(12) United States Patent
`Calias et al.
`
`USOO6521223B1
`(10) Patent No.:
`US 6,521,223 B1
`(45) Date of Patent:
`Feb. 18, 2003
`
`(54) SINGLE PHASE GELS FOR THE
`PREVENTION OF ADHESIONS
`
`(75) Inventors: Pericles Calias, Melrose; Robert J.
`Miller, Halifax, both of MA (US)
`
`(73) Assignee: Genzyme Corporation, Cambridge,
`MA (US)
`
`(*) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/503,544
`(22) Filed:
`Feb. 14, 2000
`(51) Int. Cl........................... A61K 31/74; A61 K9/00;
`A61K 47/00; A61F 13/00; A61F 2/00
`(52) U.S. Cl. .................... 424/78.08; 424/422; 424/423;
`424/400; 514/781
`(58) Field of Search .............................. 424/78.08, 400,
`424/422, 423; 524/29; 514/781
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`3,106,439 A 10/1963 Valentine et al.
`3,316.308 A 4/1967 Welch
`3,357,784. A 12/1967 Kasper
`4,178,361. A 12/1979 Cohen et al.
`4,582,865 A
`4/1986 Balazs et al. ................. 524/29
`4,605,691 A 8/1986 Balazs et al.
`4,636,524 A 1/1987 Balazs et al.
`4,713,448 A 12/1987 Balazs et al.
`4,716,154 A 12/1987 Mason et al.
`4.937.270 A 6/1990 Hamilton et al.
`5,017,229 A
`5/1991 Burns et al. ................ 424/488
`5,099,013 A 3/1992 Balazs et al.
`5,128,326 A 7/1992 Balazs et al.
`5,143,724 A 9/1992 Leshchiner et al.
`5,219,360 A 6/1993 Georgiade
`5,246,698 A 9/1993 Leshchiner et al.
`5,399,351 A 3/1995 Leshchiner et al.
`5,527,893 A 6/1996 Burns et al.
`5,676,694. A 10/1997 Della Valle et al.
`5,760,200 A 6/1998 Miller et al.
`5,783,691 A 7/1998 Mason et al.
`6,030,958 A 2/2000 Burns et al.
`
`6,066,325 A * 5/2000 Wallace et al. ............. 424/400
`6,086,907 A 7/2000 Goldberg et al.
`6,174.999 B1 * 1/2001 Miller et al................... 514/60
`6,235,726 B1
`5/2001 Burns et al.
`6.294.202 B1
`9/2001 Burns et al.
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`EP
`GB
`WO
`WO
`WO
`
`O 224987
`O 732 110
`O 939 O86
`2151 244
`WO 86 OOO79
`WO 86 OO912
`WO 86 04355
`
`6/1987
`9/1996
`* 1/1999
`7/1985
`1/1986
`2/1986
`7/1986
`
`OTHER PUBLICATIONS
`US 5,808,050, 9/1998, Mares-Guia (withdrawn)*
`Danishefsky et al., Conversion of Carboxyl Groups of
`Mucopolysaccharides Into Amides of Amino Acid Esters,
`Carbohydrate Research, vol. 16, pp. 199-205 (1971).
`Sparer et al., Controlled Release from Glycosaminoglycan
`Drug Complexes, Controlled Release Delivery Systems,
`Chapter 6, pp. 107-119 (1983).
`Larsen, N. E., et al., “Hylan Gel Biomaterial: Dermal and
`Immunologic Compatibility”, J. Biomedical Materials
`Research, 27: 1129-1134 (1993).
`Burns, J., et al., Preclinical Evaluation of Seprafilm TM
`Bioresorbable Membrane, Eur: J. Surg., Suppl. 577: 40-48
`(1997).
`
`* cited by examiner
`Primary Examiner Thurman K. Page
`Assistant Examiner Blessing Fubara
`(74) Attorney, Agent, or Firm-Isabelle A. S. Blundell
`(57)
`ABSTRACT
`Single phase gels for preventing the formation of Surgical
`adhesions are disclosed. The gels are prepared by reacting an
`aqueous Solution of a polyanionic polysaccharide, Such as
`hyaluronic acid or carboxymethyl cellulose, with divinyl
`Sulfone, to form a gel, the Solution is neutralized, and a Solid
`is precipitated from the Solution. The Solid can be redis
`Solved in water to form a gel having properties which can be
`modified to Suit a particular application
`
`15 Claims, No Drawings
`
`Exhibit 1048
`Prollenium v. Allergan
`
`

`

`1
`SINGLE PHASE GELS FOR THE
`PREVENTION OF ADHESIONS
`
`Throughout this application, various publications are
`referenced. All publications referenced herein, including
`published patent applications and issued or granted patents,
`are hereby incorporated by reference in their entireties into
`this application.
`
`BACKGROUND OF THE INVENTION
`This invention relates to Single phase gel products formed
`by the reaction of a polyanionic polysaccharide and divinyl
`sulfone (“DVS”), and preferably formed by the reaction of
`hyaluronic acid (“HA") and divinyl sulfone. The single
`phase gel products of this invention are particularly useful
`for preventing the formation of adhesions between affected
`tissue Surfaces of a Subject who has undergone a Surgical
`procedure.
`Adhesion formation is a well known complication of
`many types of Surgical procedures, and particularly abdomi
`nal and bowel Surgeries. Adhesion formation typically
`occurs as a result of the formation of a fibrin clot which
`transforms into Scar tissue connecting different tissues which
`are normally Separated. Surgical intervention is frequently
`required in order to eliminate the adhesions, although the
`adhesions can, and often do, reappear following the Surgery.
`The primary objective of adhesion prevention formulations
`is to interrupt the adhesion formation mechanism, which is
`believed to result from the diffusion of fibrinogen into the
`Space between the tissues Subject to Surgical trauma, thereby
`causing the formation of fibrin clots in the Space.
`In addition to acting as an adhesion barrier, a Successful
`anti-adhesion formulation should be “biocompatible”,
`meaning that it has no medically unacceptable toxic or
`injurious effects on the biological function of the Subject,
`and “bioabsorbable”, meaning that it can be absorbed by the
`tissue without remaining in the Subject as an implant device.
`Thus, the formulation should remain in the body for a
`Sufficient period of time to be effective in Separating the
`tissue and preventing adhesions, while being absorbed by
`the tissue once the danger of adhesion formation has ended,
`thereby minimizing any long term effects which may result
`from the use of an implant device.
`Hyaluronic acid ("HA) is a naturally occurring muco
`polysaccharide found, for example, in Synovial fluid, in
`vitreous humor, in blood vessel walls, the umbilical cord,
`and in other connective tissues. The polysaccharide consists
`of alternating N-acetyl-D-glucosamine and D-glucuronic
`acid residues joined by alternating B1–3 glucoronidic and
`B1-4 glucosaminidic bonds, So that the repeating unit
`is -(1->4)-B-D-G1cA-(1->3)-fD-G1cNAc-. In water,
`hyaluronic acid dissolves to form a highly viscous fluid. The
`molecular weight of hyaluronic acid isolated from natural
`sources generally falls within the range of from about 5x10"
`up to about 1x107 daltons.
`Hyaluronic acid, in chemically modified form, is known
`to be useful as a Surgical aid to prevent adhesions and
`accretions of body tissues during the post-operation period.
`The chemically modified hyaluronic acid gel or film is
`injected or inserted into the locus between the tissues that are
`to be kept Separate to inhibit their mutual adhesion. Chemi
`cally modified hyaluronic acid can also be useful for con
`trolled release drug delivery. See U.S. Pat. No. 4,937,270
`and U.S. Pat. No. 5,017,229, which disclose chemically
`modified versions of HA, or HA in combination with other
`polyanionic polysaccharides, Such as
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`2
`carboxymethylcellulose, which are prepared by reacting the
`HA with a carbodiimide. The chemically modified version of
`HA and carboxymethylcellulose is commercially available
`in film form as Seprafilm(R) membranes from the Genzyme
`Corporation.
`I. Danishefsky et al., Carbohydrate Res., Vol. 16, pages
`199-205, 1971, describe the modification of a mucopolysac
`charide by converting the carboxyl groups of the muco
`polysaccharide into Substituted amides by reacting the
`mucopolysaccharide with an amino acid ester in the pres
`ence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
`hydrochloride (“EDC) in aqueous solution. Danishefsky et
`al. react glycine methyl ester with a variety of
`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, Inc.,
`New York, describe modifying HA by attaching cysteine
`residues to the HA by amide bonds, and then croSS-linking
`the cysteine-modified HA by forming disulfide bonds
`between the attached cysteine residues.
`U.S. Pat. No. 5,676,964 describes the preparation of
`croSS-linked polysaccharides, including HA, wherein the
`croSS-linking reaction occurs as a result of covalent bonds
`formed between carboxyl groupS and hydroxyl groups of
`adjacent polysaccharide molecules.
`U.S. Pat. No. 4,582,865; U.S. Pat. No. 4,636,524 and U.S.
`Pat. No. 5,128,326 describe HA compositions in which the
`HA is cross-linked by reaction with divinyl sulfone, and
`further describe the use of these compositions for drug
`delivery applications. U.S. Pat. No. 4,605,691 describes a
`method for preparing cross-linked HA compositions using
`divinyl Sulfone as a croSS-linking agent in an alkali Solution.
`U.S. Pat. No. 5,143,724; U.S. Pat. No. 5,247,698 and U.S.
`Pat. No. 5,399,351 disclose biocompatible, viscoelastic
`polymeric gel Slurries prepared by reacting hyaluronic acid
`and a croSS-linking agent Such as divinyl Sulfone, which are
`used in anti-adhesion formulations. The gel Slurry is a two
`phase composition comprising discrete particles distributed
`in a polymer Solution. In one embodiment, the Slurry is
`formed from cross-linked hyaluronic acid particles con
`tained in a Solution of hyaluronic acid. The two phase
`Slurries are believed to be effective in preventing adhesion
`formation due to their ability to Separate affected tissue
`surfaces coupled with the ability to restrict diffusion at the
`Site of potential adhesion formation.
`U.S. Pat. No. 5,783,691 relates to hyaluronic acid com
`positions which are prepared by crosslinking hyaluronic acid
`with a phosphorus-containing reagent, Such as Sodium
`phosphate, in an alkaline medium to form a gel product. The
`crosslinking reagents described in this patent are not com
`pletely Soluble, resulting in a two phase System, with one
`phase containing the crosslinked product. The gels can
`contain drugs and can be used as drug release vehicles upon
`administration to a Subject.
`Two phase gel Slurries do Suffer from certain drawbacks,
`however. For instance, the material must be processed
`correctly in order to improve the handling properties of the
`material, and to permit its therapeutic application through
`the narrow openings of needles and other applicators, par
`ticularly for minimally invasive Surgical indications. Such
`processing requires the use of processing equipment and the
`
`

`

`3
`application of Shear forces to the material, which in turn can
`result in a decrease in Viscosity (thinning). Two phase
`materials contain dispersed, heterogeneous particles which
`tend to plug the narrow openings of Such delivery Systems.
`A Single phase, homogeneous composition is more useful in
`minimally invasive Surgical applications where devices are
`introduced into the body through narrow access ports.
`It would therefore be highly desirable to formulate a
`Single phase gel Solution which is capable of preventing the
`formation of adhesions, and which can be easily handled and
`Stored for future use, and which possesses the advantageous
`characteristics of two phase gels.
`SUMMARY OF THE INVENTION
`The present invention features a cross-linked polyanionic
`composition which is useful for the prevention of adhesions
`which can arise as the result of a Surgical procedure per
`formed on a Subject. The croSS-linked composition is pre
`pared by the reaction of the polyanionic polysaccharide with
`divinyl Sulfone. The reaction occurs in an aqueous Solution
`and results in the formation of a gel. The gel Solution is
`neutralized, preferably by acidifying the Solution, and a Solid
`is precipitated from the Solution. The Solid can be pulverized
`to form a powder, and Subsequently rehydrated with water to
`form a Single phase, purified gel having properties Suitable
`for use in anti-adhesion formulations.
`In one embodiment, the invention features a method for
`preparing a single phase gel for use in preventing the
`formation of Surgical adhesions. The gels of this invention
`are prepared by reacting a polyanionic polysaccharide with
`divinyl sulfone to form a cross-linked gel. Preferably, the
`polyanionic polysaccharide is hyaluronic acid or carboxym
`ethyl cellulose, and the molar ratio of divinyl sulfone to
`polyanionic polysaccharide is from about 0.1:1 to about 1:
`1, and more preferably from about 0.2:1 to about 0.6:1. The
`gel is neutralized by the addition of an acidic compound,
`Such as an inorganic acid, typically hydrochloric acid or
`Sulfuric acid, to an aqueous Solution of the gel and the
`croSS-linking agent. The gel can be precipitated as a Solid,
`preferably as a powder or fine particles, and Stored until it is
`desired to reconstitute the gel by rehydration of the powder.
`Terminal Sterilization of the gel can be accomplished by
`autoclaving the gel, and this procedure does not have any
`Substantial adverse impact on the gel Structure. Terminal
`Sterilization is a cost effective method for manufacturing a
`medical device Since it can assure a lower bioburden than
`aseptic processing, and thereby reduces the risk of infection.
`Typically, terminal Sterilization involves Steam autoclaving
`of aqueous preparations, and either ethylene oxide treatment
`or high energy bombardment (irradiation or E beam
`treatment) of the material in solid or dry form.
`In one aspect of this embodiment, the properties of the gel
`are modified by Subjecting the gel to heat treatment at a
`temperature in the range of from about 100° C. to about 150
`C. Heat treatment has the effect of modifying the properties
`of the gel, such as its viscosity. The effect of the heat
`treatment on Specific polymerS is generally not predictable
`in advance, and is based on Such factors as the relative
`degree of croSS-linking. Heat treatment of a gel material can
`be employed to alter the final viscosity of the gel by either
`causing more polymer to dissolve in Solution, which tends to
`increase the Viscosity, or by reducing the molecular weight
`of the polymer, which tends to reduce the Viscosity. Thus,
`adjustments to the gel Viscosity can be easily carried out
`using this approach.
`In another embodiment, the invention features a method
`for preventing the formation of adhesions by applying the
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`4
`croSS-linked gel prepared according to the method of this
`invention to the Surface of the tissue which is exposed during
`a Surgical procedure and which is in proximity to the Site of
`the procedure.
`The composition can be advantageously applied to the
`tissue Surfaces using non-invasive means, Such as by means
`of endoscopic instruments. Minimally invasive Surgical
`techniques are leSS traumatic to the patient, more cosmeti
`cally appealing, allow faster recovery times, and have lower
`risks of infection. The alternative to endoscopic Surgery is an
`open Surgical procedure, Such as laparotomy, involving long
`incision lines and the risk of infection.
`Sufficient material should be used to Separate the tissue
`Surfaces that may potentially develop adhesions. The gels of
`this invention remain in place for at least about 7 days, but
`no more than about 30 days, which is a sufficient period of
`time to prevent the formation of the adhesions. The gels of
`this invention are bioabsorbable, and are not toxic or inju
`rious to the patient. These gels are also water-insoluble, due
`to the croSS-linking, which enables the gels to remain in the
`body without being immediately absorbed.
`In a further embodiment, a drug Substance may be incor
`porated in the gel for delivery to the tissue at the Site of the
`Surgery. Such drug Substances include, for instance,
`NSAIDS, lidocaine, and derivatives thereof, steroids,
`growth factors, cytokines, antibiotics, etc., and the like.
`Unless defined otherwise, all technical and Scientific
`terms used herein have the same meaning as commonly
`understood by one of ordinary skill in the art to which this
`invention pertains. Although any methods and materials
`Similar or equivalent to those described herein can be used
`in the practice or testing of the present invention, the
`preferred methods and materials are now described. Unless
`mentioned otherwise, the techniques employed or contem
`plated herein are Standard methodologies well known to one
`of ordinary skill in the art. The materials, methods and
`examples are illustrative only, and are not intended to be
`limiting. Other features and advantages of the invention will
`be apparent from the following detailed description, and
`from the appended claims.
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention provides a method for preparing a
`water insoluble biocompatible composition comprising
`reacting a polyanionic polysaccharide with divinyl Sulfone
`in an aqueous Solution to form a gel, neutralizing the pH of
`the Solution, and precipitating a Solid from the Solution. The
`polyanionic polysaccharide used may be Selected from the
`group consisting of hyaluronic acid, Sodium hyaluronate,
`potassium hyaluronate, magnesium hyaluronate, calcium
`hyaluronate, carboxymethylcellulose, carboxymethyl amy
`lose and a mixture of hyaluronic acid and carboxymethyl
`cellulose. In one embodiment of the invention, the Solid
`precipitated from the Solution is then rehydrated to form a
`gel. The invention further provides that the rehydrated gel
`may then be Subjected to heat treatment. In one embodiment,
`the rehydrated gel is heated to a temperature in the range
`from about 100° C. to about 150° C.
`The present invention also provides a method for prepar
`ing a water insoluble biocompatible composition comprising
`reacting a polyanionic polysaccharide with divinyl Sulfone
`in an aqueous Solution to form a gel, neutralizing the pH of
`the Solution, and precipitating a Solid from the Solution
`wherein the polyanionic polysaccharide is hyaluronic acid.
`In one embodiment, the molar ratio of divinyl sulfone:hy
`
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`

`25
`
`S
`aluronic acid is from about 0.1:1 to about 1:1. In another
`embodiment, the molar ratio of divinyl sulfone:hyaluronic
`acid is from about 0.2:1 to about 0.6:1.
`The present invention also provides for a single phase gel
`product formed by any of the methods of the invention
`herein described. The Single phase gel product of the inven
`tion may be Sterilized. The Single phase gel product of the
`invention may further comprise a drug.
`The present invention also provides for a method for the
`prevention of adhesions in a Subject comprising applying the
`gel product of the invention to a region between two tissue
`Surfaces to be separated during the healing proceSS follow
`ing Surgery. The Subject may be a human patient.
`Additionally, the Surgery being performed on the Subject
`may be one Selected from the group consisting of abdominal
`Surgery, pelvic Surgery, gynecological Surgery, Orthopedic
`Surgery, and cardiac Surgery.
`AS used herein, and unless otherwise indicated, the term
`"polyanionic polysaccharide' denotes a polysaccharide con
`taining more than one negatively charged group, e.g., car
`boxyl groups at pH values above about pH 4.0. This includes
`hyaluronic acid ("HA), any of its hyaluronate Salts,
`including, for example, Sodium hyaluronate (the Sodium
`salt), potassium hyaluronate, magnesium hyaluronate, and
`calcium hyaluronate, carboxymethylcellulose (“CMC), and
`mixtures of hyaluronic acid and carboxymethylcellulose,
`and carboxymethyl amylose.
`A “biocompatible” substance, as the term may be used
`herein, is one that has no medically unacceptable toxic or
`injurious effects on biological function.
`A “bioabsorbable” Substance is one which is maintained
`in the body in a relatively intact form for at least about 7
`days, and is then completely absorbed by the body after
`about 30 days thereafter. A bioabsorbable substance is thus
`not considered to be an “implant', which remains in the
`35
`body for more than about 30 days without decomposition. A
`bioabsorbable Substance does not have to meet the more
`Stringent is FDA requirement imposed on implants.
`A “croSS-linked polyanionic polysaccharide' is a polya
`nionic polysaccharide which has been reacted with a divinyl
`Sulfone croSS-linking agent to form a 3-dimensional network
`by covalent bonding between the divinyl sulfone and reac
`tive sites on adjacent polymers. The degree of croSS-linking
`can be measured by the amount of cross-linking agent
`consumed in the cross-linking reaction.
`The expression "preventing adhesion formation' is
`intended to encompass not only the complete elimination of
`adhesions, but the Substantial reduction in the amount or
`number of adhesions formed as compared to the amount or
`number of adhesions formed using a control Substance Such
`as Saline, or the absence of any treatment to reduce the level
`of adhesions.
`A "gel’ is a colloidal Suspension of a dispersed Solid
`phase in a continuous phase. In the context of this invention,
`the dispersed Solid phase comprises particles of a polyan
`55
`ionic polysaccharide, and the continuous phase is water. A
`“water Soluble' gel, as that term is used herein, is a gel
`which as an aqueous 1% weight/weight (“w/w”) solution of
`the cross-linked polyanionic polysaccharide gel, when
`placed in a 50 mL beaker of distilled water maintained at
`about 20°C., and allowed to stand without stirring, dissolves
`completely into a single phase within 20 minutes. A “water
`insoluble' gel is a gel which, when prepared under the
`conditions as described for a water Soluble gel, is structur
`ally intact after 20 minutes. The gels of the present invention
`are water insoluble, enabling them to function as effective
`adhesion reduction devices.
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`A solution is “neutralized” when the pH value of the
`solution is adjusted so that the final pH of the solution is
`approximately 7.0, or alternatively, in the range of from
`about 6.0 to about 8.0. The relatively alkaline solutions of
`the present invention are adjusted by lowering the pH by the
`addition of an acidic compound to the reaction mixture of
`the polyanionic polysaccharide and divinyl Sulfone.
`The anti-adhesion formulations of this invention can be
`prepared from a polyanionic polysaccharide which is croSS
`linked by reaction with divinyl sulfone. The reactants can be
`dissolved in water at a basic pH, preferably at a pH of about
`12, and the reaction is allowed to proceed until a gel is
`formed. The mode of addition of the reagents is not critical
`to the reaction.
`The amount of divinyl sulfone consumed in the cross
`linking reaction generally varies from about 0.1 moles of
`DVS up to 1.0 mole of DVS per mole of polyanionic
`polysaccharide. Lower amounts of DVS produce lightly
`cross-linked gels which tend to be more soluble, while
`higher amounts of DVS produce more tightly cross-linked
`gels which tend to be more insoluble.
`The preferred polyanionic polysaccharides are hyaluronic
`acid and carboxymethyl cellulose. Hyaluronic acid, or its
`Salts. Such as Sodium hyaluronate, is readily Soluble in water.
`HA from any of a variety of sources can be used. For
`instance, HA can be extracted from animal tissues, Such as
`rooster combs, or harvested as a product of bacterial fer
`mentation. HA can be produced in commercial quantities
`using bioprocess technology, as described for example in
`PCT Publication No. WO 86/04355.
`The product formed from the reaction of HA and DVS is
`then mixed with water, and the pH of the mixture is adjusted
`to an approximately neutral level, e.g. about 7.0, or from
`about 6.0 to about 8.0, by the addition of an acidic com
`pound to the Solution. Suitable acids include Sulfuric acid
`and hydrochloric acid.
`The gel is then precipitated with ethanol as a Solid.
`Preferably, the gel is pulverized to form a powder, which can
`be stored until it is needed for medical use. The powder can
`be easily reconstituted by rehydration, and optionally Sub
`jected to heat treatment to adjust the rheological properties
`to achieve the desired physical characteristics.
`From the above description, one skilled in the art can
`readily ascertain the essential characteristics of the present
`invention, and without departing from the Spirit and Scope of
`thereof, can make various changes and modifications of the
`invention to adapt it to various usages and conditions.
`AS one skilled in the art will appreciate, the gels of this
`invention can be made using methods which may differ in
`certain particulars from those methods exemplified herein.
`For example, when precipitating the Solid from Solution, any
`water miscible Solvent having a lower polarity than water
`may be used. Suitable Solvents include, for example,
`ethanol, isopropyl alcohol and acetone.
`The following examples of the invention are provided by
`way of illustration only, and are not intended to limit the
`invention as Set forth in the appended claims.
`EXAMPLE 1.
`200 mL of a 0.2N Sodium hydroxide solution was added
`to 8.0 grams of hyaluronic acid (19.95 mmol), and the
`mixture was stirred at room temperature until it fully dis
`solved (about 3 hours). 266 mL of divinyl sulfone (4.0
`mmol) was added to the hyaluronic acid Solution and
`Vigorously stirred for about one minute. The reaction mix
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`7
`ture was allowed to Stand at room temperature for one hour.
`The resulting gel was placed in D.I. water for 24 hours, and
`then chopped into quarters, and allowed to stand in PBS for
`an additional 24 hours. 5 mL of PBS was added to the
`Swollen gel, and the mixture was mixed under high Sheer
`conditions. The pH of the solution was then adjusted to 7.2
`with 6 N hydrochloric acid, followed by precipitation with
`absolute ethanol (3.0 L). The white precipitate was collected
`and dried under vacuum. The powder is easily rehydrated
`upon the addition of PBS and high shear mixing.
`Following this procedure, hyaluronic acid gels can be
`Synthesized with varying amounts of divinyl Sulfone and
`reconstituted at different concentrations (expressed as %
`solid per volume, where 1% equals 1 gram/100 mL) to
`obtain a desired Theological property. The results are shown
`in Tables I, II and III below for gels having DVS:HA molar
`ratios of 0.2:1, 0.3:1 and 0.6:1, respectively, before and after
`heat treatment at 121 C. for 20 minutes, where the numbers
`in parentheses indicate the values before heat treatment.
`
`15
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`TABLE I
`
`DVS:HAMole Ratio of 0.2:1
`
`Conc.
`(%)
`1.0
`1.5
`2.0
`2.5
`
`Phase
`Angle ()
`(42.9) 49.00
`(40.7) 45.00
`(36.3) 40.00
`(2.8.8) 37.00
`
`Complex
`Modulus (Pa)
`(9.3) 6.6
`(36.9) 26
`(54.4) 42
`(269) 99
`
`Yield
`Stress (Pa)
`(3.8) 1.9
`(11.6) 7.8
`(13.6) 11.7
`(68) 19.7
`
`Viscosity
`(cP)
`(3,739) 2,189
`(20,630) 13,400
`(31,900) 24,000
`(183.580) 59,100
`
`TABLE II
`
`DVS:HAMole Ratio of 0.3:1
`
`Conc.
`(%)
`1.0
`1.5
`2.0
`2.5
`
`Phase
`Angle ()
`(42.8) 44.6
`(36.9) 45.2
`(29.0) 38.9
`(31.8) 39.9
`
`Complex
`Modulus (Pa)
`(15.44) 9.66
`(32.65) 23.01
`(81.84) 61.68
`(72.84) 56.097
`
`Yield
`Stress (Pa)
`(3.9) 1.98
`(9.72) 9.66
`(21.6) 9.66
`(19.6) 17.6
`
`Viscosity
`(cP)
`(7.267) 3,103
`(16,776) 12.937
`(53,728) 40,547
`(49,495) 35.940
`
`TABLE III
`
`DVS:HAMole Ratio of 0.6:1
`
`Complex
`Modulus (Pa)
`(19.57) 13.59
`
`(38.56) 48.02
`(55.43) 60.22
`(130.43) 141.28
`
`Yield
`Stress (Pa)
`(1.99) 3.92
`
`(11.8) 17.6
`(15.6) 23.5
`(33.3) 46.9
`
`Viscosity
`(cP)
`(9,093) 8,178
`
`(26,213) 43,885
`(45,670) 55,438
`(147,150) 160,852
`
`Phase
`Conc.
`(%) Angle ()
`1.O
`(27.4)
`34.6
`(25.2)
`27.2
`(22.4)
`26.9
`(22.4)
`26.9
`
`1.5
`2.0
`2.5
`
`The Tables above show that the gels become more resis
`tant to degradation by heat as the amount of cross-linking is
`increased. The gels made using a 0.6:1 DVS:HA molar ratio
`are quite Stable to heat treatment, which directly correlates
`to the Stability of the gels under autoclave conditions.
`EXAMPLE 2
`200 mL of a 0.2N Sodium hydroxide solution was added
`to 8.0 grams of hyaluronic acid (19.95 mmol), and the
`mixture was stirred at room temperature until it fully dis
`
`25
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`US 6,521,223 B1
`
`8
`solved (about 3 hours). 396 mL of divinyl sulfone (5.99
`mmol) was added to the hyaluronic acid Solution and
`Vigorously stirred for about one minute. The reaction mix
`ture was allowed to Stand at room temperature for one hour.
`The resulting gel was placed in D.I. water for 24 hours, and
`then chopped into quarters, and allowed to stand in PBS for
`an additional 24 hours. 5 mL of PBS was added to the
`Swollen gel, and the mixture was mixed under high Sheer
`conditions. The pH of the solution was then adjusted to 7.2
`with 6 N hydrochloric acid, followed by precipitation with
`absolute ethanol (3.0 L). The white precipitate was collected
`and dried under vacuum. The powder is easily rehydrated
`upon the addition of PBS and high shear mixing.
`EXAMPLE 3
`200 mL of a 0.2N Sodium hydroxide solution was added
`to 8.0 grams of hyaluronic acid (19.95 mmol), and the
`mixture was stirred at room temperature until it fully dis
`solved (about 3 hours). 798 mL of divinyl sulfone (12.0
`mmol) was added to the hyaluronic acid Solution and
`Vigorously stirred for about one minute. The reaction mix
`ture was allowed to Stand at room temperature for one hour.
`The resulting gel was placed in D.I. water for 24 hours, and
`then chopped into quarters, and allowed to stand in PBS for
`an additional 24 hours. 5 mL of PBS was added to the
`Swollen gel, and the mixture was mixed under high Sheer
`conditions. The pH of the solution was then adjusted to 7.2
`with 6 N hydrochloric acid, followed by precipitation with
`absolute ethanol (3.0 L). The white precipitate was collected
`and dried under vacuum. The powder is easily rehydrated
`upon the addition of PBS and high shear mixing.
`EXAMPLE 4
`Gels prepared from the rehydrated powder as shown in
`Example 1 were tested for efficacy in preventing post
`operative adhesions in a rat cecal abrasion model as
`described in Burns et al., Eur. J. Surg. 1997, Suppl. 577,
`40–48. Ten (10) rats were used in each study. Group 1 used
`a gel having a concentration of 1.5% of active ingredient,
`and Group 2 used a gel having a concentration of 2.5% of
`active ingredient. The results are shown in Table IV below
`listed as the percentage of each group with adhesions of
`grade greater than 2; the average incidence of adhesions,
`plus or minus the Standard error of the mean; and the
`percentage of each group with no adhesions.
`
`TABLE IV
`
`Group
`
`% w/ adh. 2 2
`
`Avg. Inc. : SEM % w/ no adh.
`
`Control
`Group 1
`Group 2
`
`8O
`3O
`2O
`
`1.5 + 0.4
`O.6
`O.3
`O.2 - 0.1
`
`2O
`70
`8O
`
`What is claimed is:
`1. A method for preparing a water insoluble biocompat
`ible composition comprising
`reacting a polyanionic polysaccharide with divinyl Sul
`fone in an aqueous Solution to form a gel, neutralizing
`the pH of the Solution, and precipitating a Solid from
`the neutral Solution.
`2. The method of claim 1, wherein the polyanionic
`polysaccharide is Selected from the group consisting of
`hyaluronic acid, Sodium hyaluronate, potassium
`hyaluronate, magnesium hyaluronate, calcium hyaluronate,
`carboxymethylcellulose, carboxymethyl amylose and a mix
`ture of hyaluronic acid and carboxymethylcellulose.
`
`

`

`US 6,521,223 B1
`
`9
`3. The method of claim 1 or 2, wherein the solid precipi
`tated from the Solution is then rehydrated to form a gel.
`4. The method of claim 3, wherein the rehydrated gel is
`then Subjected to heat treatment.
`5. The method of claim 4, wherein the rehydrated gel is
`heated to a temperature in the range from about 100° C. to
`about 150° C.
`6. The method of claim 2, wherein the polyanionic
`polysaccharide is hyaluronic acid.
`7. The method of claim 6, wherein the molar ratio of
`divinyl sulfone: hyaluronic acid is from about 0.1:1 to about
`1:1.
`8. The method of claim 7, wherein the molar ratio of
`divinyl sulfone: hyaluronic acid is from about 0.2:1 to about
`O.6:1.
`9. A single phase gel product formed by the method of
`claim 2, 6, 7 or 8.
`
`10
`10. The gel product of claim 9, which is sterilized.
`11. The gel product of claim 9, which further comprises
`a drug.
`12. The gel product of claim 10, which further comprises
`a drug.
`13. A method for the inhibition of adhesions in a subject
`comprising applying the gel product of claim 9 to a region
`between two tissue Surfaces to be separated during the
`healing process following Surgery.
`14. The method of