United States Patent (19)
`Burns et al.
`
`5,017,229
`11). Patent Number:
`(45) Date of Patent: May 21, 1991
`
`(54)
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`75
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`(73
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`WATER INSOLUBLE DERVATIVES OF
`HYALURONCACD
`
`Inventors: James W. Burns, Holliston; Steven
`Cox, Boston; Alan E. Walts, Reading,
`all of Mass.
`Assignee: Genzyme Corporation, Cambridge,
`Mass.
`
`Appl. No.: 543,163
`Fied:
`Jun. 25, 1990
`
`Int. Cl. ......................... A61K 47/26; CO8L 1/00
`U.S. C. .................................... 106/162; 106/157;
`106/186; 106/213; 514/777; 424/7.1; 424/488;
`536/4.1; 252/315.3
`Field of Search ..................... 252/315.3; 514/777;
`424/7.1, 488; 536/4.1; 106/157, 162, 186, 213
`
`(56)
`
`FOREIGN PATENT DOCUMENTS
`O193510 9/1986 European Pat. Off. .
`2151244 7/1985 United Kingdom.
`86/00079 1/1986 World Int. Prop. O. .
`86/00912 2/1986 World Int. Prop. O. .
`OTHER PUBLICATIONS
`Sparer et al, "Controlled Release ... ', Controlled Re
`lease Delivery Systems, Marcel Dekker, Inc., 1983.
`Laurent et al., "Cross-Linked Gels . . . ', ACTA
`Chemica Scandinavica, Ejnar Munksgaard, Copenha
`gen, 1964, pp. 274-275.
`Danishefsky et al., "Conversion . . . ', Carbohydrate
`Research, Elsevier, Amsterdam, 1971, pp. 199-205.
`Primary Examiner-Theodore Morris
`Assistant Examiner-David M. Brunsman
`Attorney, Agent, or Firm-Fish & Richardson
`57
`ABSTRACT
`A water insoluble, biocompatible gel that includes the
`reaction product of hyaluronic acid, a polyanionic poly
`saccharide, and an activating agent.
`
`References Cited
`U.S. PATENT DOCUMENTS
`4,582,865 4/1986 Balazs et al. .......
`4,774,093 9/1988 Provonchee et al.
`
`
`
`- a
`
`... 524/29
`
`424/493
`
`25 Claims, No Drawings
`
`ALL 2069
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`1.
`
`WATER INSOLUBLE DERVATIVES OF
`HYALURONCACD
`
`5
`
`10
`
`25
`
`BACKGROUND OF THE INVENTION
`This application is a continuation-in-part of Hamilton
`et al., U.S. patent application Ser. No. 07/100,104 enti
`tled "Water-Insoluble Derivatives of Hyaluronic Acid'
`filed Sept. 18, 1987 now U.S. Pat. No. 4,937,270, 26
`June 1990. The present invention relates to biocompati
`ble films and gels formed from chemically modified
`hyaluronic acid.
`Hyaluronic acid ("HA") is a naturally occurring mu
`copolysaccharide found, for example, in synovial fluid,
`15
`in vitreous humor, in blood vessel walls and umbilical
`cord, and in other connective tissues The polysaccha
`ride consists of alternating N-acetyl-D-glucosamine and
`D-glucuronic acid residues joined by alternating 31-3
`glucuronidic and g 1-4 glucosaminidic bonds, so that
`the repeating unit is -(1->4)-B-D-GlcA-(1->3)-B-D-
`20
`GlcNAc-. In water, hyaluronic acid dissolves to form a
`highly viscous fluid. The molecular weight of hyalu
`ronic acid isolated from natural sources generally falls
`within the range of 5x 10' up to 1 x 107 daltons.
`As used herein the term "HA' means hyaluronic acid
`and any of its hyaluronate salts, including, for example,
`sodium hyaluronate (the sodium salt), potassium hyalu
`ronate, magnesium hyaluronate, and calcium hyaluro
`nate.
`HA, in chemically modified ("derivatized”) form, is
`30
`useful as a surgical aid, to prevent adhesions or accre
`tions of body tissues during the post-operation period.
`The derivatized HA gel or film is injected or inserted
`into the locus between the tissues that are to be kept
`separate to inhibit their mutual adhesion. To be effec
`tive the gel must remain in place and prevent tissue
`contact for a long enough time so that when the gel
`finally disperses and the tissues do come into contact,
`they will no longer have a tendency to adhere.
`Chemically modified HA can also be useful for con
`trolled release drug delivery. Balazs et al., 1986, U.S.
`Pat. No. 4,582,865, states that "cross-linked gels of HA
`can slow down the release of a low molecular weight
`substance dispersed therein but not covalently attached
`to the gel macromolecular matrix." R. V. Sparer et al.,
`1983, Chapter 6, pages 107-119, in T. J. Roseman et al.,
`Controlled Release Delivery Systems, Marcel Dekker,
`Inc., New York, describes sustained release of chloram
`phenicol covalently attached to hyaluronic acid via
`ester linkage, either directly or in an ester complex
`including an alanine bridge as an intermediate linking
`group.
`I. Danishefsky et al., 1971, Carbohydrate Res., Vol.
`16, pages 199-205, describes modifying a mucopolysac
`charide by converting the carboxyl groups of the muco
`55
`polysaccharide into substituted amides by reacting the
`mucopolysaccharide with an amino acid ester in the
`presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodi
`imide hydrochloride ("EDC') in aqueous solution.
`They reacted glycine methyl ester with a variety of 60
`polysaccharides, including HA. The resulting products
`are water soluble; that is, they rapidly disperse in water
`or in an aqueous environment such as is encountered
`between body tissues.
`Proposals for rendering HA compositions less water
`soluble include cross-linking the HA. R. V. Sparer et
`al., 1983, Chapter 6, pages 107-119, in T. J. Roseman et
`al., Controlled Release Delivery Systems, Marcel Dekker,
`
`5,017,229
`2
`Inc., New York, describe modifying HA by attaching
`cysteine residues to the HA via amide bonds and then
`cross-linking the cysteine-modified HA by forming
`disulfide bonds between the attached cysteine residues.
`The cysteine-modified HA was itself water soluble and
`became water insoluble only upon cross-linking by oxi
`dation to the disulfide form.
`De Belder et al., PCT Publication No. WO 86/00912,
`describe a slowly-degradable gel, for preventing tissue
`adhesions following surgery, prepared by cross-linking
`a carboxyl-containing polysaccharide with a bi- or poly
`functional epoxide. Other reactive bi- or polyfunctional
`reagents that have been proposed for preparing cross
`linked gels of HA having reduced water solubility in
`clude: 1,2,3,4-diepoxybutane in alkaline medium at 50
`C. (T. C. Laurent e al., 1964, Acta Chem. Scand., vol.
`18, page 274); divinyl sulfone in alkaline medium (E. A.
`Balasz et al., U.S. Pat. No. 4,582,865, (1986); and a vari
`ety of other reagents including formaldehyde, dime
`thylolurea, dimethylolethylene urea, ethylene oxide, a
`polyaziridine, and a polyisocyanate (E. A. Balasz et al.,
`U.K. Patent Appl. No. 8420 560 (1984). T. Mälson et
`al., 1986, PCT Publication No. WO 86/00079, describe
`preparing cross-linked gels of HA for use as a vitreous
`humor substitute by reacting HA with a bi- or polyfunc
`tional cross-linking reagent such as a di- or polyfunc
`tional epoxide. T. Mailson et al., 1986, EPO 0 193 510,
`describe preparing a shaped article by vacuum-drying
`or compressing a cross-linked HA gel.
`SUMMARY OF THE INVENTION
`The invention features a method for preparing a
`water insoluble gel by combining HA, a polyanionic
`polysaccharide, and an activating agent under condi
`tions sufficient to form the gel.
`Preferred polyanionic polysaccharides include car
`boxymethylcellulose ("CMC'), carboxymethylamylose
`("CMA'), chondroitin-6-sulfate, dermatin sulfate, hepa
`rin, and heparin sulfate; CMC and CMA are particu
`larly preferred. The HA and the polyanionic polysac
`charide can be added together, followed by addition of
`activating agent, or the polyanionic polysaccharide
`may be combined with the activating agent, followed
`by HA addition. Another option is to combine the acti
`vating agent and the HA, followed by addition of the
`polyanionic polysaccharide.
`The preferred pH for carrying out the reaction is 4.0
`to 5.0. The preferred concentration for the polysaccha
`ride is 0.005-0.1M, more preferably 0.01-0.02M. The
`molar ratio of polysaccharide to activating agent is
`preferably at least 1:1, more preferably about 1:4. The
`preferred activating agent is a carbodiimide, e.g., 1
`ethyl-3 -(3-dimethylaminopropyl)carbodiimide or 1
`ethyl-3-(3-dimethylaminopropyl)carbodiimide methio
`dide.
`The gel may be provided in the form of an adhesion
`prevention composition, e.g., in the form of a membrane
`or composition suitable for incorportion in a syringe. It
`may also include a pharmaceutically active substance
`dispersed throughout it; in such cases, the gel is useful as
`a drug delivery system. Suitable substances include
`growth factors, enzymes, drugs, biopolymers, and bio
`logically compatible synthetic polymers.
`The term "film', as used herein, means a substance
`formed by compressing a gel or by allowing or causing
`a gel to dehydrate, and any gel of the invention may be
`formed into such a film.
`
`35
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`45
`
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`
`65
`
`

`

`10
`
`15
`
`35
`
`25
`
`5,017,229
`3
`4.
`A "biocompatible” substance, as that term is used
`cols that are within the method of the invention yet are
`herein, is one that has no medically unacceptable toxic
`different in particulars from those described here.
`or injurious effects on biological function.
`A sample of hyaluronic acid or a salt of hyaluronic
`A "polyanionic polysaccharide' is a polysaccharide
`acid, such as sodium hyaluronate, is dissolved in water
`containing more than one negatively charged groups,
`to make an aqueous mixture. HA from any of a variety
`e.g., carboxyl groups at pH values above about 4.0.
`of sources can be used. As is well-known, HA can be
`We have discovered that by treating HA with a suit
`extracted from animal tissues or harvested as a product
`able activating agent and a polyanionic polysaccharide,
`of bacterial fermentation. Hyaluronic acid can be pro
`a gel or film may be made having decreased water solu
`duced in commercial quantities by bioprocess technol
`bility, without the use of and separately added bi- or
`ogy, as described for example in PCT Publication No.
`polyfunctional cross-linking reagent.
`WO 86/04355. Preferably the concentration of HA in
`A "water soluble' gel or film, as that term is used
`this first aqueous mixture is in the range between 0.4%
`herein, is one which, formed by drying an aqueous
`and 2.5% weight/weight ("w/w'). Subsequent reac
`solution of 1% weight/weight ("w/w') sodium hyalu
`ronate in water, having dimensions 3 cm x3 cm x 0.3
`tions are slower and less effective at significantly lower
`mm, when placed in a beaker of 50 ml of distilled water
`concentrations, while significantly higher concentra
`at 20° C. and allowed to stand without stirring, loses its
`tions are difficult to handle owing to their high viscos
`structural integrity as a film after 3 minutes, and be
`ity.
`comes totally dispersed within 20 minutes. A "water
`The aqueous HA mixture should be acidic, preferably
`insoluble' film of the invention, as that phrase and like
`20
`having a pH between pH 4.0 and pH 5.0, more prefera
`terms are used herein, formed using a 1% aqueous solu
`bly between pH 4.3 and pH 4.75. At lower pH values
`tion of HA, modified according to the invention, having
`the preferred activating agent, EDC, is unstable, and at
`the same dimensions and similarly allowed to stand
`higher values the reaction rate is diminished. Preferably
`without stirring in a beaker of 50 ml of distilled water at
`hydrochloric acid is added to adjust the pH, although
`20 C., is structurally intact after 20 minutes; the film
`other known acids can be used.
`boundaries and edges are still present after 24 hours,
`Once the pH of the aqueous HA mixture has been
`although the film is swollen.
`adjusted, an activating agent is admixed. Preferred acti
`HA is said to be "activated', as that term is used
`vating agents include carbodiimides, most preferably
`herein, when it is treated in an aqueous mixture in a
`manner that renders the carboxyl groups on the HA
`EDC (in some references this substance is termed 1-(3-
`vulnerable to nucleophilic attack or to forming a water
`dimethylaminopropyl)-3-ethyl-carbodiimide
`O
`insoluble gel with a polyanionic polysaccharide; and an
`“DEC') or ETC (1-ethyl-3-(3-dimethylaminopropyl)-
`"activating agent' is a substance that, in an aqueous
`carbodiimide methiodide).
`mixture including HA, causes the HA to become so
`Then a nucleophilic lysine ester is admixed to the
`activated.
`aqueous HA-activating agent mixture. Preferred esters
`Because the gels and films are water insoluble, they
`include methyl, ethyl, or t-butyl esters. The lysine can
`can be thoroughly washed with water before use to
`be in the form of di-lysine, tri-lysine, or polylysine, or
`remove unreacted substances.
`their hydrochloride salts.
`Films and gels of the invention can also be prepared
`The lysine ester and the activating agent may be
`in colored form, by including a dye or stain in the reac
`admixed to the pH adjusted HA mixture in any se
`tion mixture. Such colored films and gels can be more
`quence, either all at once or gradually.
`easily seen when in place or during placement, making
`If a colored product is desired, a solution of a dye or
`them easier to handle during surgical procedures than
`stain such as the blue dye "Brilliant Blue R', also
`colorless ones.
`The polysaccharide-modified films and gels retain
`known as "Coomassie TM Brilliant Blue R-250', dis
`45
`tributed as "Serva Blue' by Serva, can be admixed to
`their strength even when hydrated. Because they ad
`the reaction mixture at this point. The resulting product
`here to biological tissues without the need for sutures,
`they are useful as postoperative adhesion prevention
`has a blue color that can provide a good contrast to the
`membranes. They can be applied to tissue even in the
`color of body tissues, making the film or gel easy to see
`presence of bleeding.
`while it is handled during surgery and once it is in place.
`50
`Other features and advantages of the invention will
`Once the reagents (and the stain or dye, if any) have
`be apparent from the following description of the pre
`been admixed, the reaction mixture can be simply al
`ferred embodiments thereof, and from the claims.
`lowed to stand for a time, or it can be continually or
`occasionally stirred or agitated.
`DESCRIPTION OF THE PREFERRED
`Upon admixing of the reagents the pH rises, and can
`EMBODIMENTS LYSINE-MODIFIED HA
`be maintained at the desired pH by addition of acid as
`The gels and films of the invention are made gener
`the reaction proceeds. We have found, however, that
`ally as follows. HA is dissolved in water and the pH of
`films and gels with various desired physical properties
`the resulting aqueous mixture is adjusted downward;
`can be obtained by simply allowing the pH to rise as the
`then the dissolved HA is activated by admixing a suit
`60
`reaction proceeds. The mode of addition of the rea
`able activating agent, and a suitable lysine ester is ad
`gents, particularly the EDC and the lysine ester, is not
`mixed with the activated HA and allowed to stand until
`critical, but the ratios of these reagents to the HA is
`the desired gel has formed. The activating agent and the
`important. We have found that the best results are ob
`ester can be admixed in any sequence.
`The preferred method of making the lysine-modified
`tained when the ratio of HA:EDC:Lysine ester ranges
`gels and films of the invention will now be described in
`from 1:2:1 to 1:4:10. Lower values typically result in
`more detail. As one skilled in the art will appreciate,
`weaker, less insoluble products, while higher values
`gels and films of the invention can be made using proto
`typically result in stronger, more insoluble products.
`
`55
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`65
`
`

`

`15
`
`5,017,229
`5
`6
`addition of 0.1N HC1. Then 314 mg of EDC (1.64
`POLYANIONIC
`mmol) was added all at once followed by 190 mg (1.05
`POLYSACCHARIDE-MODIFIED HA
`mmol) of L-leucine methyl ester hydrochloride. The
`Polyanionic polysaccharide-modified HA gels and
`pH of the reaction mixture then rose to 6.2 over two
`films are prepared generally by mixing HA (as de
`hours. The reaction mixture was kept at room tempera
`scribed above) with a polyanionic polysaccharide and
`ture for five hours, after which time it had formed a
`an activating agent to form a water-insoluble precipi
`thick insoluble hydrogel. This hydrogel could be
`tate. The precipitate can be cast into thin membranes
`washed with a 1M NaCl solution to remove residual
`useful for postoperative adhesion prevention. It can also
`reagents without loss of its physical properties.
`be colored as described above. To increase the strength
`Example 2: In this example various EDC/leucine:HA
`O
`of films cast from the precipitate, the films may be sub
`ratios were used for comparison of gel formation and
`properties.
`jected to dehydrothermal treatment in which they are
`heated under vacuum (about 30 mm Hg) at approxi
`The procedure was as in Example 1, using sodium
`mately 105 C. for 24 hr.
`hyaluronate (400 mg, 1.0 mmol of carboxyl groups) in
`The polysaccharide and HA can be mixed together,
`15 ml of water. In separate experiments the following
`after which the activating agent is added. Alternatively,
`quantities of EDC and leucine methyl ester hydrochlo
`the polysaccharide may be reacted with the activating
`ride were then added: 153 mg EDC (0.8 mmol)/182 mg
`agent, followed by addition of HA. A third option is to
`leucine methyl ester hydrochloride (1.0 mmol); 76 mg
`combine the HA with the activating agent, followed by
`EDC (0.4 mmol)/90 mg leucine methyl ester hydro
`addition of the polysaccharide. Preferred activating
`chloride (0.5 mmol); and 38 mg EDC (0.2 mmol)/45 mg
`20
`agents are as described above and include the carbodii
`leucine methyl ester hydrochloride (0.25 mmol). Strong
`mides EDC and ETC. The reaction is preferably car
`hydrogels were obtained as in example 1 for the highest
`ried out at a pH between 4 and 5. The preferred poly
`ratio of EDC and leucine methyl ester hydrochloride.
`saccharide concentration ranges from 0.005 to 0.1M,
`At the lowest ratio of reactants (0.2 mmol/0.25 mmol to
`and is more preferably in the range 0.01 to 0.02M. The
`1.0 mmol HA carboxyl groups) a weak gel was ob
`25
`preferred molar, ratio of polysaccharide to activating
`tained, which collapsed to a fluid after two weeks.
`agent is at least 1:1, more preferably about 1:4.
`Example 3: In this example the HA concentration
`was reduced by one-half for comparison of resulting gel
`FILM FORMATION
`properties.
`HA modified according to the above descriptions can
`The procedure was as in example 1 except the HA
`be cast as films in a straightforward manner. Typically
`(400 mg; 1.0 mmol of carboxyl groups) was dissolved in
`the reaction mixture is poured into a vessel having the
`30 ml of water rather than 15 ml (1-% w/w HA). A
`desired size and shape and allowed to air dry. In general
`hydrogel was formed, although it was weaker than that
`films formed by drying mixtures poured thickly, so that
`obtained in Example 1.
`they have a lower surface area/volume, possess greater
`Example 4: In this example films were prepared using
`strength than films formed by drying thinner, higher
`EDC as an activating agent and leucine methyl ester
`hydrochloride as a nucleophile.
`surface area/volume mixtures.
`Alternatively a film can be formed by compressing a
`Sodium hyaluronate (400 mg, 1.0 mmol of carboxyl
`gel under conditions that permit escape of water, as, for
`groups) was dissolved in 40 ml of distilled water. The
`example, by compressing the gel between two surfaces,
`pH of the solution was adjusted to pH 4.75 by addition
`at least one of which is porous, as described, for exam
`of 0.1N HCl. Then EDC (314 mg, 1.64 mmol) was
`ple, in EPO 0 193510.
`added in a single portion, followed by 190 mg (1.05
`If desired, a gel or film can be washed prior to use by,
`mmol) of L-leucine methyl ester hydrochloride. The
`for example, perfusion with water or 1M aqueous so
`pH of the reaction mixture rose to 6.2 during two hours,
`dium chloride. Alternatively the reaction mixture can
`after which time the solution was poured into a petri
`45
`be dialyzed to remove residual reagents prior to casting
`dish of area 6360 mm2, and allowed to dry to a film over
`as a film. Washing to remove residual reagents or rea
`a two day period. Films produced in this manner were
`gent-derived material such as substituted ureas is desir
`strong and insoluble in water and 1M aqueous NaCl.
`able if the film or gel is to be used for therapeutic appli
`The films could be washed with water or aqueous NaCl
`cations. Gels or films colored blue with Brilliant Blue R
`as in Example to remove residual reagents without loss
`50
`of their physical properties. Infrared spectroscopic
`as described above do not lose their coloration during
`such washing. The removal of reagents or reaction
`analysis of such films showed no carbodiimide absorp
`products can be monitored by high pressure liquid chro
`tion at about 2130 cmi and displayed absorptions at
`matography.
`about 1740 cm-l 1700 cm-1, 1650 cm-l, and 1550
`cm-l.
`DETAILED DESCRIPTION OF THE
`Example 5: In this example various HA concentra
`INVENTION
`tions were used in making films for comparison of re
`sulting film properties.
`The invention is described in more detail in the foll
`lowing examples. These examples are given by way of
`The procedure described in example 4 was repeated,
`illustration and are not intended to limit the invention
`using three different initial HA concentrations made by
`60
`except as set forth in the claims.
`dissolving the HA (400 mg, 1.0 mmol of carboxyl
`Example 1: In this example hydrogels were prepared
`groups) in 30 ml, 40 ml, or 100 ml of distilled water.
`using EDC as an activating agent and leucine methyl
`Films produced using each of these initial concentra
`ester 5 hydrochloride as a nucleophile.
`tions of HA were strong and insoluble in water and 1M
`Sodium hyaluronate (400 mg; 1.0 mmol of carboxyl
`65 aqueous NaCl, showing that a range of concentrations
`groups) having a molecular weight between 1 x 106 and
`of HA can be used. Each of these films could be washed
`2x 106 was dissolved in 10 ml of distilled water. The pH
`with water or aqueous NaCl without loss of its physical
`properties.
`of the aqueous solution was adjusted to pH 4.75 by
`
`30
`
`35
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`55
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`

`

`5,017,229
`8
`7
`Example 6: This example illustrates the effect of dia
`was poured into a petri dish and allowed to dry and
`lyzing the reaction mixture prior to casting to form a
`insoluble films were obtained.
`Example 13: In this example gels were prepared using
`film, as compared with washing the film after forming
`leucine ethyl ester hydrochloride.
`it.
`Sodium hyaluronate (400 mg in 40 ml of water), EDC
`A solution of HA (400 mg in 15 ml of H2O) was
`brought to pH 4.7 and EDC (314 mg; 1.6 mmol) was
`(314 mg; 1.64 mmol) and L-leucine methyl ester hydro
`added, followed by leucine ethyl ester hydrochloride
`chloride (190 mg, 1.05 mmol) were allowed to react as
`in Example 4. Upon completion of reaction (2 hours)
`(1.0 mmol). The mixture formed a thick, water insoluble
`gel within from 5 to 24 hours.
`the reaction mixture was dialyzed against water,
`Example 14: In this example films and gels were pre
`through 12,000 NMW cutoff dialysis tubing in order to
`pared using ETC as the HA activating agent.
`remove residual reagents The dialyzed mixture was
`Sodium hyaluronate (400 mg, 1.0 mmol of carboxyl
`then cast as a film as in Example 4. The film so obtained
`groups) having a molecular weight in the range be
`was strong and insoluble in water or 1M aqueous NaCl.
`tween 1 x 106 and 2x 106 daltons was dissolved in water
`Example 7: In this example films were formed by
`(10 ml and 30 ml). The pH of each aqueous solution was
`drying more thickly poured reaction mixtures, to com
`15
`adjusted to pH 4.75 by addition of 0.1N HCl. Then 475
`pare the properties of films produced from drying mix
`mg of ETC (1.6 mmol) was added all at once, followed
`tures at differing surface area/volume.
`by 190 mg (1.05 mmol) of L-leucine methyl ester hydro
`A reaction mixture obtained as in Example 4 (40 ml
`chloride. The pH of this reaction mixture rose to pH 6.2
`reaction volume) was cast into a small petri dish (area
`over the next 2 hours. The reaction mixture containing
`3330 mm). The film so obtained was insoluble in 1M
`10 ml of water formed an insoluble gel. The reaction
`aqueous NaCl and in water (100 C.; 1 hour).
`mixture containing 30 ml of water gave an insoluble film
`Example 8: In this example films were prepared using
`after drying.
`other amino acid esters and HA activated with EDC.
`Example 15. This example illustrates the preparation
`A solution of HA (400 mg in 40 ml of H2O) was
`of a colored film.
`25
`brought to pH4.7 using 0.1NHC). Then EDC (314 mg.
`A solution of HA (400 mg in 30 ml of H2O) was
`1.6 mmol) was added all at once followed by 1 mmol of
`brought to pH 4.75 as in example 13 and then ETC (475
`the amino acid derivative. The reaction mixture was
`mg; 1.6 mmol) and leucine methyl ester hydrochloride
`poured into a petri dish and allowed to dry. Insoluble
`(190 mg; 1.05 mmol) were added. A dilute solution of
`films were obtained from L-valine methyl ester hydro
`"Serva Blue' (5 mg/ml) dye in H2O (0.5 ml) was then
`30
`chloride, L-isoleucine methyl ester hydrochloride, L
`added to the reaction mixture. The resulting mixture
`proline methyl ester hydrochloride, and L-phenylala
`was poured into a Petri dish and a water insoluble blue
`nine methyl ester hydrochloride.
`film was obtained after 16 hours. The blue color was
`Example 9: In this example films were prepared using
`retained by the film when the film was washed with 1M
`a simple primary amine (aniline) as a nucleophile. .
`NaCl and then with H20.
`35
`A solution of HA (400 mg in 40 ml of H20) was
`Example 16. This example illustrates the tissue bi
`brought to pH 4.7 using 0.1N HCl. Then EDC (314 mg.
`ocompatibility of a film of chemically modified HA.
`1.6 mmol) was added all at once followed by 1 mmol of
`Four strips of films prepared according to the proce
`aniline. The reaction mixture was poured into a petri
`dure described in Example 4, and two USP negative
`dish and allowed to dry, and insoluble films were ob
`control strips were surgically implanted into the para
`40
`tained.
`vertebral muscle of White New Zealand rabbits (two
`Example 10: In this example films were prepared
`per test). The test sites were evaluated either macro
`using other esters of leucine.
`scopically after 72 hours or with complete histopathol
`A solution of HA (400 mg in 40 ml of H2O) was
`ogy after 7 days. In accordance with the USP XXI, p.
`brought to pH 4.7 using 0.1N HCl. Then EDC (314 mg;
`1237, the test material met the requirements of the USP
`45
`1.6 mmol) was added all at once followed by 1 mmol of
`Implantation Test for the Evaluation of Plastic Materi
`the leucine ester. The reaction mixture was poured into
`als.
`a petri dish and allowed to dry. Insoluble films were
`Example 17. This example illustrates the preparation
`obtained from both L-leucine ethyl ester hydrochloride
`of lysine-modified HA.
`and L-leucine t-butyl ester hydrochloride.
`A 0.4%(w/w) solution of HA in water was prepared.
`Example 11: In this example gels were prepared using
`The pH of this solution was adjusted to between 4.3 and
`other amino acid methyl esters.
`4.75 by addition of acid. To each 100 ml of this solution
`A solution of HA (400 mg in 15 ml of H2O) was
`was added 0.76 g of EDC with stirring until the EDC
`brought to pH 4.7 and EDC (314 mg, 1.6 mmol) was
`had completely dissolved. To each 100 ml of the HA
`added, followed by the amino acid derivative (1 mmol).
`/EDC solution was added 0.20 g of lysine methyl ester
`55
`The reaction mixture formed a thick gel within from 5
`(LME) with stirring until the LME had completely
`to 24 hours. Water insoluble gels were obtained using
`dissolved. The addition of HA, EDC, and LME was
`L-valine methyl ester hydrochloride, L-isoleucine
`conducted at room temperature; once the final HA
`methyl ester hydrochloride, L-arginine methyl ester
`MEDC/LME solution had been formed, it was stored at
`hydrochloride, L-proline methyl ester hydrochloride,
`4. C. until needed.
`and L-histidine methyl ester hydrochloride.
`The LME-modified HA material can be processed
`Example 12: In this example films were prepared
`into various shapes, sizes, and consistencies depending
`using an amino acid amide (leucinamide) as a nucleo
`on the end application. If a thin sheet of the material is
`phile.
`desired, the mixture can be poured onto a flat surface.
`A solution of HA (400 mg in 40 ml of H2O) was
`This material can then be turned into a solid by allowing
`65
`brought to pH 4.7 using 0.1N HCl. Then EDC (314 mg.
`the water to evaporate under ambient or elevated tem
`1.6 mmol) was added all at once followed by 1 mmol of
`peratures. An alternative method of producing sheets of
`L-leucinamide hydrochloride. The reaction mixture
`the material is to subject it to freeze drying. The pore
`
`50
`
`O
`
`60
`
`

`

`5,017,229
`9
`10
`size of the final product can be controlled by adjusting
`combining an aqueous solution of HA at a concentra
`the initial freezing temperature. Curved surfaces and
`tion in the range between 0.4% and 2.6% w/w, a
`polyanionic polysaccharide, and an activating
`other shapes can be produced in a similar manner by
`initially casting the gel onto a negative image surface
`agent under conditions sufficient to form said gel.
`and then processing as described. The dried sheet can
`2. The method of claim 1 wherein said polyanionic
`polysaccharide is chosen from the group consisting of
`be processed further, if desrired, by pressing to a de
`carboxymethylcellulose, carboxymethylamylose, chon
`fined thickness in a Carver laboratory press. This is
`particularly useful for applications requiring placing a
`droitin-6-sulfate, dermatin sulfate, heparin, and heparin
`thin film between anatomical structures where space is
`sulfate.
`3. The method of claim 2 wherein said polyanionic
`limited.
`10
`polysaccharide is carboxymethylcellulose.
`Mechanical testing of the freeze-dried material, rehy
`4. The method of claim 2 wherein said polyanionic
`drated in normal saline, resulted in force to break values
`polysaccharide is carboxymethylamylose.
`of 170-900 g/cm2. The elongation to break values for
`5. The method of claim 1 wherein said HA and said
`this material were between 33 and 62%.
`polyanionic polysaccharide are added together, fol
`Example 18. This example illustrates the preparation
`lowed by addition of said activating agent.
`of CMC-modified HA.
`15
`6. The method of claim 1 wherein said polyanionic
`HA (0.4% w/w, 0.01M) and Aqualon-type CMC
`polysaccharide is combined with said activating agent,
`having a molecular weight of 250,000 and a degree of
`followed by addition of HA.
`substitution in the range 0.65 to 0.90 (0.19% w/w,
`0.01M) were mixed together in aqueous solution at
`7. The method of claim 1 wherein said HA is con
`bined with said activating agent, followed by addition
`room temperature. The pH of the mixture was adjusted
`20
`of said polyanionic polysaccharide.
`to and maintained at pH 4.7-4.8 by addition of 1M HCl,
`8. The method of claim 1 wherein said activating
`To each 100 ml of this solution was added 0.67 g
`agent comprises a carbodiimide.
`(0.04M) EDC. During reaction with EDC, the pH of
`9. The method of claim 8 wherein said carbodiimide
`the solution was maintained at pH 4.7-4.8 by addition of
`comprises 1-ethyl-3-(3-dimethylaminopropyl) carbodi
`0.1M HCl and the reaction allowed to proceed for 1
`25
`imide, or 1-ethyl-3-(3-dimethylaminopropyl) carbodi
`hour, during which time a precipitate formed. The un
`imide methiodide.
`reacted EDC 5 was removed from the precipitate by
`10. The method of claim 1 wherein polyanionic poly
`dialysis against acidified water (pH 4.0) for 24 hours
`saccharide is present in a concentration of 0.005-0.1M.
`with 2 dialysate changes at 3 and 19 hours. The
`11. The method of claim 10 wherein said polyanionic
`HA/CMC slurry was then cast into flat molds and air
`polysaccharide is present in a concentration of
`30
`dried for 24 hours at room temperature.
`0.01-0.02M.
`HA/CMC membranes were shown to reduce the
`12. The method of claim 1 wherein said method is
`incidence of postoperative adhesion formation in exper
`carried out at a pH of 4.0 to 5.0.
`imental animal models. In experiments using the rat
`13. The method of claim 1 wherein the molar ratio of
`cecal abrasion model, HA/CMC membranes were
`said polyanionic polysaccharide to said activating agent
`placed around surgically abraded rat ceca; previous
`35
`is at least 1:1.
`studies had demonstrated that adhesions readily formed
`14. The method of claim 13 wherein the molar ratio
`to the ceca of rats which had been abraded in controlled
`of said polyanionic polysaccharide to said activating
`fashion. C