydratable crosslinked polymeric hydrogel fragments

Bovine dermis (Spears co. pa) is stirred in aqueous sodium hydroxide (SpectrumChemical co., CA) solution (0.1M-1.5M, or 0.4-1.2M) for 1-18 hours (or 1-4 hours) at 2-30 deg.c (preferably 22-30 deg.c). The dermis slurry is then neutralized with a mineral acid such as hydrochloric acid, phosphoric acid or sulfuric acid (Spectrum Chemical co., CA.), and the neutralized liquid phase is then separated from the insoluble dermis by filtration through a sieve. The dermis is then washed with pyrogen-free water and an alcohol such as isopropanol (Spectrum Chemical co., CA.). After 3-12 washes, the dermis is suspended in pyrogen-free water, and then the dermis, the aqueous slurry, may be heated to 50-90 ℃, preferably 60-80 ℃, to thermally gel the dermis. During the gelling, the pH of the dermis, the water slurry is adjusted to pH 3-pH 11, or pH 7-pH 9. The insoluble dermis in the slurry may also be disrupted by stirring and/or homogenization. The fracture may occur before or after the thermal gelation cycle. Thermal gelation was allowed to proceed for 1-6 hours. After gelling, the slurry was clarified by filtration. The gelatin slurry is dewatered by through-air drying at 15-40 deg.C, preferably 20-35 deg.C. The dried gelatin is then disintegrated by grinding, wherein drying means a moisture content of less than 20% by weight.

Dry gelatin is added to a cold (5 ℃ C. to 15 ℃ C.) aqueous solution of 0.0025% to 0.075% by weight glutaraldehyde (Amresco Inc, OH.) at pH 7-10. The gelatin concentration in the solution is 1% to 10% by weight. Glutaraldehyde is allowed to crosslink with the gelatin particles for 1-18 hours, and then the gelatin is separated from the aqueous phase by filtration or sedimentation. The gelatin particles are then added to an aqueous solution containing 0.00833% to 0.0667% by weight sodium borohydride (Spectrum Chemical co., CA.) at a gelatin concentration of again 1% to 10% by weight and a pH of 7 to 12, or 7 to 9. After 1-6 hours, the crosslinked gelatin is separated from the aqueous phase by filtration or sedimentation. The gelatin may then be resuspended in pyrogen-free water at a gelatin concentration of 1% to 10% by weight and then separated from the aqueous phase by filtration or sedimentation to remove residual crosslinking agent and reducing agent. Finally, the crosslinked gelatin is collected on a filter screen or a sieve, and finally the gelatin is washed with pyrogen-free water. The wet cross-linked gelatin is then placed in a drying chamber and dried at 15-40 ℃. The dried crosslinked gelatin (i.e., crosslinked gelatin having a moisture content of less than 20% by weight) was removed from the drying chamber and then ground with a mechanical grinder to give a powder having a typical particle size distribution of 0.020mm to 2.000 mm.

Example 8: quick-acting dry hemostatic sealant powder

A quick-acting dry hemostatic sealant powder is prepared by mixing a first crosslinkable component, a second crosslinkable component, and a hydrogel-forming component. The first crosslinked polymer (PEG-A) was PEG-succinimide based powder, the second crosslinked polymer (PEG-B) was PEG-thiol powder, and the hydrogel-forming component was crosslinked gelatin powder.

Example 9: quick-acting dry sealant pad

A quick-drying sealant pad is prepared by mixing a first cross-linkable component, a second cross-linkable component, and a hydrogel-forming component. The resulting composition, sealant matrix composition powder, was placed on a lyophilized collagen sponge and heated to 60-70 ℃ for about 1-2 minutes. The dry powder matrix slightly melts under this heat, fixing it to the surface of the collagen sponge, thereby forming a sealant matrix pad. Alternatively, the sealant matrix composition can be immobilized to the support with an adhesive or other excipient known in the pharmaceutical arts. In general, the technique used to immobilize the sealant matrix composition to the support may depend on the first and second components of the sealant matrix composition and the hydrogel-forming component. The sealant matrix pad embodiments of the present invention provide a convenient form by which to treat the sealant matrix composition and then release the sealant matrix composition to the surgical site via a sponge or other suitable support means.

Example 10: sealant powder for treating splenic artery puncture

Figures 7A-E illustrate treatment of a splenic artery puncture with a sealant matrix composition according to embodiments of the present invention. Heparinized to approximately 3-fold baseline. As shown in fig. 7A, porcine splenic artery puncture was induced by surgery with an 18g needle 700. After puncture, a major hemorrhage 705 occurs in the artery 710. As shown in fig. 7B and 7C, approximately 700mg of the sealant powder matrix composition 720 was applied to the puncture site by syringe 730 and gently pressed or placed on the site with a gloved finger 740 for 2 minutes. The sealant powder was found to form a fully hemostatic coagulum 750. 5 minutes after coating, the site is rinsed with rinsing apparatus 760 shown in FIG. 7D to wash off excess powder composition. When the coagulum was grasped with forceps, it appeared to adhere strongly to the tissue and had good integrity. After 44 minutes of coating, the coagulum 750 was removed by peeling with forceps 770, and bleeding 715 reappeared, as shown in FIG. 7E.

Example 11: sealant powder for treatment of hepatectomy

Figures 8A-E illustrate treatment of hepatectomy with a sealant matrix composition, according to embodiments of the present invention. Heparinized to approximately 3-fold baseline. As shown in fig. 8A, the tip 800 or edge of the lobe 805 of the pig's liver is cut with scissors 810. After excision, a major hemorrhage 815 occurs at the site. As shown in fig. 8B, about 6ml (2g) of the sealant base composition 820 is applied to the site, held in place with the tip of the syringe 825 for 2 minutes. The powder can be pressed or maintained on the defect with a gloved finger, as shown in fig. 8C. The sealant powder formed a coagulum 835 which was observed to completely stop bleeding. After 8 minutes of coating, the site was rinsed with rinsing device 840 as shown in FIG. 8D. When the coagulum was grasped with forceps, it appeared to be tightly adherent to the tissue and had good integrity. After 28 minutes of application, the coagulum 835 is removed by peeling with forceps 845 and bleeding 850 reappears.

Example 12: sealant powder for treating spleen damage

A6 mm biopsy needle was used to form spleen lesions on pigs by surgery and the core was removed with scissors. Pigs were heparinized to approximately 2.5-fold baseline. After tissue puncture, massive hemorrhage of the spleen occurred. Approximately 700mg (2ml) of the sealant base composition powder was applied to the puncture site with the edge of a 12ml syringe. No pressure is used to hold the material in place. The sealant powder was observed to form a fully hemostatic coagulum. After 4 minutes of coating, the site was rinsed. When the coagulum was grasped with forceps, it appeared to be tightly adherent to the tissue and had good integrity. After 25 minutes of coating, the coagulum was removed by peeling and bleeding reappeared.

Example 13: mechanical stress test

Sealant matrix barriers were prepared by reacting 0.60-0.65g of sealant matrix composite powder with 1ml of porcine plasma in a plastic mold. Mixing the mixtureCuring at room temperature for about 30 minutes, tacking two peripheral cyanoacrylate tapes of 3 × 1 × 0.3.3 cm blocks of gel to form an occlusal space (1 × 1cm) for breaking, clamping the tape at each end with a pre-installed clamp, subjecting the rectangular gel to a standard stress test with a Chatillon TCD2000 test apparatus until the gel shape breaks, measuring the tensile strength, continuing pressurization to break the gel to measure the maximum force (N) and deflection at maximum load (mm), the effective surface area of the gel being 1 × 0.3.3 cm, the original effective length of the gel being 1 cm., the tensile strength of the sealant gel being about 15.3N/cm². The same test was conducted on a gel composition containing a first crosslinkable component and a second crosslinkable component but no hydrogel-forming component, and the tensile strength was found to be about 5.1N/cm²。

Example 14: peel strength test

In certain embodiments, the blended powder comprises the first and second cross-linkable components and the hydrogel-forming component, and the powder self-polymerizes when dissolved in a physiological fluid such as blood or other bodily fluids. The material may be firmly bonded to tissue or other coating sites by covalent bonds. The mechanical clip can be used to separate the sealant matrix from the tissue, e.g., skin, and to test the mechanical strength of the tissue adhesion. In this example, after the sealant matrix barrier was formed, the following tensile test was performed a number of times. A composition containing a first crosslinking component (pentaerythritol poly (ethylene glycol) ether tetrasuccinimidyl glutarate) and a second crosslinking component (pentaerythritol poly (ethylene glycol) ether tetrasulfate) and crosslinked gelatin (Floseal) was prepared by mixing 3 different concentrations (10%, 20% and 30% crosslinking component) of the crosslinking components with crosslinked gelatin^TM) In a 3 × 1 × 0.3.3 cm plastic mold, about 0.40g to about 0.45g of the 3 component powder was added to about 0.6ml of porcine plasma placed on top of a chicken skin sample and cured at room temperature for about 60 minutesThe maximum peak force (N) was measured. It was observed that the increase in adhesive strength was almost linearly related to the increase in concentration of the PEG mixture (first and second cross-linkable component). The results are shown in Table 4A.

Example 15: preparation of fibrous collagen for sponge pad in fusion pad

The first fibrous collagen sample was prepared as follows. 40g of NaOH were dissolved in 450cc of H at 25 deg.C₂And O. Approximately 50g of bovine dermal slices were added to the NaOH solution. The dermal solution was stirred for 80 minutes. Pouring out the NAOH solution gently, and soaking the dermis with H₂And O washing. The dermis was dissolved with 2M HCl to pH 2.3-2.4. Stirring was continued for 18 hours. 1250ml of concentrated collagen solution (CIS) were titrated with 1M NaOH to pH 7.25 at 18 ℃. Collagen fibers were formed within 10 hours and filtered. 240ml were precipitated at pH 7.4 and then crosslinked with 33. mu.l of a 25% Glutaraldehyde (GA) solution at 8 ℃ for 23 hours. The fibrillar collagen was lyophilized by a Virtis lyophilizer through a defined cycle (recipecycle).

A second fibrous collagen sample was prepared as follows. The fibrillar collagen is crosslinked with 240g of viscous solution (e.g., CIS). By adding 60cc of H₂O dilute the solution. The pH was raised to 9.2 by the addition of about 1.8cc of 2M NaOH. The temperature of the solution was adjusted to 8 ℃ and 33. mu.l of 25% GA was added. The solution was stirred for 23 hours to give about 54g of precipitated fiber. The fibrillar collagen was lyophilized by a defined cycle using a Virtis lyophilizer.

Example 16: debuffering hydrogel-forming components

In certain embodiments, it may be desirable to incorporate a hydrogel-forming component such as FloSeal^TMTo enable the environmental liquid to readily affect the pH of the hydrogel-forming component.After coating, in situ crosslinking of the hydrogel-forming component can help the sealant matrix compound adhere to the tissue. In some cases, adhesion may be more effective at certain pH values. For example, certain gelatin-based materials may stick more easily at a pH of less than 6 or 7. Mixing FloSeal at a ratio of 1: 50^TMBy H₂O washes, and the pH of the slurry is adjusted or acidified to pH 2-7 with 0.01M HCl or 0.01M NaOH. The wet gelatin cake was filtered, and the cake was dried at 32 ℃ for 12-20 hours in a vented drying oven and lightly ground with a mortar and pestle. Dry gelatin powder was added to the mixed PEG solution for in situ crosslinking. The slurry was stirred for 30 seconds and then immediately applied to the surface of a weighed paper fully saturated with 25mM phosphate buffer, pH 7.4. The polymerization time was recorded and the results are shown in table 5.

TABLE 5

Example 17: preparation of PEG cake

In one embodiment, 0.8g PEG-succinimide powder and 0.8g PEG-thiol powder are mixed well by shaking, then N-impregnated is added₂100ml round bottom flask. The mixed powder was melted in an oil bath at 40-50 ℃ and slowly stirred by hand for 30 minutes, and then cooled. The solid film was removed from the flask with a spatula. In another embodiment, a mixed powder of PEG-succinimide and PEG-thiol is dissolved in an acidic solution of collagen (e.g., 0.3%) or gelatin (e.g., 2%) and lyophilized. It is believed that fibrillar collagen or gelatin can help disperse the matrix and improve the handling of the PEG cake.

In a comparative composition, 1.2g of collagen fibrils were dissolved in 100cc of pH 2HCl, heated in a water bath at 35 ℃ for 1-2 hours, and diluted with pH 2HCl to give a 0.3% CIS product. 0.2g PEG-succinimide and 0.2g PEG-thiol were dissolved in 2cc of 0.3% CIS. The resulting mixture was poured into a tray and lyophilized by 22 hour cycles to give a PEG cake. In yet another embodiment, 2g of gelatin is dissolved in 100cc of pH 2HCl in a 35 ℃ water bath. 4g of the bi-component PEG powder mixture was dissolved in 2cc of gelatin solution and lyophilized to give a PEG cake.

In a related embodiment, the PEG cake is prepared by lyophilizing a mixed solution of PEG-SG, PEG-SH, and collagen at pH 2.0. Animal studies were performed on abraded liver capsule tubes in a heparinized pig model. 2 drops of 0.2M phosphate buffer (pH 9.0) were added to the surface of the liver, which was bleeding slowly. A cake was placed in the area without applying pressure. At 5 and 10 minutes, each PEG cake was tested for adhesion to the site. It was found that during the preparation, the activity of PEG-SG was not decreased and PEG cake was bonded to the abraded liver tissue by covalent bond. The compositions and in vivo properties of the test samples are summarized in Table 6.

Example 18: comminuted PEG cake material

400mg of premixed CoSeal^TM、1g FloSeal^TM(e.g., pH 7.1-9.5; particle size 70-400 μm) and 2-3ccH₂O was mixed into a paste and frozen under 22 hour cycles to form a cake. As shown in fig. 9, the cake 900 is then broken up 910, pulverized into a powder form 920, and then placed into a syringe 930 (e.g., a 5cc or 10cc syringe). The syringe barrel tip 940 is removed with a razor blade, the mixture powder 920 is coated on the lesion site 950, and sealant activity is observed in situ. Exemplary results are described in examples 10-12. In another embodiment, a cake is prepared from the 3-component slurry described in table 7.

The test results for the exemplary formulations of certain embodiments show the following characteristics shown in table 8.

Example 19: preparation of a fused pad of a sealant matrix composition

By molten CoSeal^TMPremix PEG pads were prepared. By means of FloSeal at different pH values^TMPowder and premix CoSeal^TM(e.g., two PEG components in powder form) are mixed in various weight ratios to produce a 3-component powder.A lyophilized collagen sponge is used as a backup support pad to which the molten 3-component mixture is immobilized.in one embodiment, as shown in FIG. 10, 0.5-1g of the sealant matrix composition 1000 is placed at 3 × 3cm 3 of sponge 1010²Partial top sponge and sealant matrix were placed in an oven, baked at 60-70 deg.C for 1-2 minutes, and then placed in a desiccator to cool, isolate or minimize contact with air, the sealant matrix powder was observed to form a coarse film and stick to the sponge, forming a fused pad 1020. in a related embodiment, several sponges, each having a size of 3 × 3 × 0.3.3 cm, were prepared³. Certain sponges were coated with a 3-component mixture of first and second cross-linkable components and a hydrogel-forming component. Some sponges were coated with a two-component mixture of only the first and second cross-linkable components. All fused pads were tested in situ on the site of the liver lesion. The results are shown in Table 9.

In a related embodiment, several composition powders were prepared. Certain compositions comprise a 3-component mixture of first and second cross-linkable components and a hydrogel-forming component. Some compositions contain only a two-component mixture of the first and second cross-linkable components. All compositions were tested in situ on the site of liver damage. The results are shown in Table 10.

Example 20: effect of Gamma-radiation on in vivo Performance

A powder of the sealant base composition and the sealant base composition fixed to the sponge were prepared, and some of them were irradiated with gamma rays to measure the influence of the gamma rays on the in vivo performance. As shown in Table 11, no effect was observed.

Example 21: effect of pH on in vivo Performance

In vivo studies were conducted to evaluate the effect of pH, hand coating method of the hydrogel-forming components on in situ crosslinking. With Floseal pH 6.75 in a first sealant composition^TMAnd FloSeal pH 9.5 in a second sealant composition^TMFor comparison. In some cases, the sealant matrix composition is manually applied to the lesion, and in other cases, the sealant matrix composition is applied or placed on the lesion without external application. Containing pH 6.75Floseal^TMThe reaction time of the composition of (a) appears to be longer than that of Floseal containing pH 9.5^TMThe composition is slow for about 10-30 seconds. Exemplary study results are shown in table 12. It is therefore believed that the pH of the hydrogel-forming component may play a role early in the crosslinking reaction. The pH of the hydrogel-forming component can affect the rate of gel formation in a humid environment (e.g., where bleeding has occurred). In some cases, the sealant composition may be removed from the lesion if crosslinking does not occur sufficiently.

Example 22: SURGIFOM^TMUse as hydrogel-forming component

COH102 (pentaerythritol tetrakis- [1- (1 '-oxo-5' -succinyl valerate)) -2-poly (oxyethylene) diol in a ratio of 1: 2, 1: 4 and 1: 8 (by weight)]Ether) powder, COH206 (pentaerythritol tetrakis- [ mercaptoethyl-poly (oxyethylene) glycol)]Ether) powder and SURGIFOM^TMA mixture of absorbable gelatin powders (Ethicon, Somerville, NJ) was blended and then filled into a modified 5mL syringe.the resulting mixture was a substantially dry, free-flowing powder.2 grams of each composition was coated onto a lesion surgically formed on the pig liver (approximately 1cm × 1cm × 0.3.3 cm deep) and lightly compressed.for each composition, the compression was removed after 1 minute^TMPowdering and physically sealing the crosslinked hydrogel at the lesion site. No bleeding was observed at any site treated with the composition. After 5 minutes of coating, the treated lesions were rinsed with saline solution and no rebleeding occurred. After 2 hours, the treated site was examined and no bleeding was observed.

Example 23: in vivo performance of sealant matrix compositions containing clotting agents

Preparation of FloSeal in a 4: 1 weight ratio^TMAnd CoSeal^TM(pre-mixed) sealant base composition powder. In certain embodiments, this ratio provides a degree of crosslinking effective to achieve the desired level of chemical polymerization and adhesion of the composition to tissue. Thrombin powder was added to each concentration of the sealant base composition powder. The resulting mixture was tested in an animal study trial involving measuring the bleeding score in liver squares and comparing the resulting mixture to the hemostatic efficiency of a sealant matrix composition without thrombin.

The test material included 0.1g of pentaerythritol tetrakis [ mercaptoethyl-poly (oxyethylene)]Ether, 0.1g pentaerythritol tetrakis [1-1 '-oxo-5' -succinimidyl valerate) -2-poly (oxyethylene) diol]Ether, 0.8g crosslinked gelatin particles (Floseal)^TM) And thrombin at various concentrations (5k, 2.5k, 1.25k and 0.625k μ/g). In the mixing test, 4 components of the resulting mixture were mixed with a drum mixer. In the reconstitution experiments, 4ml of thrombin solution (1250. mu.l/ml) was mixed with 0.8g of FloSeal and then lyophilized for 22 hours. The dry mixture was then mixed with CoSeal using a peripheral mixer^TMAnd (4) mixing the powder. Without being bound by any particular theory, it is believed that the reconstituted thrombin formulation comprises an infiltrated FloSeal^TMA thrombin molecule of the matrix such that thrombin can be retained in the sealant matrix barrier to improve hemostatic efficiency. In the pad experiment, a pad was prepared by fixing the resulting 4-component mixture (sealant matrix composition + thrombin) on top of a gelatin sponge (Gelfoam) sponge, melting the mixture, cooling, and solidifying. The oven temperature was set to about 60 ℃ to about 65 ℃ for about 1 minute.

In vivo experiments, animals were heparinized to activate clotting times to more than 3-5 times baseline. The effect of the preparation on the extent of bleeding of surgically prepared cubes of pig liver (1 cm. times.1 cm. times.0.2 cm) was examined. After 5 minutes, the lesions were immediately washed and excess powder was removed. The lesion area of treatment was scored at 1, 5, 10 and 30 minutes. Upon contact with blood, the material polymerizes and then binds strongly to the lesion. The sealant matrix barrier mechanically seals the bleeding area, acting as a mechanical sealant by binding to the tissue. In vivo experiments, thrombin was heated at about 60 ℃ for 5 minutes and found to be fully active. In the prepared gelatin sponge pads, loss of thrombin activity was found.

The results of the acute in vivo effect evaluation are provided in table 13. A lesion bleed scored as "0" represents no bleeding and "4" represents severe bleeding. All samples tested showed no bleeding according to the bleeding score observed here. No significant advantage was found after adding thrombin to the sealant matrix composition. The use of thrombin does not show any benefit in primary hemostasis, although it may enhance secondary hemostasis/coagulation formation and wound healing.

Example 24: effect of PEG concentration on gel Strength

The effect of PEG concentration on gel strength according to one embodiment of the invention is shown in table 14 and figure 11. After gel formation, tensile testing was performed. The gel is prepared by reacting a 3-component powder, such as a sealant base composition comprising first and second cross-linkable components and a hydrogel-forming component, in a plastic mold (3X 1X 0.3 cm). Porcine plasma (1ml, Baxter animal number S-264) was added to the sealant base composition powder (0.60-0.65g), gel formation was initiated, and then cured at room temperature for about 30 minutes. The transparent adhesive tape was bonded to both ends of the gel with cyanoacrylate glue to prepare an engaging space (1X 1cm) for pulling apart. By the tensile test, the maximum force (N) at which the gel is stretched to break and the deflection (cm) at maximum load are measured. 1X 0.3cm is the effective surface area. The original effective length of the gel was 1.0 cm. Standard stress was applied to the rectangular gel by the chatillon tcd200 test apparatus until it broke up as a determining factor for tensile strength. The test results show that higher concentrations of polymer can increase the strength of the sealant matrix composition gel.

Example 25: effect of PEG concentration on swelling Rate

The effect of PEG concentration on swelling ratio according to one embodiment of the invention is shown in figures 12, 13 and 14. A swelling study was conducted to characterize the sealant matrix composition gel. When contacted with an aqueous environment, the hydrophilic polymer swells to form a hydrogel. Once the gel is formed, water molecules are free to diffuse into the swollen FloSeal^TMThe particles form a rather loose network. After addition of water, the COH102-COH206 junction is broken and the individual polymer molecules dissolve in the water. By mixing 4 different concentrations (5%, 10%, 20% and 30% w/w) of polymer CoSeal^TMAnd FloSeal^TMSealant base composition gels were prepared by mixing and reacting them with an equal amount of porcine plasma (1.7ml/g powder). The gel was cured for 30 minutes at room temperature and then allowed to swell in saline. At regular intervals, the buffer was aspirated and the weight of the remaining gel was determined. The weight change of the gel was monitored. The swelling ratio Q was calculated from the following equation:

Q＝W*/W

wherein W is the wet weight and W is the initial weight. The swelling ratio increases with increasing polymer concentration. Without being bound by any particular theory, the apparent decrease in swelling ratio may be explained by the loss of gel mass as the gel slowly erodes. The end point of the experiment was scored as the time when the gel disintegrated into several small pieces or became too sticky and loose to allow the free buffer to be decanted from the gel. The water continues to penetrate into the core and eventually the gel transforms into a viscous solution of PEG and gelatin particles. All materials took about 2-3 weeks to disintegrate (fig. 14). It seems CoSeal^TMIn the sealant matrix groupThe percentage of the compound powder has a significant effect on the gel stability of the sealant base composition. The dissolution rate of the sealant matrix composition gel varies depending on the degree of crosslinking of the polymer. The results show a higher concentration of CoSeal^TMCan result in greater gel stability and can also result in greater swelling. The relative persistence of this gel in vitro is expected to be similar to that in vivo.

The above examples provide a sufficient illustration that the compositions of the present invention can be effective sealants. These compositions can polymerize with physiological fluids or blood in situ and therefore seal or adhere very strongly to tissue.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the appended claims. All patents, publications, articles, books, and other references described herein are incorporated by reference.

Example 26: evaluation of hemostatic Properties of some formulations in animal models

Formulation Nos. 334 to 77

1g of PEG-A powder (pentaerythritol tetrakis [ mercaptoethyl-polyoxyethylene)]Ether, MW 10,000), 1g PEG-B powder (pentaerythrityl tetrakis [1-1 '-oxo-5' -succinimidyl valerate-2-polyoxyethylene glycol)]Ether, MW 10,000) and 8g FloSeal^TMThe mixture was put into a mixing flask (50ml volume), and the flask was mounted on an Inversina drum mixer to mix. The 3-component mixture was blended for 10 minutes to complete mixing. About 1.5g of the mixture was filled in a 6-syringe (5ml volume).

Formulation No. 334-77-1

1.5g of preparation No. 334-77 sample was fixed to a piece of gelatin sponge (3 × 4 cm)²Compressed gelatin sponge, Upjohn preparation, NDC 0009-. Putting the gelatin sponge with the sample on the upper part into a vacuum box, and drying at 60-65 ℃ for 1 min until the sample begins to meltAnd (4) transforming. The material is then cooled and solidified. The two cakes obtained on the gelatin sponge were placed into a sachet with desiccant inserted and sealed.

Formulation No. 334-77-4

Formulation No. 334-77 samples were placed on a piece of collagen sponge and baked, the collagen fibers were lightly cross-linked by glutaraldehyde solution (5k ppm) and the sponge was prepared by lyophilizing collagen solution (1.0%) with VirTis Genesis lyophilizer, collagen pad (3 × 4 cm)²) The layers were carefully separated into 1.5g of samples of formulation Nos. 334-77 and placed in a vacuum oven and heated at 60-65 deg.C for 1 minute until the samples began to melt. The material is then allowed to cool and solidify. Each of the obtained collagen pads was put into a pouch with a desiccant inserted therein, and sealed.

The method comprises the following steps:

the surgical method comprises the following steps:

animals (NZW rabbits, females, weighing approximately 3kg) were anesthetized and given a 4.000IU/kg dose of heparinization intravenously for 30 minutes prior to liver dissection.

Liver resection model:

the left lobe of the liver was exposed and clamped by a central laparotomy. A portion of the left lobe was excised. Exudation was controlled by application of the test drug. Coating and fixation times were normalized to not exceed 300 seconds. When the basic hemostasis time is expected to be completed for 5 minutes, the hemostat is removed.

Liver bruise model:

the left lobe of the liver was exposed by a central laparotomy. Grinding a shallow ring damage with the diameter of 2cm and the depth of 2mm on the surface of the liver lobe. The abrasion was accomplished with a punch with a grinding wheel attachment (hole grinder PROXXON FBS 230/E; grit size P40, speed 5.000/min). The resulting bleeding of small blood vessels or capillaries or the resulting exudation is treated with one of these preparations.

After a 15-minute observation period, the left lobe was replacedBack to the original position of the abdominal cavity. If hemostasis is achieved, the abdomen is closed and the omentum is removed: (2/0). In 2-level manner, as broken-line sutures2/0 the muscle and skin incisions were sutured separately.

After 24 hours, the animals were anesthetized by an excess of sodium pentobarbital (about 320mg i.v./animal). Necropsy was performed after the pain-free site was sacrificed. The abdomen was visually inspected for the presence of blood and/or blood clots resulting from the rebleeding. If present, the weight is determined by absorbing blood and/or blood clots with a pre-weighed amount of surgical rag. If hemostasis was not achieved, animals were sacrificed by excess sodium pentobarbital (approximately 320mg i.v./animal) and only the initial endpoint was evaluated.

As a result:

the objective of this study was to evaluate hemostatic properties of formulations #334-77, #334-77-1, and # 334-77-4). Two extreme hemostasis models were used: (1) heparinized rabbit liver resection and (2) liver surface model.

After application of the formulation #334-77 powder to a bleeding wound, it was found that pressing the formulation against the wound surface helped stop bleeding. It is difficult to apply this pressure with dry surgical latex gloves because the powder is more adhesive to the glove than to the wound. But more easily applied with wet gloves. The formulation forms a sealing membrane when in contact with the moisture of the blood. After coating, in many cases a haemostatic effect can be achieved, even in the extreme models used in this example. Complete hemostasis is difficult by simply applying more formulation #334-77 if complete hemostasis is not achieved after the first application and there is bleeding under the cambium. It may be difficult to limit the application of the formulation to only those areas where hemostasis is required, since if not taken into account enough, the formulation powder may fall into and adhere to the abdominal cavity. Thus, it is helpful to properly coat formulations # 334-77.

Instead, formulation #344-77-4 can be easily coated onto large areas of tissue as a constant thickness layer and allowed to exert sufficient pressure to achieve hemostasis. After coating, formulation #344-77-4 with a backing of native collagen pad remained adhered to the liver lobe, acting as an anti-bleeding agent and glue, adhering the pad to the wound and the liver capsule tube. Such a biodegradable backing may improve the hemostatic efficiency of the powder composition. The biodegradable backing may also impart flexibility to the formulation, allowing the formulation to shrink over the edges of the resection surface during application. Two animals were treated with this formulation, one with a surface model and the other with an excision model. Rapid hemostasis was achieved in both models. Only treated surface model animals survived with no bleeding 24h post-surgery. After 24h, the wool-like collagen was still at the coating site. Treated resection model animals bleed overnight to death and the fleece falls off. The difference between the two experiments was that in the first animal model, wool-like collagen was pressed onto the wound in the dry state, and in the second animal model, pressure was applied with a wet gauze wipe. The results are shown in Table 15.

Claims

1. A dry solid sealant matrix composition, the composition comprising:

a first crosslinkable component;

a hydrogel-forming component;

wherein the combined first and second cross-linkable components are present at a concentration of 5% to 75% by weight of the total composition and the hydrogel-forming component is present at a concentration of 25% to 95% by weight of the total composition; and

wherein the first and second cross-linkable components cross-link under conditions capable of reacting to form a porous matrix having voids, and wherein the hydrogel-forming component is capable of hydrating to form a hydrogel to fill at least some of the voids.

2. The composition of claim 1 wherein the first crosslinkable component comprises a multi-nucleophilic polyalkylene oxide having m nucleophilic groups and the second crosslinkable component comprises a multi-electrophilic polyalkylene oxide having n electrophilic groups, wherein m and n are each greater than or equal to 2, and wherein m + n is greater than or equal to 5.

3. The composition of claim 2, wherein the multi-nucleophilic polyalkylene oxide or the multi-electrophilic polyalkylene oxide, or both, is polyethylene glycol or a derivative thereof.

4. The composition of claim 1, wherein the hydrogel-forming component comprises gelatin and the hydrogel will absorb water when released to a moist tissue target site and is capable of hydrating to form a fragmented biocompatible hydrogel, wherein the hydrogel comprises subunits ranging in size from 0.01mm to 5mm when fully hydrated and has an equilibrium swelling ranging from 400% to 5000%.

5. The composition of claim 1, wherein said first crosslinkable component comprises a plurality of nucleophilic groups and is in the form of a powder, wherein said second crosslinkable component comprises a plurality of electrophilic groups and is in the form of a powder, wherein said hydrogel-forming component is in the form of a powder, and wherein said first and second crosslinkable components are capable of substantially immediate crosslinking under conditions capable of reacting.

6. The composition of claim 1, wherein the first cross-linkable component added to the second cross-linkable component provides a combined cross-linkable component composition and the first cross-linkable component or the second cross-linkable component is present at a concentration of from 0.5% to 20% by weight of the combined cross-linkable component composition.

7. The composition of claim 1, wherein the weight ratio of said first cross-linkable component to said second cross-linkable component is 45% to 55%.

8. The composition of claim 1, wherein the weight ratio between the first and second cross-linkable components and the hydrogel forming component is in the range of 10% to 30% w/w.

9. The composition of claim 1, further comprising a polysaccharide or a protein.

10. The composition of claim 1, further comprising a polysaccharide, wherein the polysaccharide is selected from the group consisting of hyaluronic acid, chitin, chondroitin sulfate a, chondroitin sulfate B, chondroitin sulfate C, keratin sulfate, heparin, and derivatives thereof.

11. The composition of claim 1, further comprising a protein, wherein the protein is collagen or a derivative thereof.

12. The composition of claim 1, wherein said first cross-linkable component, said second cross-linkable component and said hydrogel forming component are in the form of a mixed powder which is immobilized on the surface of a collagen sponge comprising native collagen fibrils.

13. The composition of claim 1, further comprising an active agent.

14. The composition of claim 13, wherein the active agent comprises thrombin.

15. A kit for achieving hemostasis or other fluid inhibition in an in vivo environment, the kit comprising:

a container; and

a first crosslinkable component comprising a plurality of nucleophilic groups, said first crosslinkable component being in powder form;

a hydrogel-forming component in powder form;

wherein the first and second cross-linkable components are capable of substantially immediate cross-linking under conditions capable of reaction to form a porous matrix having voids, and wherein the hydrogel-forming component is capable of hydrating to form a hydrogel to fill at least some of the voids.