JP2002518135A - Carbodiimide cross-linked albumin for bioadhesive surgical sealants and implantable devices - Google Patents

Abstract

  (57) [Summary] The present invention provides methods and products related to protein-based bioadhesives and surgical sealants, as well as implantable devices and prostheses for drug delivery. In particular, the invention relates to albumin-based methods and products. The methods and products of the present invention are useful for adhering biological tissues to each other or to prosthetic devices, sealing fluid or gas leaks in biological tissues, or preparing bioerodible devices. is there. The methods and products of the present invention are particularly useful for cardiovascular and pulmonary applications.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is related to US patent application Ser. No. 60 / 090,609 (June 23, 1998).
, The disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION [0002] The present invention is directed to coupling tissue to another tissue and / or prosthetic device; to seal incisions, perforations, and / or fluid or gas leaks in tissue; or to drug delivery or prostheses. For use in vitro or in vivo during an experimental or surgical procedure to form an implantable device for or to form a coating on an inert device for biocompatibility and other functions The development and use of carbodiimide cross-linked proteins.

BACKGROUND OF THE INVENTION [0003] Biomedical researchers have developed a wide range of in vitro or in vivo experimental or surgical procedures, including the implantation of devices such as prostheses for drug delivery systems. Various attempts have been made to develop methods and products for joining tissue to tissue and / or tissue to prosthetic devices. In particular, researchers have sought to develop safe and efficient means to join or seal tissue that has been cut, torn, or pierced as a result of trauma and / or surgical procedures.

[0004] Historically, suturing and stapling have been the most common methods for joining or sealing tissue. However, there are a number of disadvantages to these methods. For example, depending on the nature of the tissue and the degree of tissue damage, suturing and stapling may require significant skill and / or long surgical times. More importantly, their results often result in gaps between the sutures or staples, tearing of delicate tissue around the sutures or staples, and bonding that can result in leakage of biological fluids or bacterial infection Is not satisfactory due to the potential for progressive weakening of

For these and other reasons, safer and more efficient methods for joining or sealing tissue have been considered. In particular, bioadhesive or surgical sealants have been developed that adhere to tissue surfaces and form a bond between tissues until healing is complete.

For example, fibrin-based adhesives have received considerable care in this area (eg, Epstein et al. (1986) Ann. Otol. Rhi.
nol. Larrygol. 95: 40-45; Siedentop et al. (1983)
), Laryngoscope 93: 1310-1313). However, fibrin glues have low mechanical (bonding) strength, as well as lengthy set times, which limit their utility.

Various approaches have been developed to utilize collagen (the major connective tissue protein in animals) as a bioadhesive or surgical sealant. However, success has generally been limited by various disadvantages, including the following: exothermic crosslinking / polymerization reactions, long reaction times, and the presence of oxygen or physiological Reactions that are ineffective in the pH range (eg, Lee et al. (
1970), Addition in Biological Systems
, R .; S. Manly, Academic Press, New York,
(See Chapter 17). In addition, some prior art collagen-based adhesives contain toxic substances or by-products that make them unsuitable for in vivo biomedical use (eg, Bounocore (1970) Adhesion in Bi).
technical Systems, R .; S. Manly, Academic
Press, New York, Chapter 15, and US Patent 3,453,22.
No. 2). More recent approaches to using collagen-based bioadhesives can be found, for example, in: US Pat. No. 5,219,895, US Pat. No. 5,354,336, US Pat. No. 5,874. , 537, and PC
T publication WO 97/42986.

[0008] Non-biological adhesives such as cyanoacrylates (eg, isobutyl-2-cyanoacrylate) have also been tested for their potential adhesive properties. However, these materials are difficult for in vivo applications. This is because cyanoacrylate adhesives require application in the dry area. further,
The cured solid form of the cyanoacrylate produced with this approach is not resorbable, thus limiting its usefulness for in vivo applications. In addition, adverse tissue effects have been reported as a result of the exothermic nature of the polymerization reaction.

[0009] Bioadhesives based on gelatin-resorcinol cross-linked with formaldehyde have also been developed (eg, Koehlein et al. (1969), Surge).
ry 66: 377-382, and Bellotto et al. (1992), Sur.
g. Gynecol. Obstet. 174: 221-224) have similar disadvantages. Of particular relevance to such products are the dangerous properties of formaldehyde and resorcinol. In addition, gelatin must be applied at temperatures significantly higher than human body temperature, and requires significant care during mixing and application to achieve successful results.

[0010] Carbodiimides have been utilized as crosslinking reagents in various situations. For example, carbodiimides have been used as cross-linking reagents with polysaccharides, hyaluronic acid, and pectin (eg, Tomihata et al. (1997), J. Bio.
med. Mater. Res. 37: 243-251). further,
Carbodiimide is used as an adhesive for gelatin and poly (L-glutamic acid) (
(PLGA) was used to crosslink aqueous mixtures (eg, Otani et al. (
1996); Biomed. Mater. Res. 31: 157-166)
checking). However, one of the more disadvantageous results was the potential toxic effect of the derivatives and residual products of the reaction.

At present, there are few adhesive compositions that have utility in surgical and biomedical applications, especially those involving soft tissues. Therefore, there remains a need for safe and effective compositions for use as bioadhesives, surgical sealants, and / or implantable devices.

SUMMARY OF THE INVENTION The present invention is directed to implants for adhering biological tissue and / or prosthetic devices, sealing fluid and / or gas leaks in biological tissue, and for drug delivery. Provided are methods and compositions that are useful for preparing (including bio-erodable implants).

[0013] In one aspect, the invention encompasses proteins and cross-linked preparations that are useful for cross-linking biological tissue and / or prosthetic devices. The compositions of the present invention include a protein solution that is suitable for tissue crosslinking.

[0014] In a preferred embodiment, the protein preparation of the invention comprises an albumin preparation. In one embodiment, the albumin is bovine serum albumin (BSA)
). In a preferred embodiment, the albumin is human albumin.
Preferably, the albumin preparation is provided at a pH between 5.0 and 8.0, more preferably at a pH between 5.5 and 7.5.

[0015] In one embodiment, the albumin preparation comprises a protein that is a naturally occurring albumin protein, a recombinant albumin protein, a major fragment of the albumin protein, or a chemically modified albumin. In a preferred embodiment, the albumin is a mammalian albumin protein or a major fragment of a mammalian albumin protein. Most preferably, the albumin protein comprises an amino acid sequence of at least 100 amino acid residues having at least 60% homology to the amino acid sequence of human albumin.

In an alternative embodiment, the albumin enhances one or more physical properties such as solubility, reactivity with the carbodiimide crosslinker, stability, viscosity in aqueous solution, and immunocompatibility. Contains an amino acid sequence that has been recombinantly modified with respect to the naturally occurring albumin sequence. In a further embodiment, the albumin preparation comprises:
Includes additives to increase or decrease the rate of the crosslinking reaction or to promote the interaction between the protein solution and the tissue or material at the site of application. For example,
Albumin can be provided with one or more surfactants, lipids, and / or fatty acids.

In any one of the above embodiments, the albumin is a polysaccharide (eg, glycosaminoglycan, dextran, hyaluronic acid, chondroitin sulfate, heparan sulfate), polyether (eg, polyethylene glycol, polypropylene glycol, Polybutylene glycol), polyesters (eg, polylactic acid, polyglycolic acid, polysalicylic acid), and aliphatic, alicyclic, and aromatic agents;
It can be covalently linked to a molecule selected from the group consisting of perfluorinating or non-perfluorinating agents, and acylating or sulfonating agents. Further, the albumin preparation may include chlorinated, fluorinated, brominated, or iodinated albumin protein.

Preferably, the albumin protein is provided in an aqueous solution at a concentration of about 10-50%, more preferably about 20-40%, most preferably 35-40% by weight. In an alternative embodiment, the albumin protein is provided in a solution of a secondary or tertiary alcohol. In a preferred embodiment, the alcohol solution comprises isopropyl alcohol (IPA) or isobutyl alcohol (IBA). In a most preferred embodiment, albumin is dissolved in a 20% IPA solution or an 8% IBA solution.

In a further embodiment, the protein modifies its properties in a cross-linking reaction or promotes its interaction with a tissue or substrate at the site of application, as described and exemplified in the description and examples below. To be derivatized.

In a preferred embodiment, the crosslinker preparation comprises a carbodiimide having the general structure R ₁ —N ＝ C ＝ N—R ₂ , or a halogen, tertiary amino, ester, keto substituent or other degradable group. Wherein R ₁ and R ₂ are independently linear or branched, saturated or unsaturated, alkyl, alkenyl,
It is selected from the group consisting of an aryl, aralkyl, or aralkenyl group. More preferably, the carbodiimide is ethyldimethylaminopropylcarbodiimide (EDC-HCl); 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide; 1,3-di-p-tolylcarbodiimide; 1,3-diisopropylcarbodiimide 1,3-dicyclohexcarbodiimide; 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meth-p-toluenesulfonate; polycarbodiimide; 1-tert-butyl-3-ethylcarbodiimide;
1,3-dicyclohexylcarbodiimide; 1,3-bis (trimethylsilyl)
Carbodiimide; 1,3-di-tert-butylcarbodiimide; 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methoxide; and 1- (
3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. Most preferably, the carbodiimide is EDC.HC
l. In a preferred embodiment, the carbodiimide preparation comprises 10% and 20%.
% EDC.HCl, most preferably 15% EDC.HCl.

In another aspect, the invention provides a method for producing a cross-linked albumin composition for use in a bioadhesive surgical sealant or implantable device, the method comprising the steps of: Providing; providing a carbodiimide preparation; and mixing the albumin preparation and the carbodiimide preparation under conditions that allow albumin to crosslink. In a preferred embodiment, the albumin and carbodiimide preparations are mixed in one of the following ratios: between about 1: 1 and 1: 100, between about 1: 5 and 1:50, Between about 1:10 and 1:20, between about 100: 1 and 1: 1, between about 36: 1 and 4: 1, and about 18: 1.

[0022] A preferred method of the present invention is to perform incisions, perforations,
And / or contacting the tissue with an effective amount of an albumin preparation and a carbodiimide preparation under conditions that promote crosslinking of the albumin preparation to the tissue. , Thereby the incision,
Perforation or sealing off leakage of fluid or gas. Such methods are particularly useful for surgical procedures such as cardiovascular, lung, kidney, and liver surgery.

According to the method of the present invention, a leak of fluid or gas may be blocked by crosslinking the tissue surrounding the leak. Alternatively, the crosslinked gel of the present invention may seal off the leak by physically occluding it without crosslinking the tissue surrounding the leak.

In one or more embodiments, the albumin and carbodiimide preparation are mixed prior to applying it to a tissue location. Alternatively, the mixing step is performed at the tissue location. In an alternative embodiment, the accessory molecule
ecule) is provided and mixed with the albumin and carbodiimide preparations. The accessory molecule is preferably selected from the group consisting of viscosity enhancers, crosslinkers, buffers, hormones, growth factors, antibiotics, surfactants, lipids, fatty acids, and anti-inflammatory agents.

In a preferred embodiment, the primer solution is applied to the tissue or prosthetic device before adding the mixture of albumin and crosslinker. The primer solution can be a saline solution. Preferably, the primer solution is an albumin solution. More preferably, the primer solution is the same as the albumin preparation used in the crosslinking reaction, and most preferably, the primer solution is a dilute solution of the albumin preparation.

In any one of the above embodiments, the albumin and carbodiimide preparation preferably has a pH between 5.0 and 8.0, more preferably a pH between 5.5 and 7.5. Between about 6.0 and 7.0, most preferably between 6.0 and 7.0. However, the pH of the preparation is preferably adapted to the desired rate of crosslinking. Rapid cross-linking is useful in providing the cardiovascular system to quickly block blood leaks. Rapid crosslinking involving EDC.HCl is obtained at pH 5.0-5.5. Alternatively, for pulmonary applications, rapid crosslinking is less critical and the preparation can be provided at a higher pH. The rate of the crosslinking reaction is also described in the description below, and as illustrated by the following examples, additives,
It can be modified using derivatized albumin or by changing the ratio or concentration of albumin to carbodiimide.

In a further embodiment, the preparation can be provided in a hydrophobic solution. Preferably, the solution does not interfere with the crosslinking reaction. Preferred solutions include secondary and tertiary alcohols (including IPA and IBA). In a preferred application, a 30% solution of BSA is made using 20% IPA or 8% IBA.

In another embodiment of the present invention, the protein and crosslinker may be provided as a dry powder. The protein and crosslinker powder are preferably mixed prior to applying them to a tissue location. In a most preferred embodiment, the protein and crosslinker absorb body fluid from the site of application, and the body fluid absorption is
Initiate the crosslinking reaction.

Alternatively, additional fluids can be provided at the site of application, as needed. In a further embodiment, the site of application is primed with a solution of saline or dilute protein prior to applying the mixture of dry protein and crosslinker. The primer solution is then absorbed by the dry mixture, thereby initiating a crosslinking reaction.

In another aspect, the invention relates to synthetic materials such as vascular grafts (eg, PTFE)
Materials or a biological implant (eg, a polyethylene material).

[0031] In a related aspect, the invention provides a method for bonding a first biological tissue to a second tissue and / or a prosthetic device, the method comprising the first tissue and the first tissue. The two tissues or prosthetic devices are contacted with a mixture of an albumin preparation and a carbodiimide preparation under conditions that promote crosslinking of the albumin preparations to the first and second tissues or prosthetic devices. Process.

In a further aspect of the present invention, there is provided a method for forming an implantable device. The method comprises the steps of providing an albumin preparation, providing a carbodiimide preparation, providing a mold, and cross-linking the albumin preparation and the carbodiimide preparation in the form with albumin. And mixing under conditions that allow for

The products of the present invention also include a bioadhesive surgical sealant or implantable device made according to any of the methods described above.

In another aspect, the invention provides a kit for making a bioadhesive surgical sealant or implantable device, the kit comprising, in separate containers, an albumin preparation and a carbodiimide preparation. including. In a preferred embodiment, the kit comprises an accessory molecule, preferably from the group consisting of viscosity enhancers, crosslinkers, buffers, hormones, growth factors, antibiotics, anti-inflammatory agents, hydrophobicity enhancers, and surfactants. It further includes a molecule to be selected.

In another embodiment of the kit, the albumin preparation and the carbodiimide preparation are provided in a dual delivery device that delivers the preparation in either a predetermined or adjustable ratio.

A detailed description of certain preferred embodiments of the present invention is provided below. Other embodiments of the present invention will be apparent from a consideration of the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION In general, the present invention provides methods and products related to protein-based bioadhesive and surgical sealants, and implantable devices for drug delivery and prostheses. In a preferred embodiment, the invention relates to albumin-based methods and products. However, it should be understood that other proteins may be substituted in the following description of the albumin-based methods and products. The compositions of the present invention are formed by crosslinking albumin by reacting it with carbodiimide under certain conditions. The general reaction proceeds in two steps (see, eg, Damink et al., 1996). First, the free carboxyl group of the albumin molecule (eg, the carboxyl terminus or the side chain of a glutamic or aspartic acid residue) attacks carbodiimide,
Forming one of the following two intermediates:

[0038]

Embedded image The free amino group of the same or another albumin molecule (eg, the amino terminus, or the side chain of a lysine or arginine residue) then attacks one of these intermediates, causing cross-linked albumin and substituted forms. Urea is produced as a by-product, as follows:

[0039]

Embedded image This reaction is performed in vitro in a laboratory or manufacturing facility to produce a product containing cross-linked albumin as a bioadhesive or surgical sealant material, or as part of an implantable drug delivery device or prosthesis. obtain. Alternatively, in vitro during an experimental or surgical procedure to couple one tissue to another and / or to a prosthetic device, or to seal incisions, perforations, and / or fluid or gas leaks in the tissue. Can be done.
Optionally, and as described more fully below, the albumin starting material can be a chemically modified form of albumin, wherein the carbodiimide crosslinker is
The crosslinker may include other functional or reactive groups (including additional carbodiimide groups), such as poly (carbodiimide), additional non-carbodiimide crosslinkers may be utilized, and / or various accessory molecules may be used. Can be added to modify the course of the crosslinking reaction or the characteristics of the end product.

The method of the present invention comprises the steps of providing a suitable albumin preparation and a suitable carbodiimide and, optionally, ancillary molecules, and separating these components from the intermolecular (and molecular) nature of the carbodiimide with the albumin molecule. Inner) a step of mixing under conditions that allow for promoting crosslinking. Suitable albumin preparations, carbodiimide crosslinkers, reaction conditions, accessory molecules, and utilities are separately described in detail below. However, first, in order to more clearly and concisely describe the subject matter which applicants consider as their invention, the following definitions will be used in the following detailed description and the appended claims. Provided for terms.

As used herein, the term “albumin” refers to any mammalian protein, including allelic variants, as well as Minghetti et al. (1986)
J.). Biol. Chem. 261: 6747-6775, at least 60% homology to the human albumin sequence disclosed, preferably at least 70%
Alternatively, it refers to a modified albumin and albumin fragment comprising a sequence of at least 100 amino acid residues having 80% homology.

As used herein, the term “homology” refers to the BLAST amino acid sequence comparison program (default parameters for appropriate programs)
XBLAST (Altschul et al., (1990), J. Mol. Biol. 2).
15: 403-10). ) Means “homology” or “similarity” (see http://www.ncbi.nlm.nih.gov). Alternatively, the homology is determined by Myers and Miller, CABI.
Calculated using the OS (1989) algorithm, which is incorporated into the ALIGN program (version 2.0). Exemplary parameters for use in comparing amino acid sequences are a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4.

1. Albumin Preparation The bioadhesives, surgical sealants, and implantable devices of the present invention include a cross-linked form of the protein albumin. Preferably, the albumin is of mammalian origin, but other sources of albumin may also be used. It is believed that most albumins are easily crosslinked according to the method of the present invention. However, low immunogenic albumin is preferred for in vivo applications. Thus, for use in humans, the albumin is preferably human albumin. Bovine serum albumin (BSA) can also be used in humans and is more readily available. Alternatively, albumin can be prepared using methods known in the art.
It can be a recombinant albumin isolated from a cell expressing the recombinant albumin gene. When produced recombinantly for use in humans, the albumin gene is preferably a human or bovine gene. However, other species or biosynthetic variants may be used. Major fragments of albumin (comprising at least 100 residues of the albumin sequence), either produced by partial proteolysis or by recombinant means, can also be used in place of intact albumin. Alternatively, useful fragments comprise at least 50 fragments of the albumin sequence.
Residues, and more preferably at least 75 residues. Finally, a mixture of different forms of albumin (eg, human, bovine, recombinant, fragmented) can also be used.

[0044] Albumin can be purified directly from tissues or cells using methods well known in the art (eg, Cohn et al. (1946) J. Amer. Che.
m. Soc. 68: 459; Cohn et al. (1947) J. Am. Amer. Chem.
Soc. 69: 1753; Chen (1967) J. Am. Biol. Chem. 24
2: 173). Alternatively, albumin is purchased from a commercial manufacturer. For example, albumin preparations from various mammalian and avian species are available in Sigma Chemical C in solution or lyophilized powder form.
company (St. Louis, MO). A preferred commercial albumin preparation is a 30% human albumin solution (eg, Sigma Catalog No. A9080) or a 30% BSA solution (eg, Sigma Catalog No. A8327).

In a preferred embodiment, the albumin comprises 10 to 50% by weight, preferably 2%.
It is provided as an aqueous solution of 0-50%, and most preferably about 35-40% by weight of albumin. As described more fully below, lower concentrations of albumin may be used when a viscosity enhancer is added. Preferably, the solution is substantially purified to remove contaminants (eg, immunogens or proteases) that disrupt or interfere with the bioadhesive or sealant properties of the cross-linked albumin. On the other hand, the presence of many other proteins, such as collagen, elastin, laminin, fibrin, and thrombin, can be tolerated.

Alternatively, albumin can be provided as a dry powder. In such embodiments, the dried albumin is solubilized at the site of administration. Thus, the body fluid (eg, blood) present at the site of administration may be sufficient to solubilize the protein. Alternatively, additional fluid can be provided with the dried albumin. The crosslinks can also be provided as a dry powder solubilized at the site of administration. In a preferred embodiment, the dry protein and the crosslinker are mixed prior to administration. In a most preferred embodiment, a wetting reagent is added to the mixture of protein and crosslinker to increase liquid absorption. Preferably, the wetting reagent absorbs water from available body fluids and accelerates the solubilization of the protein and crosslinker.

[0047] Albumin can be modified or derivatized to increase viscosity. For example,
The viscosity of albumin is relatively large (10-100 kD) for substantially linear molecules (eg, polysaccharides (eg, glycosaminoglycans, dextran, hyaluronic acid, chondroitin sulfate, heparan sulfate), polyethers (eg, , Polyethylene glycol, polypropylene glycol, polybutylene glycol)
, Polyesters such as polylactic acid, polyglycolic acid, polysalicylic acid, and aliphatic, cycloaliphatic or aromatic acylating or sulfonating agents). Preferred acylating agents include aliphatic, cycloaliphatic and aromatic anhydrides or acid halides, especially the anhydrides of dicarboxylic acids. Non-limiting examples of these include glutaric anhydride, succinic anhydride, lauric anhydride, diglycolic anhydride, methacrylic anhydride, phthalic anhydride, succinyl chloride, glutaryl chloride, and lauroyl chloride. Acylating agents also include various substituents and second functionalities (eg, aliphatic, cycloaliphatic, aromatic and halogen substituents, and amino, carboxy, keto, ester, epoxy, and cyano functions). Similarly, preferred sulfonating agents useful in the present invention include aliphatic, cycloaliphatic and aromatic sulfates and sulfonyl halides, which also include, as described above, Various substituents and second functional groups may be included.

[0048] Albumin can also be modified or derivatized to increase its hydrophobicity to facilitate its interaction with hydrophobic tissue or prosthetic material. In particular, albumin is a branched or linear alkyl, alkenyl, or aromatic reagent (
It can be derivatized with long-chain alkyl or alkenyl and alkyl aldehydes or carboxylic acids, including, for example, octyl aldehyde or dodecyl aldehyde or octyl carboxylic acid or dodecyl carboxylic acid.

Finally, in order to increase its ability to interact with its hydrophobic and fluorine-containing prosthetic materials (eg, PTFE-containing materials), albumin or modified albumin is produced by standard methods well known in the art. , Halogenated, preferably fluorinated. For example, albumin can be derivatized with a polyfluorodicarboxylic anhydride (eg, hexafluoroglutaric anhydride), a polyfluoroalkyl ether (eg, perfluoroalkyl glycidyl ether), or other halogen-containing reagent.

Alternatively, the recombinant albumin may have one or more amino acid residues inserted, deleted or substituted to increase the viscosity of the albumin, alter the hydrophobicity of the protein, or more for derivatization. Or can be produced by standard techniques of site-directed mutagenesis to provide more free carboxyl or amine groups for the crosslinking reaction. As a general matter, under the conditions used, albumin contains an appropriate (and roughly equal) number of free carboxyl and free amino groups for crosslinking. Thus, alteration of the albumin sequence may involve larger and / or more hydrophobic residues and / or charged residues that may be involved by non-covalent intramolecular binding by charge or hydrophobic interactions. It is most useful for increasing the viscosity of proteins by substituting small or hydrophobic residues (eg, glycine, alanine). Alternatively, two forms of modified albumin may be produced in which the free carboxyl and amine contents of the albumin are substantially different. Such a form,
They tend to form intermolecular crosslinks rather than intramolecular crosslinks, and thus have a greater degree of viscosity when mixed in roughly equal amounts.

(II. Carbodiimide Crosslinking Agent) Carbodiimide is a cross-linking reagent having the following general formula: R ₁ —N ＝ C ＝ N—R ₂ , wherein R ₁ and R ₂ are strong nucleophiles If it does not contain, it can be essentially any chemical group. Carbodiimides are highly reactive and the presence of a nucleophilic group on either R ₁ or R ₂ has been destabilized due to intermolecular (or intramolecular) reactions between the carbodiimide molecules. In a preferred embodiment, R ₁ and R ₂ are linear or branched saturated or unsaturated alkyl, alkenyl, aryl, halogen, tertiary amine, ester, keto, or other substituents. Group, an aralkyl group, or an aralkenyl group, or a variant thereof. Further, one or both of R ₁ and R ₂ may include an additional carbodiimide group, such that the crosslinker is a polycarbodiimide.

Preferably, carbodiimides which are water-soluble are used and react with albumin under physiological conditions. However, a suspension of a water-insoluble carbodiimide (eg, ethyldimethylaminopropyl carbodiimide (EDC)) may also be useful for albumin crosslinking when well dispersed during the crosslinking reaction. By appropriate selection of the R group, the solubility and reactivity of the carbodiimide can be varied. Furthermore, the choice of the R group affects the immunogenicity and toxicity of the crosslinker, and its ability to interact with the albumin molecule. In another embodiment, the R group can be selected to have an additional bridging group (eg, a photoactivated bridging group).

[0053] Preferably, the carbodiimide is provided as a solution or suspension. However, in some embodiments, the carbodiimide may be provided in a dry form (eg, a powder). The dried carbodiimide is either solubilized or suspended prior to administration to the tissue, or solubilized or suspended by the body fluid present at the site of administration.

One preferred crosslinking agent is ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC.HCl). The cross-linking agent is water-soluble and effectively cross-links albumin under conditions that are suitable for binding tissue in vivo and / or sealing surgical leakage.

Another example of a preferred carbodiimide is 1- (3-dimethylaminopropyl)
-3-Ethylcarbodiimide (Aldrich Catalog No. 42433-1);
1,3-di-p-tolylcarbodiimide (Aldrich catalog number D219
80-0); 1,3-diisopropylcarbodiimide (Aldrich cat. No. D12540-7); 1,3-dicyclohexylcarbodiimide (Aldr
ich catalog number D8000-2); 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meth-p-toluenesulfonate (Aldric)
h catalog number C10640-2); polycarbodiimide (Aldrich catalog number 45875-9); 1-tert-butyl-3-ethylcarbodiimide (Aldrich catalog number 42639-3); 1,3-dicyclohexylcarbodiimide (Aldrich catalog number 37911). -5); 1,3-bis (trimethylsilyl) carbodiimide (Aldrich Catalog No. 34433)
-8); 1,3-di-tert-butylcarbodiimide (Aldrich Cat. No. 23556-3); 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide methiodide (Aldrich Cat. No. 16534-4); and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (A
ldrich catalog number 16146-2) (all Aldrich Chemi
cal Company, Milwaukee, Wis.).

In another preferred embodiment, the crosslinking agent is a polyethylene glycol (PEG) based aqueous solution of the general formula: R ₁ O-PEG-R ₂ -N = C = NR ₃ -PEG-OR ₄ Carbodiimide, wherein R _{1 to} R ₄ are independently selected as described above. For example, they can be -NCOs (isocyanates) that make them bifunctional PEGs, thereby producing poly (PEG carbodiimides). R ₂ and R ₃ may also include the hydrolytically or enzymatically cleavable group, as a result, cross-linked products are biodegradable through cleavage of crosslinking. PEG-based carbodiimides are synthesized by reacting an alkoxy PEG isocyanate in the presence of a suitable catalyst.

R ₁ O-PEG-R ₂ -NCO + R ₄ O-PEG-R ₃ -NCO → R ₁ O-PEG-R ₂ -N = C = NR ₃ -PEG-OR ₄ Useful Catalyst Examples are phosphorene, phosphorane (p
phospholane), a phosphorene oxide or a phosphorene sulfide, a phosphorane oxide or a phosphorane sulfide.

In another preferred embodiment, the crosslinking agent (eg, EDC-HCl) is
It may be provided in an aqueous solution of an inert polymeric material. Preferably, the crosslinker solution and albumin solution have similar viscosities and volumes to facilitate efficient mixing and delivery. Examples of inert polymers include poly (vinyl alcohol)
, PEG, non-ionic surfactants (including PEG-based surfactants such as pluronic polymers), poly (saccharides) and other inert polymers.

(III. Crosslinking Reaction Conditions) The carbodiimide crosslinking agent of the present invention can react with albumin (and other proteins) spontaneously to form a crosslink between amino acid side chains of the protein. Thus, no initiator of the crosslinking reaction is required. Importantly, this reaction can occur under conditions of pH and temperature compatible with in vivo applications.

The carbodiimide crosslinking reaction of the present invention is very pH sensitive and is preferably carried out at a pH between 5 and 7, more preferably between pH 5 and 6. At a pH value less than 5, unmodified albumin can precipitate and a pH greater than 8
By value, the rate of the crosslinking reaction is greatly reduced. Physiological pH is around 7.
0-7.4, so the reaction mixture may be allowed to occur at physiological pH, or the reaction mixture may be slightly acidified by the addition of acid accessory molecules.

The reaction can be performed at room temperature for in vitro use and at body temperature for in vivo applications.

The molar ratio of carbodiimide to albumin significantly affects both the rate of the crosslinking reaction and the characteristics of the final crosslinked product. That is, a higher ratio of carbodiimide to albumin results in a faster reaction and a higher cross-linked product.

The bioadhesives, surgical sealants and implantable devices of the present invention preferably comprise carbodiimide with albumin and 1 to 10 per mole of albumin monomer.
It is prepared by mixing in a molar ratio of carbodiimide between 0 moles. In a preferred embodiment, the carbodiimide and albumin are approximately 36: 1.
At a molar ratio of In a more preferred embodiment, this ratio is approximately 1
8: 1. In some embodiments, sufficient crosslinking can be achieved using a carbodiimide: albumin ratio of approximately 4: 1. But about 0.4
A ratio of 1: does not provide sufficient crosslinking for most applications of the present invention. In an exemplary embodiment of the invention, consider a bioadhesive or surgical sealant produced using carbodiimide. EDC · HCl is approximately 191.7
While the albumin monomer has an average molecular weight of about 69,293. Thus, a bioadhesive prepared by mixing EDC.HCl and BSA in a molar ratio of approximately 36: 1 yields (1 mole x 69.293 grams /
(Mol) 69.293 g of BSA approximately (36 mol × 191.7 g / mol) 6901.2 g EDC.HCl, or approximately 1:10 weight EDC.H
Cl: corresponds to the mixture with albumin. Similar calculations can, of course, be used for other carbodiimide reagents to determine the appropriate weight: weight ratio based on the desired molar ratio.

Thus, for example, EDC · HCl cross-linked albumin preparations can be used in
: 100, preferably about 1: 5 to 1:50, and most preferably about 1:10
EDC.HCl: albumin can be produced in a weight: weight ratio varying from 1:20. At a 1: 1 ratio, the reaction proceeds very quickly and the product
It may cure before the application is finished. The resulting product is extremely robust and relatively non-plastic or non-flexible. At a ratio of 1:20, the reaction is much slower and the product can be shaped and shaped while the reaction is in progress. The resulting product is weaker but more plastic or flexible. 1: 1
A ratio of 00 is considered useful in some applications, but a ratio of 1: 100
Ratios as low as 0 are expected to be ineffective due to insufficient crosslinking.

For example, for applications involving sealing of fluid and gas leaks in lung tissue, 1: 5
A weight: weight ratio of EDC.HCl: albumin of at present is presently preferred. For applications involving larger films to seal wound openings, lower EDC HC
A 1 ratio, perhaps 1:10 to 1:20, may be appropriate, but this produces a weaker and gel-like sealant. A 1:20 ratio is predicted to be too weak for cardiovascular applications, while a 1: 1 ratio is predicted to be very inelastic.

As described above, in a preferred embodiment, both albumin and carbodiimide are provided in an aqueous preparation. Alternatively, however, an aqueous preparation of albumin can be mixed with a suspension of insoluble carbodiimide. Because the two components (albumin and carbodiimide) begin to crosslink upon mixing, and because this reaction can be very rapid for some formulations, mixing them immediately prior to application, or making them bioadhesive It can be important to mix at the site where it is desired to form a seal or surgical seal. Furthermore, since carbodiimides are not stable in aqueous solutions for long periods of time, they can be prepared by dissolving carbodiimide powder or lyophilized carbodiimide in aqueous solution just before mixing with albumin. Alternatively, the carbodiimide can be provided in an inert, water-miscible, biocompatible organic solvent. Thus, dual delivery devices, having separate compartments to hold the albumin preparation and carbodiimide crosslinker prior to dispensing and mixing, can be particularly useful. Thus, in one preferred method, a dual barrel syringe (dou) that simultaneously dispenses and mixes these compartments
ble-barreled syringes) are used. Such a dual barrel syringe can be very convenient for in vivo applications where a bioadhesive or surgical sealant is applied to the site of tissue injury or incision. In one embodiment, a dual barrel syringe comprises a first barrel containing an aqueous solution of albumin and a second barrel containing carbodiimide powder separated from the aqueous solution by a fragile membrane. For the use of such a syringe, the membrane is first broken and the carbodiimide is dissolved in the aqueous solution of the second barrel, and the syringe is then used as described above. In another embodiment, the double barrel comprises a first barrel comprising an aqueous albumin solution at a pH at which a cross-linking reaction can occur (eg, 5.0-6.0), and an aqueous solution adjusted to alkaline pH with water in the solution. A second barrel containing a carbodiimide solution that reduces the conversion of the carbodiimide to a disubstituted urea compound. In this embodiment, the pH and / or buffer system in the two barrels is selected such that upon mixing, the pH of the resulting solution is sufficiently acidic to allow this crosslinking reaction to proceed effectively. It must be. In an alternative embodiment, the single barrel syringe contains an albumin solution separated from the carbodiimide powder by a fragile membrane. This cross-linking reaction is initiated by disruption of the membrane, and the resulting mixture is applied as described above. For surgical use, the two components and the syringe may be provided as a sterile and disposable kit. Alternatively, the two components can be applied as a spray from a device that has separate reservoirs for the two components. Finally, although not preferred, the two components can be applied sequentially. This method has the disadvantage that the components are not thoroughly mixed, and only a thin film of cross-linked albumin can be formed at these interfaces.

IV. Ancillary Molecules In some embodiments, ancillary molecules are added to the reaction. Such accessory molecules may include viscosity enhancers, solubility enhancers, non-carbodiimide crosslinkers, anti-inflammatory agents, hormones, growth factors, antibiotics, buffers and the like.

[0068] In some preferred embodiments, a viscosity enhancer is added to the mixture,
Thus, the concentration of albumin utilized can be reduced. However, the concentration of albumin is preferably at least 10%, and more preferably at least 20%.
%. In a preferred embodiment, the viscosity enhancer is itself crosslinked during the reaction. Viscosity enhancers include substituted or unsubstituted polysaccharides (eg, glycosaminoglycans or heparin sulfate), fibrous proteins (eg, collagen, elastin, fibrin, fibrinogen, thrombin, laminin), or Other compounds that polymerize under physiological conditions or in the presence of the carbodiimides of the invention (eg, polyacids and polyamines)
May be mentioned. Preferred viscosity enhancers include glycosaminoglycans, dextran, hyaluronic acid, collagen, chondroitin sulfate, and elastin.

In some preferred embodiments, accessory molecules are added to alter the rate and / or extent of crosslinking. In general, carboxylic acids can reduce the rate or extent of crosslinking in the first step of the carbodiimide crosslinking reaction by competing with protein carboxyl groups. Similarly, amines can reduce the rate or extent of crosslinking in the second stage of the carbodiimide crosslinking reaction by competing with protein amine groups. However, polycarboxylic acids, polyamines, poly (carboxy / amino) compounds (ie, compounds having multiple carboxyl and amino groups), and mixtures thereof, increase the rate of gel formation by reacting with carbodiimides. Such polycarboxylic acids, polyamines, and / or poly (carboxy / amino) compounds are capable of forming crosslinks with two or more protein molecules and participating in this gel formation with relatively high density of carboxy groups and And / or have an amino group. Thus, such an accessory molecule is
It preferably has a molecular weight of less than 1,000 daltons per carboxy and / or amino group, more preferably less than 500 daltons, and most preferably less than 250 daltons. For example, an accessory molecule having a molecular weight of 2,000 daltons and having four carboxy and / or amino groups has a molecular weight of 500 daltons per carboxy or amino group. Polycarboxylic acids include citric acid and poly (acrylic acid). Polyamines include poly (lysine)
And chitosan.

In an alternative embodiment, the other acid is EDC by lowering the pH
Included to facilitate the mediated protein crosslinking reaction, and other bases
Can be used to slow down this crosslinking reaction. The optimum pH for the carbodiimide crosslinking reaction is between about 5.0 and 6.0. However, the reaction can also be increased at lower pH due to denaturation of the protein at lower pH.

In a further embodiment, the hydrophobicity of the albumin solution is increased by dissolving the albumin in a solution that is more hydrophobic than water. In a preferred embodiment, the albumin is solubilized in a solution containing a secondary or tertiary alcohol. Preferably, the albumin is isopropyl alcohol (
IPA) or isobutyl alcohol (IBA) in solution. Most preferably, a 30% solution of BSA is prepared with 20% IPA or 8% IBA.

Finally, they react with carbodiimide, so that phosphate buffer and Tr
Buffers such as is buffer are not preferred. Currently preferred buffers are inorganic acids such as hydrochloric acid.

V. Utility The crosslinked albumin preparations of the present invention have many applications. For example, they can be utilized as surgical sealants, bioadhesives, hemostats, coating materials, and drug delivery vehicles. When formed into films ex vivo and then implanted, they can also be applied to prevent surgical adhesion by forming a resorbable barrier between the healing tissues. For use as a surgical sealant, crosslinked albumin blocks air and blood leaks in the lungs, blood in the liver, spleen and kidneys, cerebrospinal fluid in the dura mater, and blood in the heart and vasculature It is expected to be particularly useful in this regard.

For cardiovascular applications, the carbodiimide cross-linked albumin must be designed to have sufficient flexibility for pulsatile elongation, but also strong enough to withstand cardiovascular pressure Must have. In a preferred embodiment, the reaction mixture quickly forms a blockade when applied to vascular leaks. Preferably, the crosslinking is such that the reaction mixture is applied to the site of vascular application (eg, a blood vessel).
Occurs before it can slide down or be washed away. Further, cross-linking preferably occurs before the reaction mixture is diluted or washed away by blood or other bodily fluids. In a preferred embodiment, the crosslinked gel forms within 5 minutes of the time the components are mixed and applied to the vascular site. In a most preferred embodiment, the crosslinked gel forms within 30-45 seconds.

Rapid gel formation is also important to prevent excessive blood loss. This is particularly important when the patient (eg, a trauma patient in an emergency) has multiple vascular injury sites. In one embodiment, the reaction mixture may be applied to stop bleeding if the exact source of blood loss has not been identified. In this embodiment, the gel formation time should be slow enough that the reaction mixture is applied over a relatively wide area of bleeding, but before the reaction mixture is diluted or washed out, Should be fast enough to block off.

In pulmonary applications, the gel reaction mixture is used to seal off air or blood leaks in the lungs. In a preferred embodiment, the reaction mixture is designed to take longer to form a gel than the reaction mixture used for vascular applications. Preferably, the reaction mixture can spread beyond the surface of the lung before cross-linking to form a gel. In addition, the reaction mixture can include additives (eg, lipids or surfactants) that help spread the mixture over the lung surface. In pulmonary applications, the mixture does not appear to slide or flush out of the application site. This is because the lungs provide a relatively large and flat area when compared to the surface of blood vessels.

In a preferred embodiment, the primer solution is applied to the tissue before the gel mixture. Preferably, the primer solution is a dilute solution of BSA. More preferably, the primer solution is a dilute solution of the crosslinking reaction mixture without a crosslinking agent. In this embodiment, the primer mixture is hydrophobic. In one embodiment, a hydrophobic derivative of albumin is used. In another embodiment, albumin is mixed with a fatty acid. For example, albumin can be complexed with palmityl anhydride. In a preferred embodiment, the molar ratio of fatty acid to albumin is greater than 1: 1 and preferably 3: 1. Alternatively, albumin can be purchased in combination with fatty acids. For example, BSA can be purchased in combination with octanoic acid (Sigma Catalog No. A3174, 1 g protein)
10-15 mg of octanoic acid per). In a preferred embodiment, the primer solution is applied to tissue to replace body fluids and provides a good environment for a crosslinking reaction. A brush is a useful applicator for spreading this primer at the tissue site where the crosslinking reaction mixture is to be administered.

[0078] In some embodiments, the albumin solution that is cross-linked includes additional reagents to facilitate interaction with tissue at the site of application. In a preferred embodiment, the albumin solution comprises a surfactant and / or lipid when used in a pulmonary setting. Preferably, the surfactant and lipid components are similar to the natural surfactant and lipid composition of the lung. Alternatively, synthetic surfactants and lipids can be used.

[0079] The cross-linked albumin compositions of the present invention can also be used to produce implantable drug delivery devices. In particular, because the cross-linked albumin composition is slowly eroded and absorbed by the body, the cross-linked albumin composition is bioerodible with the drug dispersed throughout the cross-linked albumin matrix. Can be used to generate an implant. The degree of crosslinking determines both the ability of the drug to diffuse into and out of the matrix, and the rate at which the device erodes. Furthermore, the degree of crosslinking determines the stiffness or flexibility of the device. Thus, by controlling the degree of cross-linking, devices can be delivered that deliver drugs at different rates and have different degrees of flexibility. The device comprises introducing an albumin preparation of the invention and a carbodiimide, optionally with ancillary molecules such as pharmaceuticals, into a mold under conditions that promote albumin crosslinking to form an implantable device. Generated.

EXAMPLES Example 1. Effect of pH on the Rate of BSA Crosslinking with EDC.HCl A 37.5% solution of BSA was placed in sterile water (10 ml) for the following crosslinking experiments. Prepared by dissolving BSA (6 g) and stirring for several hours until a clear solution was obtained. Aliquots of 1 ml of this solution were taken at various pH values and taken at 0.
Crosslinking was carried out with 25 ml of an 8% aqueous solution of EDC.HCl.

(Experiment 1: pH 7.0) A 1 ml solution of 37.5% BSA (pH = 7.09) was added to 0.2 ml as follows.
It was mixed with 5 ml of 8% EDC.HCl (pH = 7.0). The protein solution was stirred with a small magnet stir bar using a magnetic stirrer at a fixed setting. The solution was viscous and the bar was stirred slowly. The stirring became faster when the EDC.HCl solution was added. This is probably due to the addition of more water and the resulting dilution of BSA. After 10 minutes, the stirring became progressively slower. This is probably due to EDC
Due to the fact that HCl promoted the crosslinking of BSA. At 15 minutes, stirring was completely stopped. The crosslinked gel was soft but elastic and showed significant cohesion.

(Experiment 2: pH 6.02) A solution of BSA at pH 6.02 was added to 1 ml of 37.5% BSA diluted in water.
. Prepared by dropwise addition of 5N HCl. As described above, 0.25 ml of 8% EDC.HCl was added while stirring the solution. One minute after the completion of the addition of EDC.HCl, stirring was stopped and a very cohesive, elastic, non-sticky gel was formed.

(Experiment 3: pH 5.5) A solution of BSA at pH 5.5 was added to 1 ml of 37.5% BSA diluted in water.
Prepared by dropwise addition of 5N HCl. As described above, 0.25 ml of 8% EDC.HCl was added while stirring the solution. After 31 seconds, stirring was stopped and, in addition, a very cohesive gel formed.

Experiment 4: A similar experiment was performed using a 1 ml solution of 37.5% BSA at pH 5.3. In this experiment, a strong gel formed within 26 seconds.

The total volume of the gel was 1.25 ml. This gel is. 0.375 g of B
SA, 0.020 g of EDC.HCl, and 0.855 g of water. Thus, the solid represented 31.6% of the gel weight and EDC.HCl was only 1.6% of the gel weight. The cross-linked protein has an ED of about 5.3% by weight.
C. HCl was included.

[0086] These experiments show that within the range of pHs tested, as pH decreases, E
This suggests that the rate of BSA crosslinking by DC.HCl is increased. Various pH
The relative cross-linking rates in were ranked as follows: 5.3>5.5> 6.
01 >> 7.

Example 2 Crosslinking of a Commercial 30% BSA Solution Using EDC.HCl A 30% BSA solution was purchased from Sigma (catalog number A8327).
The pH of 1.5 ml of this BSA solution is brought to 5.38 and 5 with 3 drops of 0.5N HCl.
. Adjusted to 40. A 1 ml aliquot of the pH adjusted BSA was stirred with a magnetic bar using a stationary stirrer. 0.25 ml 8% EDC
HCl solution (80 mg EDC.HCl dissolved in 1 ml water) was added. Stirring was slowed down after 1 minute and stopped after 1.5 minutes. The resulting gel was less rigid than the gel obtained using a 37.5% BSA solution.

Example 3. Two Syringe Mixture System for Delivering BSA / EDC.HCl Bioadhesive A two syringe mix system delivering a 9.3: 1 ratio of BSA: EDC.HCl was tested. A 3 ml volume of BSA (pH 5.5) was collected in the first syringe (10 cc syringe). A 0.5 ml volume of 18.12% EDC.HCl (90 mg of EDC.HCl dissolved in 0.5 ml of distilled water) was collected in a second syringe (1 cc syringe). These syringes were modified with a modified Micromedix (Eagan, MN).
It was connected to the applicator mixing nozzle. The ends of the syringe plungers were tied together with a plastic tub and the mixture of BSA and EDC.HCl was extruded into a PE weigh boat. The mixture became a non-viscous solution that rapidly gelled to a non-flowing gel in about 40 seconds. The gel still has a soft feel. After about 1.5 minutes, the mixture was completely recovered as a rubbery, highly sticky crosslinked gel.

Example 4. Effect of BSA and EDC.HCl concentration on BSA crosslinking rate using EDC.HCl. A 1 ml aliquot of a 30% BSA solution was added to 0.125 ml of 16.67% aqueous EDC.HCl. Crosslinking was performed with a solution (pH 7). The solution gelled within 11-12 minutes.

A similar experiment performed with a 35% BSA solution resulted in a slightly slower gelation time of 13-15 minutes.

In another experiment, a 1 ml aliquot of a 30% BSA solution was
Crosslinking with 1 of 23.07% aqueous EDC.HCl solution (pH 7). The solution gelled within 7-8 minutes. However, this experiment was performed with 50% BSA (pH
When performed in 7), gelation was faster and gels were obtained in 4-6 minutes.

A 1 ml aliquot of the 30% BSA solution was cross-linked with 0.125 ml of a 33.34% aqueous EDC.HCl solution (pH 7). The solution gelled within 5-6 minutes. Thus, this indicates that higher concentrations of crosslinker result in shorter gel times.

Example 5. Effect of additives on the rate of gel formation 1 ml volume of 35% BSA solution and glutaric anhydride derivatized BSA (polyacid) (ratio 3.6: 1, pH 5.8) With 0.125 ml of 16.6% aqueous ED
Mixing with the C.HCl solution resulted in a self-standing rubbery gel after 15-25 seconds. In a similar reaction, the 35% BSA solution (without any glutaric anhydride derivatized BSA) gelled after 55-65 seconds.

In another experiment, a 1 ml volume of a 35% BSA solution (pH 5.5) was added to a 0.1% solution.
Mixed with 1 ml deionized (DI) water and 0.15 g solid citric acid (final BSA concentration was 28-30%). 0.125 ml aqueous 16.67% E
When mixed with the DC.HCL solution, a hard gel was obtained within 4-8 seconds. In a similar reaction without citric acid, the gel time was 30-40 seconds.

As discussed above, polyacids such as dicarboxylic acid, citric acid, polyacrylic acid, glutaric anhydride derivatized BSA, and other polyacids can alter the local pH or increase Reacts with EDC.HCl at a faster rate than modified BSA, or
Gelation can be accelerated by either denaturing the SA, thereby increasing the BSA crosslinking rate. However, high proportions of monocarboxylic acids can result in longer gel times by preventing protein cross-linking reactions.

In a further experiment, 1 ml of 35% BSA was added to 0.1 ml of EDC-HC
And a hard gel was formed in 45 to 50 seconds. In a similar experiment, 0.6% ethylenediamine dihydrochloride was added along with the crosslinker, and gelation slowed to 70-85 seconds. Thus, diamines can reduce the rate of gelation.
However, polyamines such as polyethyleneamine, chitosan, poly (l-lysine) can be expected to accelerate the reaction. Other additives, such as N-hydroxysuccinimide (NHS), water-soluble analogs (sulfo NHS, sulfonate of NHS), or hydroxybenzotriazole (HOBT) have been shown to reduce the rate of gelation. These effects are pH sensitive.

In one experiment, a 4% solution of dextran (molecular weight: 3-7M) was added to 35%
% BSA solution (pH 5.5). When mixed with a 16.67% aqueous solution of EDC.HCl in an 8: 1 ratio, this very thick solution produced a hard gel within 25 seconds.

In another experiment, 2 g of BSA was dissolved in a 5 ml slurry of 25% hydroxyapatite. The pale yellow, milky solution was treated with 16.67% EDC.HC
The aqueous solution was mixed and quickly poured into a cylindrical mold. The solution gels rapidly to yield a strong flexible material that can be used, for example, as a matrix for plastic surgery, tissue engineering applications, or drug delivery systems.

Example 6 Shelf Life of EDC.HCl Designed to evaluate the shelf life of EDC.HCl both at room temperature (RT) and refrigeration, and as both solid and aqueous solutions. In an experiment performed, 30% (pH 5.5) using 16.67% EDC.HCl aqueous solution.
Gelation of the BSA solution was achieved. Gel time was taken as a direct measure of the activity of the crosslinker. Thus, the crosslinker can be provided as a powder that can be stored at room temperature for more than six months. Alternatively, the aqueous solution of the crosslinking agent should be refrigerated,
And it is completely active for less than a month. Solid crosslinker at RT (24 ° C.) in an airtight container stored in a desiccator:

[0100]

[Table 1] Aqueous solution of crosslinking agent (16.67%) in refrigerator:

[0101]

[Table 2] Aqueous solution of crosslinking agent (16.67%) at RT (24 ° C.):

[0102]

[Table 3] Example 7 End-to-Side of ePTFE Graft into Artery
e) Adhesion of arterial anastomosis) In the first experiment, a mixture of BSA and EDC.HCl sealant was added to the end of ePTFE (extended polytetrafluoroethylene) graft on porcine aorta.
Delivered in vitro over a lateral anastomosis. The mixture was delivered via a static mixing nozzle and contained a 9.3: 1 ratio of 35% BSA (pH 5.55): 40% EDC.HCl. The adhesive mixture was slowly extruded onto all sides of the anastomosis. This mixture crosslinked very quickly. In fact, the gel could be cut clean with scissors about 30 seconds after this application. The arteries were pressurized by introducing saline via a large syringe, and the graft treated with adhesive provided good closure.

In another experiment, a 40% solution was prepared using a 25/10 ratio of BSA and glutaric anhydride-derivatized BSA. This solution (pH 6) was used on porcine lungs (ex vivo) with an aqueous solution of 16.67% EDC.HCl at a ratio of 8: 1 (vol / vol) to block the planar wedge resection. This solution adheres to the lungs during treatment and has a static pressure of more than 60 mm Hg (mean lung pressure during surgery is 20-25 m
mHg range).

A 9.3: 1 ratio of BSA solution: EDC.HCl solution was made as follows.
A 5 ml volume of 35% BSA (pH 5.5) was collected into a 10 cc syringe and a 1 ml volume of 40% EDC.HCl (400 mg EDC.HC in 1 ml of water).
was dissolved in a 1 cc syringe. The two syringes were connected to a static mixing nozzle, which delivered a 9.3: 1 ratio of BSA and EDC.HCl solution. Assuming total delivery of the BSA solution, the resulting gel contains 5.0 ml, ie, 5 × 35% = 1.75 g of BSA. This gel also has 5 / 9.3 =
0.538 ml = 0.538 × 40% = 0.215 g containing EDC · HCl (
The EDC.HCl syringe does not empty and only 0.538 ml of EDC.HCl solution is delivered with a 5 ml volume of BSA solution). The total weight of this adhesive is 5.
538 g, so the weight percent of EDC.HCl in the adhesive is 0.215 ×
100 / 5.538 = 3.88%. The weight percent of EDC.HCl in albumin is 0.215 × 100 / 1.75 = 12.3%.

In a second experiment, the same composition was applied to a 2 × 20 mm suture in a pig carotid artery.
For use in vivo on ePTFE patches. The albumin sealant adhered to the blood vessels and sealed the leaky suture.

Example 8 Effect of Derivatization of BSA BSA was derivatized with a reactive molecule having a hydrophobic tail to increase its hydrophobicity.

In one experiment, 10 g of BSA was dissolved in 200 ml of 0.05N phosphate buffer and the pH was adjusted to 8.5. Immediately, 6.89 ml of hexanoic anhydride in acetone solution (this acetone solution is saturated with hexanoic anhydride)
Was added. There was no apparent change in the pH of this solution. The reaction was allowed to stir at low temperature for 2 days. This mixture is diafiltrated (diafi
liter), adjusted the pH to 6.0, and dried. This dried derivatized BSA
Showed more hydrophobic properties, as revealed by contact angle studies.

In one experiment, 10 g of BSA was added to 90 ml of deionized water and 100 m
Dissolved in 1 l of 0.05N phosphate buffer and adjusted the pH to 8.5. 13 g of pyromellitic dianhydride were added dropwise in acetone solution. The pH was maintained at 8-9 using dilute NaOH. The reaction was allowed to stir at low temperature (about 4 ° C.) overnight. The mixture was diafiltered, the pH was adjusted to 6.0 and dried. This dried derivatized BSA exhibited more hydrophobic properties, as revealed by contact angle studies. This solution was also highly viscous as compared to a BSA solution of similar concentration.

In one experiment, 10 g of BSA was dissolved in 67 ml of 0.05N phosphate buffer. The pH of the resulting solution was adjusted to 8.5 and 6 g of tetrafluorophthalic anhydride was added as a solution in acetone. The pH is adjusted to 8 using dilute NaOH.
９9. After stirring for several hours, the reaction mixture was diafiltered,
The pH was adjusted to 6.0 and dried. This dried derivatized BSA exhibited more hydrophobic properties, as revealed by contact angle studies.

In one experiment, 20 g of BSA was dissolved in 500 ml of a mixture of 65/35 deionized water and methanol. The pH of the pale yellow solution was adjusted to 9.0 and 8 ml of (2,2,3,3,4,4,5,5,6,6,7,7,8,8,9
, 9,9-Heptadecafluorononyl) -oxirane was added in one portion with a 50% acetone solution. The reaction was stirred for 2 days while maintaining the pH at 9.
The slightly turbid solution was centrifuged, dialyzed, adjusted to pH 6.0, and dried. This dry modified BSA solution shows higher viscosity and improved hydration for ePTFE grafts and adheres very well to grafts and natural tissues upon crosslinking with an appropriate amount of EDC.HCl did.

Example 9 Dry Powder Formulation In one experiment, gelatin (300 Bloom, Sigma) was loaded with EDC.
-Thoroughly mixed with HCl in a ratio of 10: 1. This powder was applied to a planar wedge resection model dog lung. This material absorbed surrounding body fluids, rapidly gelled into a rubbery mass, and stopped leakage.

Example 10. Pulmonary application A 40% (pH 6) BSA solution was mixed with tyloxapol and dipalmitoyl, phosphatidylcholine (DPPC), so that each 1 mg
/ Ml and 14 mg / ml. This dispersion is treated with an aqueous solution of EDC · HCl (
20%) and applied (10: 1) to porcine lung in a wedge resection model. This site was previously primed with a 30% BSA solution (pH 5.5). The material was allowed to reach maximum strength (about 4-5 minutes) and then tested. This material withstood a dynamic pressure of about 100 mm Hg until lung tissue rupture occurred.

In another experiment, gelatin (300 Bloom) was mixed with DPPC and tyloxapol in similar ratios. This material was a gel at room temperature. This material was warmed to about 45 ° C. and mixed appropriately with aqueous EDC.HCl. Upon application of lung tissue, the material gelled rapidly and adhered well to the wound site.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS , JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Pendercar, Sanyog Manaher United States Mass. 01890 , Winchester, Swanton Street 200, Apartment 510 (72) Inventors Wilkie, James A. United States Massachusetts 02176, Melrose, Mount Hood Terrace 11F term (reference) 4C081 AC04 BA14 BA15 BA16 CA231 CD171 CE01 CE02 CE11 DA12

Claims (48)

[Claims] 1. A method for producing a cross-linked albumin composition for use in a bioadhesive surgical sealant or implantable device, comprising: (a) providing an albumin preparation; (B) providing a carbodiimide preparation; and (c) mixing the albumin preparation and the carbodiimide preparation under conditions that allow the albumin to crosslink. 2. The albumin preparation comprises a protein selected from the group consisting of a naturally occurring albumin protein, a recombinant albumin protein, a major fragment of an albumin protein, and a chemically modified albumin. Item 1. The method according to Item 1. 3. The albumin is selected from the group consisting of a mammalian albumin protein and a major fragment of the mammalian albumin protein.
The method according to claim 2. 4. The method of claim 2, wherein said albumin preparation comprises a protein comprising an amino acid sequence of at least 100 amino acid residues having at least 60% homology to the amino acid sequence of human albumin. 5. The method of claim 5, wherein said albumin is recombinantly modified with respect to a naturally occurring albumin sequence, and wherein said albumin is soluble, reactive with carbodiimide crosslinks, stability, viscosity in aqueous solution, and immunocompatibility. 3. The method of claim 2, wherein the method comprises an amino acid sequence in which one or more selected physical properties are enhanced. 6. The albumin may be a polysaccharide (eg, glycosaminoglycan, dextran, hyaluronic acid, chondroitin sulfate, heparan sulfate), a polyether (eg, polyethylene glycol, polypropylene glycol, polybutylene glycol), a polyester (eg, , Polylactic acid, polyglycolic acid, polysalicylic acid), and aliphatic agents, alicyclic agents, aromatic agents, perfluorinating agents, non-perfluorinating agents, acylating agents or sulfonating agents. 3. The method of claim 2, comprising albumin covalently linked to the molecule. 7. The method according to claim 2, wherein the albumin preparation is chlorinated, fluorinated, brominated or iodinated. 8. The method according to claim 1, wherein the albumin preparation is an aqueous solution having a concentration of about 10 to 50% by weight. 9. The method according to claim 1, wherein the albumin preparation is an aqueous solution having a concentration of about 20 to 40% by weight. 10. The method according to claim 1, wherein the albumin preparation is an aqueous solution having a concentration of about 35 to 40% by weight. 11. The carbodiimide preparation is a carbodiimide having the general structure R ₁ —N ＝ C ＝ N—R ₂ or a halogen, tertiary amino, ester, keto-substituted or variant thereof having another decomposable group. Wherein R ₁ and R ₂ are independently selected from the group consisting of linear or branched saturated or unsaturated alkyl, alkenyl, aryl, aralkyl, or aralkenyl groups.
0. The method according to any one of 0. 12. The carbodiimide is ethyl dimethylaminopropyl carbodiimide (EDC · HCl); 1- (3-dimethylaminopropyl) -3-
1,3-di-p-tolylcarbodiimide; 1,3-diisopropylcarbodiimide; 1,3-dicyclohexylcarbodiimide; 1-cyclohexyl-3- (2-morpholinoethyl) carbodiimide meth-p-toluenesulfonate; polycarbodiimide 1-tert-butyl-3-ethylcarbodiimide; 1,3-dicyclohexylcarbodiimide; 1,3-bis (trimethylsilyl) carbodiimide; 1,3-di-tert-butylcarbodiimide; 1- (3-dimethylaminopropyl)- 12. The method according to claim 11, wherein the method is selected from the group consisting of 3-ethylcarbodiimide methoxide; and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. 13. The method of claim 1 wherein said albumin and carbodiimide are present in an amount of about 1: 1 and 1:
The carbodiimide: albumin weight ratio between 100 and 100 is mixed.
0. The method according to any one of 0. 14. The method of claim 1, wherein said albumin and carbodiimide are present in an amount of about 1: 5 and 1:
A carbodiimide: albumin weight ratio of between 50 and 50.
The method according to any one of claims 1 to 4. 15. The method according to claim 15, wherein said albumin and carbodiimide are present in an amount of about 1:10 and 1: 1.
: 20 in a weight ratio of carbodiimide: albumin of between 1 and 20.
0. The method according to any one of 0. 16. The method of claim 1, wherein the albumin and carbodiimide are mixed at a carbodiimide: albumin weight ratio of between about 100: 1 and 1: 1.
0. The method according to any one of 0. 17. The method according to claim 16, wherein the albumin and the carbodiimide are about 36: 1 and 4
11. The carbodiimide: albumin weight ratio of between 1: 1.
The method according to any one of claims 1 to 4. 18. The method of claim 1, wherein the albumin and the carbodiimide are mixed at a carbodiimide: albumin weight ratio of about 18: 1. 19. The method according to claim 1, wherein the mixing step is performed at a tissue location. 20. The method according to any one of claims 1 to 10, wherein an accessory molecule is provided and mixed with said albumin and carbodiimide preparation. 21. The accessory molecule is selected from the group consisting of viscosity enhancers, crosslinkers, buffers, hormones, growth factors, antibiotics, surfactants, lipids, fatty acids, and anti-inflammatory agents. 20. The method according to 20. 22. The method of any one of claims 1 to 10, wherein the albumin preparation comprises a secondary or tertiary alcohol. 23. The method of claim 21, wherein said albumin preparation comprises IPA or IBA. 24. A method for adhering a first biological tissue to a second tissue and / or a prosthesis device, the method comprising the following steps: the first tissue and the second tissue A tissue or prosthesis device under conditions that promote crosslinking of the albumin preparation to the first tissue and the second tissue or prosthesis device with a mixture of the albumin preparation and the carbodiimide preparation;
Contacting. 25. An incision, perforation, or cut in biological tissue during a surgical procedure.
And / or a method for blocking fluid or gas leakage, comprising the steps of: combining the tissue with an effective amount of an albumin preparation and a carbodiimide preparation to promote crosslinking of the albumin preparation to the tissue; Contacting under conditions whereby the incision, perforation, or fluid or gas leak is sealed off. 26. The method of claim 25, wherein the surgical procedure is selected from the group consisting of cardiovascular surgery, lung surgery, kidney surgery, and liver surgery. 27. A method for forming an implantable device, comprising the steps of: (a) providing an albumin preparation; (b) providing a carbodiimide preparation; (c) providing a mold. And d) mixing the albumin preparation and the carbodiimide preparation under conditions that permit crosslinking of the albumin in the mold. 28. A bioadhesive surgical sealant or implantable device produced by any of the foregoing methods. 29. A kit for producing a bioadhesive surgical sealant or implantable device, comprising in separate containers: a) an albumin preparation; and b) a carbodiimide preparation. kit. 30. The kit of claim 29, further comprising an accessory molecule. 31. The accessory molecule is selected from the group consisting of viscosity enhancers, crosslinkers, buffers, hormones, growth factors, antibiotics, and anti-inflammatory agents.
The kit according to 1. 32. The albumin preparation and the carbodiimide preparation are provided in a dual delivery device that delivers the preparation in either a predetermined ratio or an adjustable ratio. The kit according to 1. 33. The method of any one of claims 24-26, wherein any one of said tissue or prosthetic device is first contacted with a primer solution. 34. The method according to claim 3, wherein the primer solution is a physiological saline solution.
3. The method according to 3. 35. The method according to claim 3, wherein the primer solution is an albumin solution.
3. The method according to 3. 36. The method of claim 35, wherein said albumin solution is the same as said albumin preparation. 37. The method of claim 36, wherein said albumin solution is a diluted albumin preparation. 38. The albumin and carbodiimide preparation is provided at a pH between 5.0 and 8.0.
38. The method according to any one of 37. 39. The method of claim 38, wherein said pH is between 5.5 and 7.5. 40. The method of claim 39, wherein said pH is between 6.0 and 7.0. 41. The method of claim 1, wherein the crosslinking reaction results in gel formation in less than 10 minutes. 42. The method of claim 41, wherein said gel formation occurs in 5 minutes or less. 43. The method of claim 41, wherein said gel formation occurs in less than one minute. 44. The method of claim 41, wherein said gel formation occurs in 30 seconds. 45. The carbodiimide preparation has an E between 10% and 20%.
13. The method of claim 12, comprising an aqueous solution of DC.HCl. 46. The method of claim 45, wherein the carbodiimide preparation comprises a 15% aqueous solution of EDC.HCl. 47. An albumin preparation for cross-linking with EDC.HCl, wherein the preparation comprises albumin and derivatized albumin, wherein the ratio of albumin to derivatized albumin is specific to a particular tissue application. An albumin preparation that is appropriate for the ratio of crosslinks to be adapted. 48. The albumin preparation of claim 47, wherein said particular tissue application is a cardiovascular application.