liii
`
`III
`
`Ill
`
`111111
`
`11111
`
`11111
`
`1111
`
`HIll
`
`liii
`
`11111
`
`US00783798682
`
`1110
`
`IIIH
`
`IH
`
`111111
`
`1111
`
`12 United States Patent
`Chenault
`
`10 Patent No
`45 Date of Patent
`
`US 7837986 B2
`Nov 23 2010
`
`54 PROTEIN-BASED POLYMER TISSUE
`ADHESWES FOR MEDICAL USE
`
`75 Inventor
`
`73 Assignee
`
`Henry Keith Chenault Hockessin DE
`US
`
`du Pont de Nemours and
`Company Wilmington DE US
`
`Notice
`
`Subject
`
`to any disclaimer the term of this
`is extended or adjusted under 35
`patent
`U.S.C 154b by 747 days
`21 Appl No 11/289169
`22 Filed
`
`Nov 29 2005
`
`65
`
`Prior Publication Data
`
`US 2006/0115531 Al
`
`Jun 12006
`
`Related U.S Application Data
`60 Provisional application No 60/63 2272 flIed on Dec
`12004
`
`51
`
`52
`58
`
`56
`
`mt Cl
`A61J 31/00
`U.S Cl
`
`2006.01
`
`424/78.2
`
`Field of Classification Search
`424/78.2
`See application file for complete search history
`
`References Cited
`
`U.S PATENT DOCUMENTS
`
`5830986
`6413742 Bl
`6620125
`6696089 B2
`2002/0022588
`
`11/1998
`
`Merrill et al
`
`7/2002 Olsen et al
`9/2003 Red
`Kabanov et al
`2/2004
`
`2/2002 Wilkie et al
`
`2004/0063613 Al
`
`4/2004
`
`Rolkeet al
`
`FOREIGN PATENT DOCUMENTS
`
`liP
`
`WO
`WO
`WO
`
`004 597
`W097/3003
`WO 99/66964
`WO01/4576
`
`5/2000
`
`8/1997
`
`12/1999
`6/200
`
`OTHER PUBLICATIONS
`
`el al Journal of Biomedical Materials Research
`Otani
`157-166 1996
`Biomaterials vol 17 No 14 1387-1391 1996
`Ota.ni et
`for tnternaliona Application Serial No PCT/US2005/
`Search Report
`043389
`
`vol 31
`
`Primary ExaminerMichael
`Assistant ExaminerJames
`
`Hartley
`
`Rogers
`
`57
`
`ABSTRACT
`
`Tissue adhesives formed by crosslinking albumin andlor
`gelatin with certain
`andlor polycarboxylates
`pulyamines
`carbodiimide are disclosed The use of
`using water-soluble
`the tissue adhesives for medical and veterinary applications
`such as topical wound closure and surgical procedures
`such
`as intestinal anastomosis vascular anastomosis tissue repair
`and ophthalmic
`procedures
`drug delivery anti-adhesive
`applications and as
`nence are described
`
`bulking agent to treat urinary inconti
`
`3236834
`
`2/1966 Sofifer
`
`11 Claims No 1rawings
`
`EXHIBIT
`
`

`

`US 7837986 B2
`
`PROTEIN-BASED POLY1VIER TISSUE
`ADHESIVES FOR MEDICAL USE
`
`TO RELATED
`CROSS-REFERENCE
`APPLICATION
`
`This application claims priority under 35 U.S.C 119 from
`U.S Provisional Application Ser No 60/632272 filed Dec
`12004
`
`FIELD OF THE INVENTION
`
`The invention relates to the field of medical adhesives
`More specifically the invention relates to protein-based tis
`sue adhesives formed by crosslinking albumin and/or gelatin
`polyamines
`with certain
`and/or
`certain polycarboxylates
`water-soluble carbodiimide
`
`using
`
`BACKGROUND OF THE INVENTION
`
`Tissue adhesives have many potential medical applica
`tions including topical wound closure supplementing
`or
`replacing sutures or staples in internal surgical procedures
`adhesion of synthetic onlays or inlays to the cornea drug
`and as anti-adhesion
`devices
`barriers to preveot
`delivery
`adhesions Conventional
`tissue adhesives are
`post-surgical
`generally not suitable for wide range of adhesive applica
`tions For example cyanoacrylate-based adhesives have been
`used for topical wound closure but
`the release of toxic deg
`radation products limits their use for internal applications
`are slow curing have poor mechani
`Fibrin-based
`adhesives
`cal strength and pose
`risk of viral
`infection Additionally
`adhesives do not covalently
`the Fibrin-based
`bind to the
`
`underlying tissue
`Protein-based tissue adhesives using albumin or gelatin are
`known For example Wilkie et al U.S Patent Application
`et al WO
`Publication No 2002/0022588
`and Tammishetti
`describe tissue adhesives formed by crosslinldng
`99/66964
`albumin with
`carbodiimide The addition of
`polyamine
`
`10
`
`15
`
`20
`
`25
`
`30
`
`specifically polylysine or chitosan or
`polycarboxylate 40
`specifically citric acid or polyacrylic acid to increase the
`is also described in those disclosures
`rate of crosslinking
`However
`for use as
`
`tissue adhesive for in vivo applications
`such as intestinal anastomosis adhesives with lower toxicity
`and enhanced adhesive strength are needed The use of car-
`bodiimides in the adhesive composition causes some toxicity
`problems The toxicity problem is exacerbated by the use of
`toxic polyamine such as polylysine Chitosan is not soluble
`enough to be effective in increasing the adhesive properties of
`the adhesive
`
`50
`
`less
`
`Otani et al
`
`Biotnoteniols
`
`.1 Biomed Mo/en Res 31157-1661996 and
`171387-1391 1996 describe
`tissue adhe
`gelatin and polyL-glutamic
`sive prepared by erosslinking
`acid with water-soluble earbodiimide Although the adhe
`toxic than the albumin-polylysine
`sive is
`adhesive ss
`described above it
`lacks adhesive strength
`Therefore in the continuing search for new tissue adhesives
`for in vivo applications such as intestinal anastomosis
`the
`protein-based tissue
`problem to be solved is
`to provide
`toxicity and higher adhesive strength so
`adhesive with lower
`than those currently available
`have addressed the stated problem by discov
`Applicants
`tissue adhesive formed by crosslinking albumin and/
`ering
`or gelatin with certain polyamines and/or certain polycar
`earbodiimide
`The as
`water-soluble
`boxylates
`using
`and polycarboxylates of the invention have low
`polyamines
`toxicity and provide
`tissue adhesive with improved adhesive
`
`strength Additionally the polyamines and polycarboxylates
`lower earbodiimide con
`of the invention permit the use of
`centration than can be used in the absence of the polyamines
`or in the presence of the polyamines or
`or polycarboxylates
`known in the art thereby further
`polyearboxylates
`reducing
`the toxicity of the adhesive
`
`SUMMARY OF THE INVENTION
`
`The invention provides
`kit comprising
`first vessel containing an aqueous solution comprising
`albumin gelatin or mixture thereof
`second
`vessel
`an undissolved water-
`containing
`soluble carbodiimide and
`the contents of which comprise water
`third vessel
`
`provided that
`
`polysac
`
`the contents of at least one of the first or third vessels
`least one of
`further comprise at
`least one polyamine selected from the group con
`at
`sisting of
`water-dispersible multi-arm polyether
`amine
`aminoalkylated
`water-dispersible
`charide and water-dispersible
`aminoalkylated poly
`vinyl alcohol
`the total polyamine concentration
`being in the range of about 0.5% to about 20% by
`weight or
`selected from the group
`at least one pnlyearboxylate
`cellulose carboxym
`consisting of carboxymethyl
`ethyl dextran and earboxymethyl
`starch the total
`being in the range of
`concentration
`polyearboxylate
`about 0.5% to about 5% by weight or
`mixture of said polyamine of 0a having
`polyamine concentration in the range of about 0.5% to
`about 20% by weight
`of
`and said polycarboxylate
`ib having
`concentration in
`total polycarboxylate
`the range of about 0.5% to about 5% by weight
`
`total
`
`or
`
`ii
`
`the kit
`
`at
`
`fourth vessel containing
`
`at
`
`further comprises
`least one of
`least one polyamine selected from the group con
`sisting of
`water-dispersible multi-arm polyether
`amine
`aminoalkylated
`water-dispersible
`eharide and water-dispersible
`aminoalkylated poly
`neat liquid or
`vinyl alcohol as
`an aqueous pnlyamine solution comprising
`least
`one polyamine selected from the group consisting of
`amine
`water-dispersible multi-arm polyether
`
`polysac
`
`at
`
`aminoalkylated
`
`water-dispersible
`and
`
`polysaceharide
`polyvinyl
`water-dispersible
`aminoalkylated
`the total polyamine concentration being in
`alcohol
`the range of about 0.5% to about 20% by weight or
`an aqueous polyearboxylate
`solution comprising at
`least one polycarboxylate
`selected from the group
`earboxym
`consisting of carboxymethyl cellulose
`ethyl dextran and earboxymethyl
`starch the total
`being in the range of
`concentration
`polycarboxylate
`about 0.5% to about 5% by weight or
`solu
`an aqueous mixed polyamine/polycarboxylate
`least one polyamine of iib
`tion comprising said at
`total polyamine concentration of about 0.5%
`having
`to about 20% by weight and said at
`least one poly
`carboxylate of iie having
`total polyearboxylate
`concentration of about 0.5% to about 5% by weight
`
`or
`
`iii
`
`combination of
`
`and ii
`
`provided further that
`
`

`

`US 7837986 B2
`
`if
`
`corn-
`
`com
`iv if
`the aqueous solution in the first
`vessel of
`prises albumin but not gelatin then the concentration of
`albumin in the aqueous solution is about 25% to about
`40% by weight
`the aqueous solution in the first vessel of
`prises gelatin but not albumin then the concentration of
`gelatin in tbe aqueous solution is about 15% to about
`35% by weight and
`com
`vi if the aqueous solution in the first vessel of
`prises mixture of albumin and gelatin then the total
`concentration of albumin and gelatin combined is about
`15% to about 40% by weight
`In another embodiment
`the invention provides
`for forming
`coating on an anatomical
`site on tissue of
`
`method
`
`living organism comprising
`
`at
`
`ii
`iii
`
`at
`
`forming on said site an aqueous mixture comprising
`least one of albumin or gelatin
`carbodiimide and
`water-soluble
`at least one of
`least one polyamine selected from the group con
`sisting of
`water-dispersible multi-arm polyether
`amine
`water-dispersible
`aminoalkylated
`charide and water-dispersible
`vinyl alcohol or
`selected from the group
`at least one polycarboxylate
`consisting of carboxymethyl cellulose carboxym
`ethyl dextran and carboxymethyl starch and
`allnwing said aqueous mixture tn cure thereby fnrming
`said coating or
`
`polysac
`
`aminoalkylated
`
`poly
`
`ii
`iii
`
`forming an aqueous mixture comprising
`at least one of albumin or gelatin
`water-soluble carbodiimide and
`least one of
`at least one polyamine selected from the group con-
`sisting of
`water-dispersible multi-arm polyether
`
`at
`
`amine
`aminoalkylated
`water-dispersible
`charide and water-dispersible
`
`aminoalkylated poly
`
`polysac
`
`vinyl alcohol or
`selected from the group
`at least one polycarboxylate
`consisting of carbuxymethyl cellulose carboxym
`ethyl dextran and carboxymethyl starch and
`applying said mixture to the site before the mixture com
`pletely cures and allowing said aqueous mixture to cure
`completely thereby forming said coating
`
`ii
`
`provided that
`if the aqueous mixture comprises albumin but not gela
`of albumin in the aqueous
`tin then the concentration
`mixture is about 20% to about 36% by weight
`if the aqueous mixture comprises gelatin but not albu
`min then the concentration of gelatin in the aqueous
`mixture is about 12% to about 32% by weight
`if the aqueous mixture comprises mixture of albumin
`and gelatin then the total concentration uf albumin and
`gelatin combined is about 12% to about 36% by weight
`iv the concentration of the water-soluble carbodiimide in
`the aqueous mixture is about 1% to about 10% by
`weight
`
`iii
`
`if
`
`the polyamine but
`the aqueous mixture comprises
`the polycarboxylate then the concentration of the
`not
`polyamine in the aqueous mixture is about 0.4% to about
`20% by weight
`vi if the aqueous mixture comprises the polycarboxylate
`the polyamine then the concentration
`but not
`of the 65
`in the aqueous mixture is about 0.4% to
`polycarboxylate
`about 5% by weight and
`
`60
`
`vii
`if the aqueous mixture comprises both the polyamioe
`and the polycarboxylatc then the concentration of the
`polyamine in the aqueous mixture is about 0.4% to about
`20% by weight and the concentration of the polycar
`boxylate is about 0.4% to about 5% by weight
`In another embodiment the invention provides
`for bonding at least two anatomical
`sites together comprising
`
`method
`
`least two
`
`forming an aqueous mixture in contact with at
`anatomical sites comprising
`at least one of albumin or gelatin
`water-soluble carbodiimide and
`least one of
`at least one polyamine selected from the group con
`sisting of
`water-dispersible multi-arm polyether
`
`ii
`
`iii
`
`at
`
`amine
`water-dispersible
`amiooalkylated
`charide and water-dispersible
`vinyl alcohol or
`at least one polycarboxylate selected from the group
`consisting of carboxymethyl cellulose carboxym
`ethyl dextran and carboxymethyl starch and
`
`aminoalkylated
`
`poly
`
`polysac
`
`to
`
`tS
`
`20
`
`allowing said aqueous mixture to cure
`
`provided that
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`ii
`
`if the aqueous mixture comprises albumin but not gela
`of albumin in the aqueous
`tin then the concentration
`mixture is about 20% to about 36% by weight
`if the aqueous mixture comprises gelatin but not albu
`mm then the concentration
`of gelatin in the aqueous
`mixture is about 12% to about 32% by weight
`if the aqueous mixture comprises mixture of albumin
`and gelatin then the total concentration of albumin and
`gelatin combined is about 12% to about 36% by weight
`iv the concentration of the water-soluble carbodiimide
`in
`the aqueous mixture is about 1% to about 10% by
`weight
`
`iii
`
`if
`
`the aqueous mixture comprises the polyamine but
`of the
`the polycarboxylate then the concentration
`not
`polyamine in the aqueous mixture is about 0.4% to about
`20% by weight
`vi if the aqueous mixture comprises the polycarboxylate
`the polyamioe then the concentration
`but not
`of the
`in the aqueous mixture is about 0.4% to
`polycarboxylate
`about 5% by weight and
`vii
`if the aqueous mixture comprises both the polyamioe
`and the polycarboxylate then the concentration of the
`polyamine in the aqueous mixture is about 0.4% to about
`20% by weight and the concentration of the polycar
`boxylate is about 0.4% to about 5% by weight
`compo
`In another embodiment the invention provides
`sition resulting from forming an aqueous mixture compris
`ing
`
`at least one of albumin or gelatin
`water-soluble carbodiimide and
`
`at least one of
`
`at
`
`least one polyamine selected from the group con
`sisting of
`water-dispersible multi-arm polyether
`amine
`polysac
`water-dispersible
`aminoalkylated
`charide and water-dispersible
`vinyl alcohol or
`ii at least one polycarboxylate selected from the group
`consisting of carboxymethyl cellulose carboxym
`ethyl dextran and carboxymethyl starch and
`
`aminoalkylated poly
`
`allowing the aqueous mixture to cure
`
`

`

`ii
`
`iii
`
`provided that
`if the aqueous mixture comprises albumin but not gela
`of albumin in the aqueous
`tin then the concentration
`mixture is about 20% to about 36% by weight
`if the aqueous mixture comprises gelatin but not albu
`min then the concentration of gelatin in the aqueous
`mixture is about 12% to about 32% by weight
`if the aqueous mixture comprises mixture of albumin
`and gelatin then the total concentration of albumin and
`gelatin combined is about 12% to about 36% by weight
`iv the concentration of the water-soluble carbodiimide
`in
`the aqueous mixture is about 1% to about 10% by
`weight
`the polyamine but
`the aqueous mixture comprises
`the polycarboxylate then the concentration of the
`not
`4% to about
`polyamine in the aqueous mixture is about
`20% by weight
`vi if the aqueous mixture comprises the polycarboxylate
`of the
`but not
`the polyamine then the concentration
`in the aqueous mixture is about 0.4% to
`polycarboxylate
`about 5% by weight and
`vii
`the aqueous mixture comprises both the polyamine
`and the polycarboxylate then the concentration of the
`polyamine in the aqueous mixture is about 0.4% to about
`20% by weight
`and the concentration
`of the polycar
`boxylate is about 0.4% to about 5% by weight
`Methods for using the protein-based polymer tissue adhe
`sive of the invention for topical wound closure intestinal and
`vascular anastomoses
`sealing corneal
`incisions preventing
`adhesions and drug delivery are also provided
`
`if
`
`if
`
`DETAILED DESCRIPTION
`
`OF THE INVENTION
`
`The invention
`relates to
`tissue adhesive
`protein-based
`formed by crosslinking albumin and/or gelatin with certain
`polyamines and/or certain polycarboxylates
`water-
`using
`soluble carbodiimide The tissue adhesive of the invention is
`useful as an adhesive for medical and veterinary applications
`limited to topical wound closure and
`including but not
`surgical procedures
`such as intestinal anastomosis
`vascular
`anastomosis tissue repair and ophthalmic procedures Addi
`tionally the tissue adhesive may have utility
`in drog delivery
`and as
`anti-adhesive applications
`bulking
`urinary incontinence
`are used herein and should be
`The following definitions
`referred to for interpretation of the claims and the specifica
`
`to treat
`
`agent
`
`at
`
`compound having at least
`
`compound having
`
`tion
`The term polyamine refers to
`two primary amine groups
`The term polycarboxylate
`refers to
`least two carboxylic acid groups
`The term water-dispersible multi-arm polyether amine
`branched polyether wherein at
`least three of the
`refers to
`branches arms are terminated by primary amine group
`which is water soluble or able to be dispersed in water to form
`second
`colloidal suspension capable of
`reacting with
`in aqueous solution
`reactant
`The term polyether refers to
`wherein
`unit
`is
`carbon atoms
`to
`ing
`The term hydrocarbylene group refers to
`divalent
`group formed by removing two hydrogen atoms one from
`each of two different carbon atoms from hydrocarbon
`The term branched polyether refers to
`one or more branch points arms including star deodritic
`comb and hyperbranched polyethers
`
`polymer having the repeat
`group hay-
`
`hydrocarbylene
`
`polyetherhaving
`
`20
`
`30
`
`so
`
`60
`
`65
`
`hydroxyl hydrogens replaced by hydrocarbyl
`groups bearing
`at least one primary amino group wherein the hydrocarbyl
`ts groups are optionally substituted or optionally contain het
`eroatoms and wherein the aminoalkylated
`polysaccharide is
`water soluble or able to be dispersed in water
`to form
`second reac
`colloidal suspension capable of reacting with
`in aqueous solution
`tant
`The term water-dispersible aminoalkylated
`polyvinyl
`alcohol refers to polyvinyl alcohol which has at least two
`of its hydroxyl hydrogens replaced by hydrocarbyl
`groups
`bearing at least one primary amino group wherein the hydro
`carbyl groups are optionally substituted or optionally contain
`25 heteroatoms
`and wherein
`polyvioyl
`the aminoalkylated
`is water soluble or able to be dispersed in water
`alcohol
`form colloidal suspension capable of reacting with second
`reactant
`in aqueous solution
`The term hydrocarbyl group refers to
`univalent group
`formed by removing
`hydrogen atom from hydrocarbon
`The term
`by weight as used herein refers to the weight
`relative to the total weight of the solution unless
`percent
`otherwise specified
`The term anatomical site refers to any external or internal
`35 part of the body of humans or animals
`The term tissue refers to any tissue both living and dead
`in humans or animals
`The term hydrogel
`matrix consisting of
`40 molecules
`held together
`crosslinks
`that can absorb
`form an elastic gel
`The term gene refers to
`effects the production of
`specific protein including regula
`and fol
`non-coding sequences
`tory sequences
`preceding
`non-coding sequences the coding sequence
`lowing
`The term recombinant host cell as used herein refers to
`that has been transformed using genetic engineeriog
`cell
`techniques to produce albumin or gelatin
`By medical application is meant medical applications
`related to humans and for veterinary purposes
`The invention
`tissue adhesive formed
`by
`provides
`crosslinking albumin and/or gelatin with certain polyamines
`using water-soluble carbo
`and/or certain polycarboxylates
`55 diimide Because these proteins contain hoth amine and car
`boxylic acid groups they can be crosslioked with polyamines
`and/or polycarboxylates
`using carbodi
`imide crosslioking
`The polyamines and polycarboxylates
`of the invention have
`low toxicity and provide
`tissue adhesive with improved
`adhesive strength Additionally the polyamines and polycar
`lower carbodi
`boxylates of the invention permit the use of
`imide concentration
`than can be used in the absence of the
`or in the presence of the
`known io the art thereby
`polyamioes or polycarboxylates
`further reducing the toxicity of the adhesive The crosslioking
`reaction forms hydrogel which has many desirable charac
`tissue adhesive including but not
`limited to
`as
`
`US 7837986 B2
`
`The term dendritic polyether
`free-like strocture
`polyether having
`
`refers to
`
`highly braoched
`
`The term comb polyether
`refers to
`polyether having
`main chain with multiple trifinctional branch points from
`each of which
`linear arm emanates
`The term star polyether
`refers to
`polyether having
`from svhich linear arms emanate
`single branch point
`The term hyperbranched polyether
`refers to
`highly
`branched polyether having fewer branches and less regular
`to branching than
`dendritic polyether
`The term water-dispersible aminoalkylated
`ride refers to
`polysaccharide which has at
`
`polysaccha
`least two of its
`
`to
`
`refers to water-swellable polymeric
`network of macro-
`three-dimensional
`
`covalent
`
`or non-covalent
`by
`substantial amount of water
`
`to
`
`nucleic acid fragment
`
`that
`
`as
`
`polyamines or polycarboxylates
`
`teristics
`
`

`

`US 7837986 B2
`
`and cohesion
`
`adhesion
`
`properties crosslinks
`improved
`readily at body temperature maintains dimensional
`stability
`initially does not degrade rapidly and has
`lower toxicity to
`cells than other tissue adhesives that use carbodiimides
`
`Albumin
`Albumins are water-soluble
`that are found in
`proteins
`tissues and fluids such as milk blood serum
`many animal
`and eggs Most albumins are believed to be suitable for use in
`the invention Of particular use are mammalian serum albu
`mins and egg albumins ovalbumins Suitable mammalian
`serum albumins include but are not
`limited to bovine serum
`albumin ovine sheep serum albumin porcine pig serum
`albumin human serum albumin equine hone serum albu
`min lapine rabbit serum albumin rat serum albumin and
`murine mouse serum albumin Suitable
`egg albumias
`include but are not limited to chicken egg albumin Mixtures
`of these albumins may also be used
`Albumin may be purified directly from tissues or fluids
`using methods known in the art for example organic solvent
`or chromatographic methods such
`as ion
`precipitation
`exchange or affinity chromatography Additionally albumin
`from many sources is available commercially from compa
`nies such as Sigma-Aldrich St Louis Mo.
`recombinant albumin produced
`The albumin may also be
`suitable recombinant host cell that expresses an albumin
`by
`gene using standard recombinant DNA and molecular clon
`and Maniatis
`ing techniques Sambrook
`Fritsch
`Laboratory Manual Second Edition
`Molecular Cloning
`Cold Spring Harbor Laboratory Press Cold Spring Harbor
`N.Y 1989 Silhavy
`Bennan
`and Enquist
`Experiments with Gene Fusions Cold Spring Harbor Labo
`ratory Cold Press Spring Harbor NY 1984 and Ausubel
`et al Current Protocols
`in Molecular Biology published
`by Greene PublishingAssoc and Wiley-lnterscience 1987
`the genes
`sequences of
`For example the nucleotide
`serum albumin GenBank Accession No
`encode
`bovine
`AF542068 and M73993 human serum albumin Lawn et al
`Nucleic Acids Res 96103-6114 1981 and ovalbumin
`Woo et al Biochemistry 206437-6446 1981 are known
`These gene sequences may be expressed in suitable host cell
`to produce the desired recombinant albumin For example
`human serum albumin may be expressed in
`recombinant
`Escherichia coli as described by Lawn et al Nucleic Acids
`Res 96103-6114 1981 in Saccharomyces
`as
`cerevisiae
`described by Kalman et al NucleicAcids Res 186075-6081
`1990 or in transgenic mice as described by Shani et al
`1195-208 1992 Additionally
`Trangenic Research
`human serum albumin is available
`commer
`recombinant
`as GTC Biotherapeutics
`such
`from companies
`cially
`Framingliam Mass and Delta Biotechnology
`Limited
`Nottingham UK
`recombinant albumin variant may be used
`Additionally
`in which
`one or more amino acid residues
`are inserted
`deleted or substituted using standard techniques
`such as
`site-directed mutagenesis Suitable albumin variants have an
`identity of at
`amino acid sequence
`that has
`least
`percent
`about 70% or at least about 80% or at
`least about 85% or at
`least about 90% or at
`least about 95% relative to the native
`albumin sequence The percent
`identity which is
`rela
`tionship between two or more polypeptide
`sequences can be
`readily calculated by known methods including but not lim
`ited to those described in Computational Molecular Biology
`ed Oxford University Press NY 1988 Bio
`Lesk
`computing Informatics and Genome Projects Smith
`ed Academic
`Press NY 1993 Computer Analysis of
`Sequence Data Part Griffin
`eds
`and Griffin
`
`that
`
`20
`
`25
`
`30
`
`Humana Press N.J 1994 SequenceAnalysis
`in Molecular
`ed Academic Press 1987 and
`Biology von Heinje
`Sequence Analysis Primer Gribskuv
`and Devereux
`eds Stockton Press NY 1991 Preferred methods to deter
`mine identity are designed to give the best match between the
`tested Methods to determine identity are codified
`sequences
`in publicly available computer programs Sequence align
`ments and percent
`identity calculations may be performed
`using the Megalign program of the LASERGENE bioinfor
`suite DNASTAR Inc Madison Wis.
`10 maties computing
`Multiple alignment of the sequences may be performed using
`the Clustal method of alignment Higgins and Sharp 1989
`CABIOS 5151-153 with the default parameters GAP PEN
`ALTY 10 GAP LENGTH PENALTY 10 Default param
`15 eters for pairwise alignments using the Clustal method are
`WINDOW
`KTUPLE
`GAP PENALTY
`and DIAGO
`NALS SAVED
`In the invention the albumin is used in the form of an
`aqueous solution The albumin is added
`to water
`to give
`concentration of about 25% to about 40% by weight
`relative
`to the total weight of the solution The optimal concentration
`to be used depends on the application and on the concentra
`carbodiimide and the polyamine
`tions of the water-soluble
`used as described below and can be
`and/or polycarboxylate
`readily determined by one skilled in the art using routine
`experimentation
`For use on living tissue it
`the aqueous
`is preferred that
`solution comprising the albumin be sterilized to prevent
`infection Any suitable sterilization method known in the art
`that does not degrade the protein may be used including but
`limited to gamma irradiation ethylene oxide steriliza
`not
`0.2 pm pore membrane
`tion or ultra-filtration
`through
`The aqueous solution comprising the albumin may further
`comprise various additives depending on the intended appli
`35 cation For example the solution may optionally include at
`least one pH modifier to adjust the pH ofthe solution Suitable
`pH modifiers are well known io the art The pH modifier may
`be an acidic or basic compound Examples of acidic pH
`limited to carboxylic acids
`modifiers include but are not
`inorganic acids and sulfonic acids Examples of basic pH
`modifiers include but are not limited to hydroxides alkox
`compounds other than primary and
`ides nitrogeo-containing
`secondary amines and basic carbonates and phosphates
`The aqueous solution comprising the albumin may option-
`least one viscosity modifier The viscosity
`45 ally include at
`modifier may be selected from among known viscosity modi
`and
`limited to polysaccharidds
`fiers including but not
`
`40
`
`55
`
`derivatives thereof such as starch or hydroxyethyl cellulose
`The aqueous solution comprising the albumin may option
`so ally include at least one antimicrobial agent Suitable antimi
`crobial agents are well known in the art Examples of suitable
`antimicrobials include but are not limited to antibiotics such
`cipro
`as tetracycline ampicillin vancomycin polymyxin
`floxacin teicoplanin cefoxitin gentamicin and tobramycin
`The aqueous solution comprising the albumin may also
`to enhance
`optionally include at least one colorant
`the visibil
`ity of the solutioo Suitable colorants include dyes pigments
`and natural coloring agents Examples of suitable colorants
`limited to FDC dyes and FDC lakes
`include but are not
`60 such as FDC Violet No.2 FDCYellow No FDC Red
`FDC Blue No
`No
`chlo
`beetroot red canthaxanthin
`rophyll rosin saffron and carmine
`The aqueous solptioo comprising the albumin may also
`optionally include at least one surfactant Surfactant
`as used
`65 herein refers to
`compound that lowers the surface tension of
`water The surfactant may be an ionic surfactant
`such as
`neutral surfac
`sodium lauryl sulfate and octanoic acid or
`
`

`

`US 7837986 132
`
`10
`
`esters
`
`tant such as polyoxyethylene
`ethers polyoxyethylene
`and polyuxyethylene sorbitan
`Additionally the aqueous solution comprising the albumin
`may optionally include anti-inflammatory agents such as
`indomethacin salicylic acid acetate ibuprophen
`sulindac
`piroxicam and naproxen
`thrombogenic
`agents
`such
`as
`and estramustine
`heal
`thrombin fibrinogen homocysteine
`and radio-opaque com
`ing prumoters such as chitosan
`such as barium sulfate and gold particles
`pounds
`
`Gelatin
`
`Gelatins are water-soluble proteins that are obtained from
`tissues such as bone and skin by acid or alkaline
`aoimal
`treatment Gelatins suitable for use in the invention include
`limited to gelatins obtained from bovine skin
`but are not
`porcine skin and fish skin Gelatins are available commer
`cially from companies such as Sigma Aldrich Commercial
`gelatins are designated by Bloom number which is ao indi
`cation of the strength of the gel produced The higher
`the
`Bloom number the stronger
`the gelatin gel
`The gelatin may also be
`recombinant gelatin produced by
`suitable recombinant host cell that expresses
`gelatin gene
`as described above For example recombinant gelatin may be
`prepared as described by Olsen et al in U.S Pat No 6413
`742 Additionally
`recombinant gelatin variant may be used
`in which
`one or more amino acid residues are inserted
`deleted or substituted
`such as
`using standard techniques
`site-directed mutagenesis Suitable gelatin variants have an
`amino acid sequence
`that has
`identity of at
`least
`percent
`about 70% or at least about 80% or at least about 85% or at
`least about 90% or at least about 95% relative to the native
`gelatin sequence
`In the invention the gelatin is used in the form of an
`aqueous solution The gelatin is added to water
`to give
`concentration of about 15% to about 35% by weight
`relative
`to the total weight of the solution The optimal concentration
`to be used depends on the application and on the concentra
`and the polyamine
`tions of the water-soluble
`carbodiimide
`used as described below and can be
`andlor polycarboxylate
`readily determined
`by one skilled in the art using routine
`
`experimentation
`For use on living tissue it
`the aqueous
`is preferred that
`solutioo comprising the gelatin be sterilized to prevent infec
`tion Any of the methods described above for sterilizing the
`albumin solution may be used
`The aqueous solution comprising the gelatin may further
`comprise various additives Any of the additives described
`above for the albumin solution may be used
`Additionally an aqueous solution comprising mixture of
`albumin and gelatin may be used
`in the invention In the
`mixture the total concentration of albumin and gelatin com
`bined is from about 15% to about 40% by weight
`relative to
`the total weight of the solution Preferably the concentration
`of albumin in the mixture is from about 1% to about 39% by
`weight and the concentration of gelatin is from about 1% to
`about 34% by weight The aqueous solution comprising
`the
`albumin the aqueous solution comprising the gelatin and the
`aqueous solution comprising mixture of albumin and gela
`tin are herein referred to as the protein solution
`
`Polyamines
`least two pri
`The polyamines of the invention contain at
`based on their low
`mary amine groups and were selected
`toxicity and their ability
`to produce
`
`tissue adhesive with
`
`good adhesive
`strength Additionally
`concen
`lower carbodiimide
`polyamines enables the use of
`tration than can be used in the absence of the polyamines or
`or in the presence of the polyamines or
`
`the
`
`use of
`
`these
`
`polycarboxylates
`
`dispersible aminoalkylated
`
`to
`
`is
`
`known in the art thereby further
`reducing
`polycarboxylates
`the toxicity of the adhesive The polyamines of the invention
`water-dispersible multi-arm polyether amine
`are
`water-
`and water-dis
`polysaeeharide
`polyvinyl alcohol Mixtures of
`persible
`aminoalkylated
`these polyamines may also be used
`Water-Dispersible Multi-Arm Polyether Amines
`amines
`The multi-arm polyether
`are water-dispersible
`polyethers having the repeat unit 0RI- wherein
`carbon atoms The multi-
`group having
`10 hydrocarbylene
`arm polyether amines of the invention include but are not
`limited to dendritic comb and star polyethers wherein at
`primary amine
`three of the arms are terminated by
`least
`group The multi-arm polyether amines have
`weight-aver
`ts age molecular weight of about 450 to about 200000 Daltons
`in addition from about 2000 to about 100000 Daltons Suit
`able examples of water-dispersible multi-arm polyether
`amines include but are not limited to amino-terminated
`star
`polyethylene oxides amino-terminated
`dendntic polyethyl
`comb polyethylene oxides
`20 ene oxides amino-terminated
`oxides amino-termi
`amino-terminated
`
`star polypropylene
`oxides amino-terminated
`nated dendritic
`polypropylene
`comb polypropylene
`oxides amino-terminated
`star copoly
`mers of ethylene oxide and propylene oxide amino-termi
`25 nated dendritic copolymers of ethylene oxide and propylene
`comb copolymers of ethylene oxide
`oxide amino-terminated
`and propylene oxide amino-terminated
`star copolymers of
`ethylene oxide and trimethylene oxide amino-terminated
`copolymers of ethylene oxide and trimethylene
`dendritic
`comb copolymers of ethylene oxide
`30 oxide amino-terminated
`and trimethylen