||||||||

USOO5583114A
United States Patent (19) 11 Patent Number: 5,583,114
Barrows et al. 45) Date of Patent: Dec. 10, 1996
54 ADHESIVE SEALANT COMPOSITION Two-Component Fibrin Sealant Tisseel(R) Kit Brochure.
75) Inventors: Thomas H. Barrows, Cottage Grove; Abstract, Abdella et al., “A New Cleavable Reagent for
Terry W. Lewis, Woodbury; Myhanh Cross-linking and Reversible Immobilization of Proteins',
T. Truong, Blaine, all of Minn. Biochem. Methods, 91:16228v, 1979.
Kalman et al., "Synthesis of a Gene for Human Serum
73 Assignee: Minnesota Mining and Albumin and Its Expression in Sacchromyces cerevisiae',
Manufacturing Company, Saint Paul, Nucleic Acids Research, vol. 18, No. 20, 1990, pp.
Minn. 6075-6081.
Sleep et al., "The Secretion of Human Serum Albumin from
21 Appl. No.: 281,473 the Yeast Saccharomyces cerevisiae Using Five Different
22 Filed: Jul. 27, 1994 Leader Sequences', Bio/Technology, vol. 8, Jan. 1990, pp.
42-46.
(51) Int. Cl.' .......................... A61K 38/00; A61K 39/00; Sijmons et al., “Production of Correctly Processed Human
CO7K 1/OO
52 U.S. Cl. ..................................... 514/21; 514/2; 514/4; Serum Albumin in Transgenic Plants', Bio/Technology, vol.
530/362; 530/363; 530/366; 530/830; 424/77; 8, Mar. 1990, pp. 217-221.
424/78.02; 424/78.06; 424/179.1; 424/193.1; Quirk et al., “Production of Recombinant Human Serum
424/1941; 424/422; 424/423; 424/428; Albumin from Saccharomyces cerevisiae', Biotechnology
52.5/54.1 and Applied Biochemistry, 11,273-287 (1989).
58 Field of Search ..................... 514/21, 2, 4, 530/350, D'Urso et al., "New Hydrogel Based on Polyethylene Gly
530/362,363, 366, 830; 424/77, 78.02, col Cross-Linked with Bovine Serum Albumin', Biotech
78.06, 422,423, 428; 524/54.1 nology Techniques, vol. 8, No. 2 (Feb. 1994) pp. 71-76.
56 References Cited
U.S. PATENT DOCUMENTS
Primary Examiner-Elizabeth C. Weimar
Assistant Examiner Abdel A. Mohamed
2,688,610 9/1954 Elins et al. .............................. 260F117 Attorney, Agent, or Firm-Gary L. Griswold; Walter N.
4,101,380 7/1978 Rubinstein et al. ... 530/350 Kirn, Paul W. Busse
4,63,097 7/1979 Baumann et al. .. ..., 52.5/531
4,356,819 11/1982 Potaczek ................................. 28/56
4,416,814 1/1983 Battista ................................... 260F117 57) ABSTRACT
4,670,417 6/1987 Iwasaki et al. ............................. 514/6 This invention is related to an adhesive composition which
4,692,462 9/1987 Banerjee ................................. 514,449
4,839,345 6/1989 Doi et al. .................................. 514/21 may be used to bond or seal tissue in vivo. The adhesive
4,851,513 7/1989 Devore et al. ... 530/356 composition is readily formed from a two component mix
5,162,430 11/1992 Rhee et al. .. ... 525/54. ture which includes a first part of a protein, preferably a
5,385,606 1/1995 Kowanko ................................ 106/124 serum albumin protein, in an aqueous buffer having a pH in
FOREIGN PATENT DOCUMENTS the range of about 8.0-11.0 and a second part of a water
compatible or water-soluble bifunctional crosslinking agent.
0.194807A3 9/1988 European Pat. Off.. When the two parts of the mixture are combined, the mixture
2551660 3,985 France. is initially a liquid which cures in vivo on the surface of
WO90/13540 11/1990 WIPO. tissue in less than about one minute to give a strong, flexible,
WO92/O2238 2/1992 WIPO. pliant substantive composition which bonds to the tissue and
WO94/03155 2/1994 WIPO is absorbed in about four to sixty days. The adhesive
OTHER PUBLICATIONS composition may be used either to bond tissue, to seal tissue
or to prevent tissue adhesions caused by surgery.
Abuchowshi et al, The Journal of Biological Chemistry, vol.
252, No. 11, pp. 3578–3581, Jun. 10, 1977.
D'Urso et al, Biotechnology Techniques, vol. 8, No. 2, pp.
71-76, Feb. 1994. 17 Claims, 3 Drawing Sheets
U.S. Patent Dec. 10, 1996 Sheet 1 of 3 5,583,114

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1. 2
ADHESIVE SEALANT COMPOSITION SUMMARY OF THE INVENTION

The present invention is generally related to an adhesive The present invention is a nontoxic, absorbable adhesive
sealant composition which may be used to bond or seal sealant composition which may be used to bond and/or seal
tissue. The adhesive composition is readily formed from a
tissue in vivo and is particularly related to a two component, two component mixture which includes a first part of a
liquid adhesive composition which is mixed together as it is protein, preferably a serum protein such as albumin, in an
applied to tissue and then cured in vivo in order to bond aqueous buffer having a pH in the range of about 8.0-11.0
tissue, to seal tissue to prevent or control pulmonary system and a second part of a water-compatible or water-soluble
air leaks, or to prevent tissue adhesions caused by surgery. bifunctional crosslinking agent. When the two parts of the
10 mixture are combined, the mixture is initially liquid. The
combined mixture then cures in vivo on the surface of tissue
BACKGROUND in less than about one minute to give a strong, flexible, pliant
substantive composition which securely bonds to the tissue
A variety of techniques have been used to bond or seal and is readily absorbed in about four to sixty days, prefer
tissue. For example, different types of tissues have been 5 ably in about four to twenty-eight days.
mechanically bound or sealed with a number of procedures, In a preferred embodiment of the invention, an adhesive
materials and methods including sutures, staples, tapes and sealant composition is formed from a two part mixture that
bandages. In some applications, these materials are made of includes a proportion of a volume of a buffered basic serum
absorbable materials which are intended to bond and/or seal
tissue as it heals and then to be absorbed over a period of albumin protein solution to a volume of a polyethylene
time.
20 glycol disuccinimidoyl succinate crosslinking agent in a
range of from about 1:10 parts albumin solution by volume
The common use of a medical adhesive or “tissue glue' to about 10:1 parts by volume crosslinking agent. In order to
has not found widespread application. To date, some adhe facilitate the mixing of the two parts of the present adhesive
sive materials are known which may be used to adhere or composition, the volume to volume ratio of albumin solution
stick tissue such as skin. For example, cyanoacrylate adhe 25 to crosslinking agent is preferably a ratio of 1:1.
sives such as HISTOACRYL adhesive available from B.
Braun, Melsungen, Germany or VETBOND tissue adhesive Preferred serum albumin proteins are selected to prevent
available from 3M, St. Paul, Minn. may be used to bond adverse tissue or unwanted immunological responses. When
tissue. In addition to cyanoacrylate adhesives, other types of the present adhesive mixture is used to bond or seal human
materials have been reported to adhere or stick to skin. For 30
tissue, a preferred serum albumin is purified human serum
example, U.S. Pat. No. 4,839,345 to Doi et al. reports a albumin which has been sterilized, dialyzed with a basic
hydrated crosslinked protein adhesive gel that is used as a buffer having a pH value of about 8.0-11.0, concentrated by
cataplasm or cosmetic mask that will externally adhere to ultrafiltration through a membrane having about a 50,000
skin but can be easily removed or pulled off and then molecular weight cut-off to yield a concentrated, buffered
readhered to the skin. Other crosslinked protein hydrogels 35
aqueous mixture having about 20-60 wit/vol %, preferably
have been reported to serve as a proteinaceous substrate to about 35–45 wt/vol %, human serum albumin.
deliver therapeutic agents such as enzymes or drugs through Preferred bifunctional crosslinking agents include poly
skin or mucous membranes. See, for example, International ethylene glycol derived crosslinking agents having a
Patent Application Ser. No. PCT/US93/07314 filed Aug. 4, molecular weight (weight average) in a range of about
1993. Still other materials have been used as hemostatic 40 1,000-15,000 and preferably in a range of about 2,006–4,
agents to stop or prevent bleeding. In particular, mixtures of 000. When the molecular weight of the crosslinking agent is
fibrinogen and thrombin such as TISSEEL sealant available in the range of about 1,000-5,000 the crosslinking agent is
from Immuno AG, Vienna, Austria or BERIPLASTP hemo generally dissolved in water at a concentration of about
static agent or sealant available from Behringwerke, Mar 50–300 mg/ml. Similarly, when the molecular weight of the
burg, Germany, have been used in vascular surgery to seal 45 crosslinking agent is in the range of about 5,000-15,000 the
tissue such as blood vessels and thus prevent blood leakage. crosslinking agent is generally dissolved in water at a
In sum, there are few available adhesive compositions concentration in the range of about 300-800 mg/ml.
that have sufficient strength, biocompatibility and bioab The adhesive composition of this invention may be used
sorbability as well as other desired properties that would in a variety of applications. Some applications include using
allow such compositions to be readily used in current 50 the adhesive sealant composition to bind tissue together
medical procedures or practices. The unavailability of a either as an adjunct to or as a replacement of sutures, staples,
suitable tissue adhesive or sealant may be related to the tapes and/or bandages. In another application, the present
stringent requirements that a suitable, useful tissue adhesive adhesive may be used to prevent post-surgical adhesions. In
must meet. Importantly, a tissue adhesive must provide this application, the adhesive composition is applied and
substantial bonding strength for either internal or external 55 cured as a layer on surfaces of internal organs or tissues in
tissues. The adhesive should be made of a biocompatible order to prevent the formation of adhesions at a surgical site
material which does not interfere with normal healing or as the site heals. Additional applications include sealing
regeneration processes. A suitable tissue adhesive must also tissues to prevent or control blood or other fluid leaks at
be easily administered in a liquid form and then rapidly suture or staple lines as well as to preventor control air leaks
cured, ideally in less than a minute, once applied. In addi 60 in the pulmonary system.
tion, a tissue adhesive must remain flexible, pliant and have BRIEF DESCRIPTION OF THE DRAWINGS
good mechanical strength after being cured. Finally, a tissue
adhesive must be completely absorbed or broken down in FIG. 1 is a graphical representation of a measured peel
vivo, without producing an allergic response, adverse tissue force of an adhesive composition of this invention.
reaction or systemic toxic effects, in an acceptable time 65 FIG. 2 is a graphical representation of peel force mea
period. Preferably a suitable adhesive would also be readily surements of different adhesive composition samples which
absorbed after it is applied. are used to adhere excised guinea pig skin strips together.
 5,583,114
3 4
FIG. 3 is a schematic diagram of an apparatus used to and the leaving group portion -G. Crosslinking agents
measure burst strength of an adhesive sealant composition. include compounds of the formula
DETAILED DESCRIPTION
The present invention is related to an adhesive composi in which -PEG- is a diradical fragment represented by the
formula
tion which has high mechanical strength, flexibility, fast cure
rate and sufficient adhesion needed to bond and/or seal tissue
in vivo. The adhesive composition is made of two compo
nents, a buffered basic protein solution and a bifunctional O where a is an integer from 20-300; --LM- is also a
crosslinking agent. The buffered protein solution and the diradical fragment such as a carbonate diradical represented
bifunctional crosslinking agent are typically prepared using by the formula, -CO)-, a monoester diradical repre
commercially available materials and established synthetic sented by the formula, -(CH2)C(O)- where b is an
methods. The use of known, commercially available mate integer from 1-5, a diester diradical represented by the
rials in the preparation of the adhesive composition provides 15 formula, -C(O)-(CH2)-C(O)- where c is an integer
a benefit in the practice of this invention because most of from 2-10 and where the aliphatic portion of the radical may
these materials generally have a history of clinical safety be saturated or unsaturated, a dicarbonate represented by the
and/or use. formula -C(O)-O-(CH)-O-C(O)- where d is an
Suitable proteins for use in the present adhesive compo integer from 2-10, or an oligomeric diradical represented by
sition include nonimmunogenic, water soluble proteins. 20 the formulas -R-C(O)-, -R-C(O)-(CH2)-
Serum lipoproteins are particularly well suited for this C(O)-, or -R-C(O)-O-(CH2)-O-C(O)- where c
purpose because these proteins bind to lipids and also is an integer from 2-10, d is an integer from 2-10, and R is
exhibit a relatively high elasticity in the natured or semi a polymer or copolymer having 1-10 monomeric lactide,
natured state. These properties are believed to provide a glycolide, trimethylene carbonate, caprolactone or p-diox
cured matrix which is strong as well as pliant and elastic. 25 anone fragments, and -G is a leaving group such as a
Other soluble proteins, in addition to serum lipoproteins, are succinimidyl, maleimidyl, phthalimidyl, or alternatively,
also suitable for use in the present invention. Aqueous nitrophenyl, imidazolyl or tresyl leaving groups.
mixtures of proteins such as derivatives of elastin, fibrino The -PEG-portion of the crosslinking agent is pref
gen and collagen may be used in the present invention. erably derived from commercially available compounds
Preferred buffered protein solutions which may be used in 30 having a weight average molecular weight in the range of
the present adhesive composition include concentrated about 1,000-15,000, preferably having a weight average
aqueous serum albumin protein mixtures that are buffered to molecular weight in the range of about 2,000-4,000. These
a pH of between about 8.0-11.0 where the buffer concen compounds have been used in different types of biomedical
tration is in a range of about 0.01-0.25 molar. Suitable buffer materials because they have been demonstrated to be non
systems include buffers which are physiologically and/or 35 toxic as well as rapidly excreted from the body when the
clinically acceptable such as known carbonate or phosphate molecular weight is below about 30,000.
buffer systems, provided the buffer does not adversely react The leaving group, -G, portion of the crosslinking agent
with or otherwise alter the crosslinking agent. A preferred is an activated leaving group which allows the crosslinking
buffer system is a carbonate?bicarbonate buffer system at a agent to react or chemically bind to free primary or second
pH value of about 9.0-10.5 at a concentration in the range 40 ary amine groups of a protein. Suitable leaving groups
of 0.05-0.15 molar. include succinimidyl, other imides such as maleimidyl and
phthalimidyl, heterocyclic leaving groups such as imida
Serum albumin protein is readily isolated from serum Zolyl, aromatic leaving groups such as a nitrophenyl, or
using known isolation processes. In addition, it is possible to fluorinated alkylsulfone leaving groups such as tresyl
produce human serum albumin from genetically trans 45 (CF-CH2-SO-O-). A preferred leaving group is the
formed cells. See, for example, the reports of Quirk et al., succinimidyl group because studies of the mutagenicity,
Biotechnology and Applied Biochemistry, 11:273-287 oncogenicity and teratogenicity of this group suggest that
(1989), Kalman et al., Nucleic Acids Research, the small amount of this activating group which is released
18:6075-6081 (1990), Sleep et al., Biotechnology, 8:42-46 as the crosslinking reaction and/or the adhesive composition
(1990), and Sijmons et al., Biotechnology, 8:217-221 50 cures does not present a local or systemic toxicology risk.
(1990). The ability to produce human serum albumin recom When used in the present composition the linking moiety,
binantly provides the benefit that protein produced by this -LM-, may be several different types of divalent com
method will be free of pathogens, viruses or other contami pounds. For example, commercially available compounds
nants that might contaminate albumin that is isolated having the -PEG- portion and the -G portion linked
directly from serum. 55 with a saturated dicarboxylic acid such as succinic acid to
When used in the present buffered mixtures it has been give a saturated diester linking moiety. Alternatively, an
found that the serum albumin is not denatured. Because the unsaturated dicarboxylic acid such as fumaric, maleic,
albumin is not denatured before it is used it is believed that phthalic or terephthalic acid may be used to give an unsat
the albumin proteins retain their natured, coiled conforma urated diester linking moiety. Alternatively, the linking
tion and thus, after being crosslinked during the curing 60 moiety may be a readily hydrolyzable compounds such as
process to provide a gel-like solid, the cured adhesive retains oligomer derivatives of polylactic acid, polyglycolic acid,
sufficient flexibility to provide a suitable adhesive matrix. polydioxanone, polytrimethylene carbonate, or polycapro
A variety of suitable crosslinking agents may be used in lactone as well as copolymers made using suitable mono
the present invention. Preferred crosslinking agents include mers of these listed polymers.
a polyethylene glycol or polyoxyethylene chain portion 65 In another embodiment of this invention an activated
(-PEG-), an activated leaving group portion (-G) and a leaving group may be attached directly to a carbonate ester
linking moiety (-LM-) which binds the -PEG-portion of polyethylene glycol. In this embodiment the linking
 5,583,114
5 6
moiety, -LM-, would be a carbonate group, -CO)- application, a fine spray of the adhesive composition is
between the -PEG- and -G portions of the crosslinking deposited on tissue as the plungers of the syringe are
agent. In still other embodiments of this invention the depressed.
linking moiety may be a dicarbonate such as ethylene The adhesive composition of the present invention may be
carbonate which is prepared by linking the -PEG and -G used in a variety of current medical procedures and prac
portions with ethylene bischloroformate. tices. In one application, the present adhesive composition
The crosslinking agents may be prepared using known may be used to eliminate or substantially reduce the number
processes, procedures or synthetic methods such as the of sutures normally required using current practices as well
procedures reported in U.S. Pat. Nos. 4,101,380 or 4,839, as eliminate the need for subsequent removal of certain
345, the procedure reported in International Application Ser. 10 sutures. In another application, this adhesive composition
No. PCT/US90/02133 filed Apr. 19, 1990 or the procedure may be used to attach skin grafts and to position tissue flaps
reported by Abuchowski et al., Cancer Biochem. Biophys, or free flaps during reconstructive surgery. In still another
7:175-186 (1984). Briefly, polyethylene glycol and a suit application, this adhesive composition may be used to close
able acid anhydride are dissolved in a suitable polar organic gingival flaps in periodontal surgery. In all of these appli
solvent in the presence of base and refluxed for a period of 15 cations, the present adhesive composition is a thin layer of
time sufficient to form a polyethylene glycol diester diacid. cured material which is effectively sandwiched between two
The diester diacid is then reacted with a leaving group such adjacent layers of living tissues. Due to bioabsorbability and
as an N-hydroxy imide compound in a suitable polar organic lack of toxicity of the adhesive composition, the healing and
solvent in the presence of dicyclohexylcarbodiimide or other subsequent reattachment of the two layers of tissue to each
condensing agents and stirred at room temperature to form 20 other is not hampered.
the desired bifunctional crosslinking agent. In addition to the use of the present adhesive composition
Alternatively, polyethylene glycol and a suitable dicar as an adhesive per se, the present composition may also be
boxylic acid chloride or bischloroformate may be dissolved used as a sealant. When used in this application, this
in a suitable polar organic solvent for a period of time composition may be used to prevent air leaks now associated
sufficient to form the mixed acid chloride polyethylene 25 with pulmonary surgery or to inhibit or prevent bleeding in
glycol ester or mixed chloroformate polyethylene glycol other surgical procedures. When used in this manner, the
ester. The mixed esters may then be reacted with a com underlying tissue may be coated with a relatively thick layer
pound such as an N-hydroxy imide compound in a suitable of adhesive since the tissue itself needs to only heal on one
polar organic solvent and stirred at an elevated temperature side. The other side of the of the adhesive, when cured,
for a period of time sufficient to form the desired bifunc 30 simply presents a lubricous gel which will be absorbed in
tional crosslinking agent. vivo in a relatively short period of time from about four to
It has also been found that the cure time of the present sixty days. In view of this property of the present adhesive
adhesive compositions may be tailored by use of buffers composition, it may also be used to preventunwanted tissues
having different pH values. For example, by varying the pH adhesions which are associated with current surgical proce
of the buffer it is possible to change the cure rate time from 35 dures.
about 10 seconds to less than about 10 minutes. Briefly,
mixing concentrated aqueous serum albumin and crosslink
ing agent mixtures with higher concentrations of buffer EXAMPLES
provides the fastest cure times. It has also been found that The following examples are intended to describe and
higher concentrations of protein and crosslinking agent illustrate the practice of the claimed invention. The
provide a relatively stronger, cured matrix. However, if the examples, however, should not be construed to limit the
mixtures are too concentrated and viscosity becomes too scope of the present invention which is defined by the
great, these adhesive compositions are not as readily applied appended claims.
or may provide adhesives with undesired properties. For The following procedures were used to prepare several
example, mixtures which are too viscous may not be readily 45
applied using available applicators such as syringes or spray different types of bifunctional crosslinking agents. The fol
apparatus. In addition, if the concentration of crosslinking lowing procedures are modifications of procedures reported
agent is too high, the resulting cured adhesive matrix may in U.S. Pat. No. 4,101,380 and Abuchowski et at, cited
swell to such an extent that the strength of the matrix in the above.
presence of water or other fluids is lowered. Further, ability 50
to adequately mix the two components using injecting Example 1
and/or spraying apparatus may be reduced.
The two component adhesive composition of the present Synthesis Of Polyethylene Glycol Disuccinimidyl Succinate
invention may be applied to tissue in a number of different PEG-SS2
ways. For example, the adhesive may be quickly mixed 55 Polyethylene glycol, PEG, (50 g, Aldrich Chemical Com
together and then applied using common applicators. Alter pany, Milwaukee, Wis., sold as 3,400 average molecular
natively the two components may be mixed together and weight, GPC analysis M. was 2,980, M, was 3,480) was
then applied as spray. In another application method, the two dissolved in 1,2-dichloroethane (250 ml) containing succinic
parts of the adhesive are added to a dual syringe. The two arthydride (14.7 g) and anhydrous pyridine (12 ml). The
barrels of the syringe are attached to a "Y' connect which is mixture was refluxed under nitrogen for three days. After
fitted to a spiral mixer nozzle. As the two components are filtration and evaporation of the solvent, the residue was
pressed out of the syringe, they are mixed in the nozzle and dissolved in 100 ml water and treated with the cation
may be directly applied to the tissue as needed in a relatively exchange resin DowexTM50 (H) (50 g) for 30 minutes. The
uniform, controlled manner. Alternatively, a spray nozzle mixture was then filtered and the DoweXTM 50 was washed
tip, such as a TISSEEL spray tip sold by Immuno AG, 65 with water (50 ml 1X). The combined filtrate was washed
Vienna, Austria for use with a two-component fibrin sealant with anhydrous diethyl ether (50 ml 2x). The PEG-disucci
kit, may be used in place of the spiral mixer nozzle. In this nate was then extracted from the water phase with two 100
 5,583,114
7 8
ml chloroform washes. Evaporation of chloroform yielded then precipitated from the filtrate upon cooling and collected
about 49 g of PEG-disuccinate. by filtration. The copolymer was placed in a flask with 500
The PEG-disuccinate was dissolved in 200 ml N,N- ml of dichloromethane and 7 g of succinyl chloride. The
dimethylformamide (DMF) at 37° C. and 4.23g of N-hy solution was refluxed under nitrogen overnight (16 hours).
droxysuccinimide (NHS) were added to the solution. The 8.5g of N-hydroxysuccinimide was added to the flask and
mixture was cooled to 0°C. 7.58 g of dicyclohexylcarbo refluxing was continued for another overnight period. A
diimide (DCC) were dissolved in 50 ml DMF and added white solid was obtained by precipitation upon cooling the
dropwise to the above solution with continuous stirring. The solution. The product was then purified by redissolving in
mixture was left at room temperature for 24 hours and toluene and reprecipitating with petroleum ether several
filtered. 100 ml of toluene were added to the filtrate and the 10 times. The final precipitate was dried under vacuum and
solution was placed in an ice bath. The desired polyethylene stored in a desiccator. The structure of the product was
glycol disuccinimidyl succinate product, PEG-SS2, was confirmed by NMR analysis.
precipitated by slowly adding petroleum ether. The precipi
tate was collected on a 10-20 micron sintered glass filter. Example 4
Dissolution in toluene and precipitation with petroleum 15 Synthesis of Polyethylene Glycol-dimaleimidyl Succinate
ether was repeated three times. The PEG-SS2 was further About 12 g of PEG-disuccinate and 1 g N-hydroxymale
purified by dissolving in 100 ml of 0.1M pH 2.2 citrate/ imide (Aldrich Chemical Co.) were placed in a 250 ml three
phosphate buffer and filtering through a 4-8 micron sintered neck round bottom flask with 50 ml of anhydrous DMF
glass filter. The PEG-SS2 was extracted with chloroform under nitrogen. The mixture was dissolved at 60° C. with
(100 ml 2X) and the solvent was evaporated under reduced 20
mechanical stirring and cooled to 0°C. A solution of 1.82 g
pressure in a rotary evaporator. The PEG-SS2 was then dicyclohexylcarbodiimide in DMF (5 ml) was added drop
dissolved in toluene and precipitated with petroleum ether, wise to the flask. The reaction was allowed to mix overnight
dried under vacuum overnight at room temperature, and under nitrogen at room temperature. Dicyclohexylurea was
stored in a refrigerator. removed by filtration and the product was obtained by
25
Example 2 adding toluene and precipitating with petroleum ether. Dis
solution in toluene and reprecipitation with petroleum ether
Synthesis of N-hydroxysuccinimide Ester of Dicarboxym were repeated three times. The purified product was dried
ethyl Polyethylene Glycol under vacuum and stored in a desiccator.
Dicarboxymethyl poly(ethylene glycol) (mol. wt. 3400) 30 Example 5
purchased from Shearwater Polymers, Inc., Huntsville, Ala.
(5 g) and N-hydroxysuccinimide purchased from Sigma Synthesis of Polyethylene Glycol-diphthalimidyl Succinate
Chemical Co., St. Louis, Mo. (1 g) were dissolved in 30 ml About 15 g of PEG-disuccinate and 1.65 g N-hydroxyph
of anhydrous DMF with mechanical stirring under nitrogen. thalimide (Aldrich Chemical Co.) were placed in a 250 ml
The solution was cooled to 0°C. and a solution of dicyclo 35 three neck round bottom flask with 30 ml of anhydrous DMF
hexylcarbodiimide (1.79g) in 5 ml DMF was added drop under nitrogen. The mixture was dissolved at 60° C. with
wise. The stirring was continued in the cold for 3 hours then mechanical stirring and cooled to 0°C. A solution of 1.82 g
at room temperature overnight (16 hrs). Dicyclohexylurea dicyclohexylcarbodiimide in DMF (5 ml) was added drop
which precipitated was removed by filtration. Toluene (100 wise to the flask. The reaction was allowed to mix overnight
ml) was added to the filtrate and cooled to 0°C. The product under nitrogen at room temperature. Dicyclohexylurea was
was then precipitated by addition of petroleum ether. The removed by filtration and the product was obtained by
precipitate was collected on a sintered glass filter. Dissolu adding toluene and precipitating with petroleum ether. Dis
tion in toluene and reprecipitation with petroleum ether was solution in toluene and reprecipitation with petroleum ether
repeated three times. The product was dried under vacuum were repeated three times. The purified product was dried
in a desiccator. under vacuum and stored in a desiccator,
45
Example 3 Example 6
Synthesis of Polyethylene Glycol-di-oligoglycolide Disuc Preparation of Two Component Adhesive
cinimidyl Succinate The following procedure was used to prepare a two
A 500 ml three neck round bottom flask was flame dried 50 component adhesive using a variety of protein sources, and
under nitrogen. 50 g of PEG (mol. wt. 3400), 300 ml of bifunctional crosslinking agents. Aqueous solutions of a
Xylene, and drop of 0.33M stannous ottoate solution in protein and a crosslinking agent as listed in Table 1 were
Xylene were charged into the flask with a continuous nitro pipetted (0.2 ml of each solution) into a porcelain test well
gen purge. The flask was heated to boil the solution and 50 and mixed continuously with a stainless steel rod. The cure
ml of xylene were removed by distillation. The solution was 55 time and physical consistency of each of the two component
then cooled to room temperature. 17 g of glycolide (Boeh adhesives are also listed in Table 1.
finger Ingleheim KG, Ingleheim, Germany) was added to the The data indicated that fish and bovine gelatin, egg and
flask and the reaction mixture was refluxed under nitrogen serum albumin as well as casein protein crosslinked with
for 16 hours. The copolymer reaction mixture was filtered PEG-SS2 provided an adhesive which was very elastic, had
hot to remove polyglycolide homopolymer. The copolymer good adhesive strength and a relatively rapid cure rate.
 5,583,114
10

TABLE 1.
Bifunctional Cure
Protein Crosslinking agent Time Consistency
Fish Gelatin 130 mg/ml 40 sec Strong gel, very
Lot 23H0307 PEG-SS2 3400 mw elastic, slightly
Sigma sticky
40% 0.1 M pH 10
Carb/Bicarb
Fish Gelatin 260 mg/ml 40 sec Strong gel, very
Lot 23H0307 PEG-SS2 3400 mw elastic, slightly
Sigma sticky
40% 0.1 M pH 10
Carb/Bicarb
Fish Gelatin 130 mg/ml 120 sec Soft gel, very
Lot 23H0307 PEG-SS2 10,000 mw sticky
Sigma
40% 0.1 M pH 10
Carb/Bicarb
Fish Gelatin 260 mg/ml 110 sec Soft gel to
Lot 23H0307 PEG-SS2 10,000 mw elastic,
Sigma moderately
40% 0.1 M pH 10 sticky
Carb/Bicarb
Gelatin Bovine 130 mg/ml 40 sec Soft gel, not
Skin Lot 53H0271 PEG-SS2 3400 mw elastic
Sigma
40% 0.1 M pH 10
Carb/Bicarb
Gelatin Bovine 260 mg/ml 40 sec Soft gel, not
Skin Lot 53HO271 PEG-SS2 3400 mw elastic
Sigma
40% 0.1 M pH 10
Carb/Bicarb
Gelatin Bovine 130 mg/ml 40 sec Soft gel, not
Skin Lot 53HO271 PEG-SS2 10,000 mw elastic
Sigma
40% 0.1 M pH 10
Carb/Bicarb
Gelatin Bovine 260 mg/ml 120 sec Soft gel, not
Skin Lot 53HO271 PEG-SS2 10,000 mw elastic
Sigma
40% 0.1 M pH 10
Carb/Bicarb
Casein 130 mg/ml 40 sec Strong gel,
pH 9.4 12.6% PEG-SS2 3400 mw elastic, not sticky
Carb/Bicarb
Poly-L-Lysine 130 mg/ml 20 sec Waxy, no
50 mg/ml HO PEG-SS2 3400 mw adhesive strength
300,000 mw
Carb/Bicarb
Poly-L-Lysine 260 mg/ml 15 sec Waxy, no
50 mg/ml H2O PEG-SS2 3400 mw adhesive strength
300,000 mw
Carb/Bicarb
Poly-L-Lysine 130 mg/ml 10 sec Waxy, no
50 mg/ml HO PEG-SS2 10,000 mw adhesive strength
300,000 mw
Carb/Bicarb
Poly-L-Lysine 260 mg/ml 10 sec Waxy, no
50 mg/ml HO PEG-SS2 10,000 mw adhesive strength
300,000 mw
Carb/Bicarb
Chicken Egg Albumin 130 mg/ml 210 sec soft, tacky
40% 0.08M pH 10 PEG-SS2 3400 mw
Carb/Bicarb
Rabbit Serum Albumin 130 mg/ml 20 sec Very elastic,
(RSA) Sigma PEG-SS2 3400 mw good adhesive
Lot 19F930
40% 0.1 M pH 10
Carb/Bicarb
Human Serum Albumin 130 mg/ml 20 sec Very elastic,
(HSA) Sigma PEG-SS2 3400 mw good adhesive
Lot 63H9041 strength, not
40% 0.1 M pH 10 sticky
Carb/Bicarb
HSA 130 mg/ml 20 sec Very elastic,
Sigma PEG-SS2 3400 mw good adhesive
 5,583,114
11 12

TABLE 1-continued
Bifunctional Cure
Protein Crosslinking agent Time Consistency
Lot 63H9041 strength, not
40% 0.1 M pH 8.44 sticky
CarbBicarb
HSA 260 mg/ml O sec Very elastic,
Sigma PEG-SS23400 milw good adhesive
Lot 63H9041 strength, not
40% 0.1 M pH 8.44 sticky
Carbi Bicarb
HSA 130 mg/ml 30 sec Very elastic,
Sigma PEG-SS2 10,000 mw slight adhesive
Lot 63H904 strength, very
40% 0.1 M pH 8.44 sticky
Carbi Bicarb
HSA 260 mg/ml 25 sec Very elastic,
Sigma PEG-SS2 10,000 mw slight adhesive
Lot 63H904 strength, very
40% 0.1 M pH 8.44 sticky
CarbBicarb
HSA 130 mg/ml 20 sec Turned brown
Baxter Healthcare PEG-dimaleimidyl upon curing,
Corp. succinate hard gel, not
Lot 2837A238AA Example 4 sticky
Carbi Bicarb
HSA 130 mg/ml 10 sec Turned red upon
Baxter PEG-diphthalimidyl curing, hard gel,
Lot 2837A238AA succinate not sticky
Carbi Bicarb Example 5
HSA 130 mg/ml 8 sec Hard gel, not
Baxter PEG-dicaboxymethyl sticky, no color
Lot 2837A238AA disuccinimidyl change
Carbi Bicarb Example 2
HSA 130 mg/ml 40 sec Hard gel, not
Baxter PEG-dioliglycolide sticky, no color
Lot 2837A238AA disuccinimidyl succinate change
Carbibicarb Example 3
HSA 130 mg/ml 30 sec Hard gel, not
Baxter PEG-disuccinimidyl sticky, no color
Lot 2837A238AA propionate change
Carbibicarb PEG(SPA)2
HSA 260 mg/ml 40 sec Hard gel, not
Baxter PEG-disuccinimidyl sticky, no color
Lot 2837A238AA propionate change
Carbibicarb PEG(SPA)2
HSA 130 mg/ml 48 hrs Hard gel, not
Baxter PEG-dioxycarbonyl (cure) sticky, no color
Lot 2837A238AA imidazole change
Carb/Bicarb
HSA 130 mg/ml 140 sec Hard gel, not
Baxter PEG-dinitrophenyl sticky, changed
Lot 2837A238AA carbonate to bright yellow
Carbi Bicarb color
HSA 260 mg/ml 140 sec Hard gel, not
Baxter PEG-dinitrophenyl sticky, changed
Lot 2837A238AA carbonate to bright yellow
Carbi Bicarb color
HSA 130 mg/ml 8 hrs Hard gel, not
Baxter PEG-ditresylate (viscous) sticky, no color
Lot 2837A238AA PEG(tres)2 24 hrs change
CarbiBicarb (cure)
HSA 130 mg/ml 72 hrs Hard gel, not
Baxter PEG-diglycidyl ether (cure) sticky, no color
Lot 2837A238AA PEG(epox)2 change
Carbibicarb
HSA 130 mg/ml O. cle Liquid
Baxter PEG-dialdehyde
Lot 2837A238AA PEG(ald)2
CarbfBicarb

mw = weight average molecular weight

 5,583,114
13 14
Example 7 protein from ICN Biomedical, Inc. were dissolved in 0.1M
Effect of Buffer and pH pH 10 carbonate/bicarbonate buffer and concentrated to 40%
Two component adhesives were prepared according to the by the same method used for HSA.
process described in Example 6 except that the pH of the Various concentrations of PEG-SS2 (3,400 mw and
buffer in the protein solution was changed as listed in Table 10,000 mw) were prepared in deionized water. The albumins
2. The data indicate that a preferred pH range is about and crosslinking agent solutions were delivered in equal
8.44-10.0. volume using a 1 ml dual syringe. The syringe tips were
fitted with a Y connector which connected to a specially
TABLE 2 machined TEFLQN adaptor inserted into a 1.8 in.x0.187 in.
10 (4.57 cm×0.475 cm) dia. spiral mixer nozzle (TAH Indus
Crosslinking tries, Inc., Robbinsville, N.J., part no. 150-312). The adhe
agent Cure sive mixture was injected through the mixer directly onto the
Protein PEG-SS2 Time Consistency
test substrate for adhesion testing.
HSA 130 mg/ml 10 min Initially softer Freshly excised guinea pig skin was cut into strips and a
Baxter
Lot 2837A238AA
3400 mw adhesive, hardens
with aging
15 polystyrene window with an opening of 0.5x1.0 inches (1.27
40% 0.1 M pH 7.4 cmx2.54 cm) was placed on one end of the strip to contain
Carbibicarb the glue in a specific region. Upon filling the window with
HSA 130 mg/ml 20 sec Very elastic, good glue it was covered with another strip of guinea pig skin. A
Sigma 3400 mw adhesive strength, 500 g steel weight was placed on top of this assembly for
Lot 63H9041 not sticky 20 about one minute. The sample was peeled apart in the jaws
40% 0.1 M pH 8.44
CarbiBicarb of a computer controlled mechanical testing machine (880
HSA 130 mg/ml 10 sec Hard gel, not sticky Material Test System, MTS System, Inc., Minneapolis,
Sigma 3400 mw Minn.) set at a strain rate of 0.8 in./min. (2 cm/min.) with a
Lot 63H9041
40% 0.15 M pH 9.07 gage length of 1 in. (2.54 cm) and a 5 lbs. (2.27 kg) load cell.
Carbibicarb 25 Peel force was recorded after the initiation of adhesive
HSA 130 mg/ml 5 sec Hard gel, not sticky failure as the constant force require to continue peeling as
Sigma
Lot 63H904
3400 Imw shown in FIG. 1. Four replicates were performed for each
test condition. The results of this test are listed in FIG. 2.
40% 0.2 M pH 9.52
Carb/Bicarb
HSA 260 mg/ml 5 sec Hard gel, not sticky 30
Sigma 3400 inw
Example 9
Lot 63H9041
40% 0.2M pH 9.52 Measurement of Adhesive Sealant Burst Strength
CarbiBicarb A pressurization assembly illustrated in FIG. 3 was used
HSA 130 mg/ml 7 sec Elastic to hard gel, to test the bursting strength of materials used to seal stan
Sigma
Lot 63H904
10,000 mw slightly sticky 35 dardized holes or slits in test membranes. This assembly
40% 0.2 M pH 9.52 included an aluminum pressure vessel (1) having a 35mm
CarbfBicarb inside diameter fitted with a millivolt output type pressure
HSA 260 mg/ml. 7 sec Elastic to hard gel, transducer (2) with a range of 0 to 15 psig (MODELPX236,
Sigma
Lot 63H9041
10,000 mw slightly sticky Omega Engineering, Inc., Stamford, Conn.) and a pressure
40% 0.2 M pH 9.52
inlet port (3). To perform a test, about a 5 mm diameter hole
Carb/Bicarb (4) (or other standardized defect) was cut in the center of a
HSA 130 mg/ml 25 sec Very elastic, not test membrane (5) using a die cutter. The membrane was
Baxter
Lot 2837A238AA
3400 nw sticky then placed on a piece of 0.4 mm thick TEFLON film with
40% 0.1 M pH 10 the hole in the membrane centered in a larger (14 mm
CarbiBicarb 45 diameter) hole in the TEFLON film. The TEFLON film was
HSA 130 mg/ml 25 sec Very elastic, not then placed on a flat surface with the membrane side down
Sigma
Lot 63H9041
3400 nw sticky and adhesive sealant test material was applied to fill the hole
40% 0.1 M pH 10 in the film. A solid TEFLON block was then quickly placed
CarbiBicarb over the sealant prior to cure so that the TEFLON film
50 served as a spacer to create a layer of sealant exactly 0.4 mm
mw = weight average molecular weight thick. After the desired cure time elapsed, the TEFLON
block was inverted and the membrane was carefully peeled
Example 8 off to obtain a circular patch of sealant (6) covering the hole
in the membrane. The test membrane with sealed defect was
Effect of Crosslinking Agent on Adhesive Strength 55 then mounted onto the open end of the pressure vessel (7) by
A 30% HSA (Human Serum Albumin) solution from placing it between two rubber washers (8) and then between
Sigma Chemical Co. and a 25% HSA solution from Baxter two metal washers (9). An air tight seal was obtained by
Healthcare, Inc. were dialyzed against 0.1M carbonate/ screwing the threaded cover (10) onto the matching threads
bicarbonate pH 10 buffer at 4°C. overnight and concentrated (11) of the pressure vessel. The opening in the cover (12)
to about 40% by ultra-filtration through a 50,000 molecular 60 was also 35 mm in diameter which, in combination with the
weight cut-off cellulose ester disc membrane (Spectrum 35 mm inside diameter washers, provided a fixed membrane
Medical Industries, Inc.) in a pressure filtration cell under surface area for pressure testing.
nitrogen at 60 psig. The final concentration was calculated Two types of membranes were used, either a collagen
based on the volume of collected filtrate. The maximum membrane or a freshly excised porcine pericardium sample.
concentration obtained under these conditions during over 65 The porcine pericardium sample was either used immedi
night ultra-filtration was typically 42-45%. The RSA (Rab ately upon harvest or after storage in a moisture-proof
bit Serum Albumin) from Sigma and RSA crystallized container at 4 C. for no longer than 24 hours. Under these
 5,583,114
16
conditions there was no discernible difference in Sealant The same process was repeated for additional 25% HSA
performance based on storage time of that tissue. solutions by dialyzing against 0.08M carbonate/bicarbonate
The pressurization sequence was initiated by injecting air buffers at pH 9 and pH 8. A pH 7 solution of HSA was
into the pressure inlet at a fixed rate of one cubic centimeter
per second using a syringe pump (Sage Instruments Model obtained by concentration of the original 25% HSA solution
351, Orion Research, Inc.). The pressure transducer was to 40% by ultrafiltration. The crosslinking agent solution
connected to a digital strain gauge meter (Omega Model PEG-SS2 (3400 mw) was 130 mg dissolved in one ml
DP205-S, Omega. Engineering, Inc.) programmed to read
pressure (ram mercury) and to display the peak pressure deionized water. The albumin and crosslinking agent solu
value at the time of adhesive sealant rupture. Replicate tests 10 tions were delivered in equal volume using a one ml dual
gave reproducible peak pressure values and the standard syringe as in Example 8. The pressure tests were performed
deviation was reported in each case. as above using collagen membrane except that the Sealant
Pressure tests were performed with an adhesive compo
sition of 40% HSA (or RSA) in 0.08M carbonate/bicarbon hydrogel was aged before testing. The results are also listed
ate buffer at different pH values with 3,400 m.wt. PEG-SS2 15 in Table 4. These data demonstrate that optimal pressure test
(130 mg/ml) on collagen and pericardium membranes. The values are achieved faster with increasing pH of the albumin
results listed in Table 3 demonstrate excellent sealant per solution. Moreover, the resultant cured sealant obtained after
formance with typical peak pressure values of about 130 mm
Hg. complete curing has taken place is unexpectedly higher with
In addition, the peak pressure for the above sealants after 20 higher pH of the albumin solution.
soaking in saline solution was measured. The test was
performed as described above except that the surface of the TABLE 3
sealant coated membrane was flooded with saline for up to Burst
a time period of 90 minutes before pressurization. Although Adhesive Pressure
the sealant hydrogel swelled to about double in thickness, 25 Tissue Tissue Opening Composition (mm Hg)
substantial retention of sealant performance was retained. Collagen 4.56 mm dia. hole HSAPEG-SS2 50
Table 4 shows the data obtained by testing a variety of Collagen 5 mm slit HSAPEG-SS2 12
proteins including fish skin gelatin, chicken egg albumin, Collagen 4.56 Inn dia, hole RSA:PEG-SS2 130
and fibrinogen. Fibrinogen mixed with thrombin ("fibrin Collagen 5 mm slit RSAPEG-SS2 125
glue', BERIPLASTP sealant, Behringwerke, Marburg, Ger 30 Porcine 4.56 mm dia, hole HSAPEG-SS2 155
Pericardium
many) was also used as a control sealant material. None of Porcine 5 mm slit HSAPEG-SS2 130
these materials performed as well as the serum albumin Pericardium
examples. The main disadvantage was the cure and aging Porcine 4.56 mm dia, hole RSA:PEG-SS2 125
time required to achieve significant strength. In particular, Pericardium
Porcine 5 Inn slit RSA:PEG-SS2 130
chicken egg albumin required twenty-five minutes of post 35 Pericardium
cure aging to achieve the same burst strength obtained from
serum albumin aged for less than five minutes.
TABLE 4
Pressure Test of Different Proteins Using Collagen and Pericardium
HSA: 40%, 0.08M CarfBicarb Buffer in Saline Lot #2837a328AA
RSA: 40% 0.08M Carb/Bicarb Buffer in Saline Lot #82-45-0050 NC
PEG-SS2: 3400 mw lot #103128-110 (130 mg/ml)
Defect: 4.56 mm hole
Air Fow Rate: 1 ccis
Pressure
(mm Hg)
Protein Crosslinker Membrane Ave Stdev Comments

HSA pH 10 PEG-SS2 Collagen 149 9 Nobubbles

Pericardium 154 4 5 min after curing
Pericardium 196 5 10 min after curing
HSA pH 10 PEG-SS2 Collagen 44 5 min after curing
155 10 min after curing
162 20 min after curing
HSA pH 9 PEG-SS2 Collagen 08 5 min after curing
14 10 min after curing
116 20 min after curing
HSA pH 8 PEG-SS2 Collagen 36 5 min after curing
78 10 min after curing
90 20 min after curing
HSA pH 7 PEG-SS2 Collagen 30 10 min after curing
52 20 min after curing
RSA pH 10 PEG-SS2 Collagen 134 5 Nobubbles
Pericardium 126 10 5 min after curing
Pericardium 194 9 10 min after curing
Fish Gelatin pH 10 PEG-SS2 Collagen 34 2 10 min after curing
40%
(Sigma)
 5,583,114
17 18
TABLE 4-continued
Pressure Test of Different Proteins Using Collagen and Pericardium
HSA: 40% 0.08M Carb/Bicarb Buffer in Saline Lot #2837a328AA
RSA: 40% 0.08M Carb/Bicarb Buffer in Saline Lot #82-451-0050 INC
PEG-SS2: 3400 mw lot #103128-110 (130 mg/ml)
Defect: 4.56 mm hole
Air Flow Rate: 1 ccfs
Pressure
(mm Hg)
Protein Crosslinker Membrane Ave Stdev Comments
Chicken Egg PEG-SS2 Collagen 14 3 10 min after curing
Albumin pH 10 151 5 45 min after curing
40%
(Sigma)
Fibrin Glue Pericardium 8 2 5 min after curing
(BERIPLAST-P) with saline, glue
Used according to slid off easily
mfg. instructions 39 2 5 min after curing
without saline,
leaked underneath
Bovine Fibrinogen PEG-SS2 Collagen 8 2 5 min after curing
pH 10 8 2 60 min after
15% curing, glue slid
(Sigma) off easily
25
Example 10 Under these conditions it was observed that anastomotic
lines coated with the sealant were air tight whereas anasto
Use of a Two Component Adhesive Sealant in General and motic lines not sealed were not air tight.
Thoracic Surgery Example 11
An anesthetized pig was used as an experimental model 30
for thoracic surgical complications such as staple line leaks Use of Two Component Adhesive to Prevent Post-Surgical
during lung and bronchus resections, bronchopleural fistu Adhesions
las, and other conditions resulting in pneumothorax. The tissue sealant hydrogel tested was a two part liquid
The two component adhesive included Part A, a 40% HSA system. Part A was a sterile 40% (w/v) solution of human
prepared by dialysis of commercially available HSA (25% 35 serum albumin in isotonic pH 10 carbonate buffer (0.1M).
Solution, BUMINATE 25%, Baxter Healthcare Corp., Part B was a 400 mg/ml solution of 10,000 molecular weight
Hyland Division, Glendale, Calif.) against 0.08M pH 10 PEG-SS2 (polyethylene glycol disuccinimidyl succinate) in
carbonate?bicarbonate buffer followed by concentration to sterile distilled water prepared just prior to use. Solutions A
40% by ultrafiltration at 50 psi using a 50,000 molecular and B were mixed in equal volumes with a dual syringe
weight cut-off cellulose ester disc membrane and Part B, a system connected to a static mixing head (Tah Industries,
130 mg/ml solution of 3,400 m.wt. PEG-SS2 dissolved in Inc.).
sterile distilled water no more than 30 minutes prior to use. Post-surgical adhesion prevention evaluation of this seal
ant formulation was initiated in a series of ten female
The PEG-SS2 was synthesized and purified as described in rabbits. A 2x2 cm area of the abdominal wall was excised
Example 1. down to the fascia on each side of the abdominal cavity
A stab wound was made on the lung of an anesthetized pig 45
exposed by a midline laparotomy incision. The uterine horns
with a scalpel which resulted in significant air leakage were injured by scraping 20 times with a no. 10 scalpel
during inspiration as evidenced by bubbling of air through blade. Each animal served as its own control by randomly
irrigation fluid administered to the site. The wound was applying test material to only one of the abdominal wall
blotted with gauze to remove blood and fluid. The respirator injuries. The uterine horns were then attached with two
was turned off and the adhesive was applied as a sealant 50 stitches to the abdominal wall within a few millimeters of
using a dual syringe (Behring PANTAJECT syringe, the edge of the wound closest to the laparotomy incision.
Behringwerke, Marburg, Germany) equipped with a spiral Two weeks after surgery the rabbits were examined in
mixing tip. After a 20 second cure time ventilation was order to evaluate and score the extent, type, and tenacity of
restored and the lung was again covered with irrigation fluid. adhesions present on the abdominal wall injury sites. These
No air leaks were observed. 55 results are shown in Table 5. The rating system used to
A functional end-to-end anastomosis in pig intestine was obtain these scores is shown in Table 6. Although technical
conducted using a standard stapling procedure. The adhesive difficulties were encountered as noted in Table 5, the test
material described above was applied to the staple lines. material clearly provided an unexpected benefit in both the
This resulted in a clear, adherent hydrogel coating which prevention of adhesions and a reduction in their severity
appeared to seal the anastomotic line. without the presence of a known active ingredient.
 5,583,114
19 20
TABLE 5
Scoring of Adhesions Formed in Material Evaluation
Characteristic
Extent Type Tenacity
Animal Control Treatment Control Treatment Control Treatment
BAM 8, 2 0+ 3 0-- 3 0
BAM9 3 1 3 3 1
BAM 10 0- 1. 0. 3 0+ 2
BAM 1 Ok O 0k O 0k O
BAM 12 4 4 3 3 3 3
BAM 13 2 1 3 2 3 2
BAM 14 1* O 3-k O 3-k O
BAM 15 1 0kk 1 0k+ 2 0**
BAM 16 1 Ok l 0* 2 0*
BAM 17 1 Ok 1. 0* 2 O*
Average 1.5 0.7 1.8 0.9 2.1 0.8

*Uterine horn tacked to abdominal wall with only one suture

**Uterine horn no longer sutured to abdominal wall
+ Fascia removed with peritoneum and muscle layers

diester diradical of the formula, -C(O)-(CH2)-

TABLE 6 C(O)- where c is an integer from 2-10 and where the
Adhesion Scoring 25 aliphatic portion of the diradical may be saturated or
Adhesion Scoring unsaturated, a dicarbonate diradical of the formula
Characteristic Adhesion Score -C(O)--O-(CH2)-O-C(O)- where d is an inte
ger from 2-10, and an oligomeric diradical represented
Extent (% sidewall involvement) by the formulas -R-C(O)-, -R-C(O)-
None O 30 (CH2)-C(O)-, or -R-C(O)-O-(CH2)-O-
s l C(O)- where c is an integer from 2-10, d is an integer
250 2 from 2-10, and R is a polymer or copolymer having
s75 3 1-10 monomeric fragments selected from the group
>75 4. consisting of lactide, glycolide, trimethylene carbonate,
Type 35 caprolactone and p-dioxanone, and
None O wherein -G is a leaving group selected from the group
Filmy, no vessels (transparent) 1. consisting of succinimidyl, maleimidyl, phthalimidyl,
Opaque, no vessels (translucent) 2 imidazolyl, nitrophenyl or tresyl, and
Opaque, small vessels present grossly 3 - - o
Opaque, larger vessels present grossly 4. wherein al combination of the first and second mixtures is
Tenacity 40 initially liquid and then cures on the surface of tissue to
give a flexible, substantive matrix which bonds to the
None O tissue and has a burst strength greater than about 10
Adhesions essentially fell apart 1 mmHg.
Asian Riccion for lysis : 2. The adhesive mixture of claim 1 wherein the protein in
eq arp y the first mixture is about 35–45 wt/vol% serum albumin.
3. The adhesive composition of claim 1 wherein the serum
We claim: . - - - albumin is human serum albumin.
1. An adhesive composition consisting essentially of 4. The adhesive composition of claim 1 wherein the buffer
i) a first aqueous mixture of about 20-60 wit/vol% serum is 0.05-0.15 molar carbonate/bicarbonate buffer at a pH of
albumin in about 0.01-0.25 molar buffer at a pH in a about 9.0-10.5.
range of about 8.0-11.0, 50 5. The adhesive composition of claim 1 wherein the
ii) crosslinking
a second aqueous mixture of about 50-800 mg/ml of a
agent having amolecular weight in a range
second aqueous mixture is about 50–300 mg/ml of a
crosslinking agent having a molecular weight in a range of
of about 1,000-15,000, wherein the crosslinking agent about 1,000–5,000.
is of the formula 6. The adhesive composition of claim 1 wherein the ratio
55 of a volume of the first mixture to a volume of the second
G-LM-PEG-LM-G mixture is in a range of about 1:10 to about 10:1.
7. The adhesive composition of claim 1 wherein-LM
wherein -PEG- is a diradical fragment represented by is an oligomeric diradical -R-C(O)-(CH2)-C(O)-
the formula where c is an integer from 2-10 and R is a polymer or
60 copolymer having 1-10 monomeric fragments selected from
-O-(CH-CH-O-)- the group consisting of lactide, glycolide, trimethylene car
bonate, caprolactone and p-dioxanone.
where a is an integer from 20-300; 8. The adhesive composition of claim 1 wherein -G is
wherein-LM- is a diradical fragment selected from the succinimidyl.
group consisting of a carbonate diradical of the for- 65 9. An in vivo method of adhering tissue comprising the
mula, -CO)-, a monoester diradical of the formula, steps of topically applying and bonding an adhesive mixture
-(CH2)C(O)- where b is an integer from 1-5, a of claim 1 to the tissue.
 5,583,114
21 22
10. An in vivo method of sealing air leaks in pulmonary
tissues comprising the step of topically applying and curing
the adhesive mixture of claims 1 to an air leak site in the wherein -PEG- is a diradical fragment represented by
pulmonary tissue. the formula
11. An in vivo method to prevent post-surgical adhesions
comprising the step of topically applying and curing the
adhesive mixture of claims 1 to tissue surrounding a surgical
site. where a is an integer from 20-300;
12. An in vivo method to seal tissue comprising the step wherein-LM- is a diradical fragment selected from the
of topically applying and bonding the adhesive mixture of 10 group consisting of a carbonate diradical of the for
claims 1 to tissue to prevent or control blood or other fluid mula, -C(O)-, a monoester diradical of the formula,
leaks.
13. The adhesive composition of claim 1 wherein the -(CH2)C(O)- where b is an integer from 1-5, a
second aqueous mixture is about 300-800 mg/ml of a diester diradical of the formula, -C(O)-(CH2)-
crosslinking agent having a molecular weight in a range of 15 C(O)- where c is an integer from 2-10 and where the
about 5,000-15,000. aliphatic portion of the diradical may be saturated or
14. The adhesive composition of claim 13 wherein unsaturated, a dicarbonate diradical of the formula
-LM- is a diester diradical of the formula -C(O)- -C(O)-O-(CH2)-O-C(O)- where d is an inte
(CH)-C(O)-. ger from 2-10, and an oligomeric diradical represented
15. The adhesive mixture of claim 1 wherein -LM- is 20 by the formulas -R-C(O)-, -R-C(O)-
a diester diradical of the formula, -C(O)-(CH2)- (CH2)-C(O)-, or -R-C(O)-O-(CH2)-O-
C(O)- where c is an integer from 2-10 and where the C(O)- where c is an integer from 2-10, d is an integer
aliphatic portion of the diradical may be saturated or unsat from 2-10, and R is a polymer or copolymer having
urated. 1-10 monomeric fragments selected from the group
16. The adhesive composition of claim 15 wherein 25 consisting of lactide, glycolide, trimethylene carbonate,
-LM- is a oligomeric diradical derived from polyglycolic caprolactone and p-dioxanone; and
acid. wherein -G is a leaving group selected from the group
17. A method of making a tissue adhesive consisting of consisting of succinimidyl, maleimidyl, phthalimidyl,
the step of forming a mixture of imidazolyl, nitrophenyl or tresyl, and
30
i) a first aqueous mixture of about 20-60 wit/vol% serum wherein a combination of the first and second mixtures is
albumin in about 0.01-0.25 molar buffer at a pH in a initially liquid and then cures on the surface of tissue to
range of about 8.0-11.0, give a flexible, substantive matrix which bonds to the
ii) a second aqueous mixture of about 50-800 mg/ml of a tissue and has a burst strength greater than about 10
crosslinking agent having a molecular weight in a range 35
mmHg.
of about 1,000-15,000, wherein the crosslinking agent
is of the formula