(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`1111111111111111 IIIIII IIIII 11111 IIIII IIII I II Ill lllll lllll lllll lllll lllll llll 1111111111111111111
`
`( 43) International Publication Date
`1 December 2005 (01.12.2005)
`
`PCT
`
`(10) International Publication Number
`WO 2005/112888 A2
`
`(51) International Patent Classification 7: A61K 9/06, 47/36
`
`(21) International Application Number:
`PCT/US2005/0l 7641
`
`(22) International Filing Date:
`
`19 May 2005 (19.05.2005)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`(30) Priority Data:
`60/572,944
`
`English
`
`English
`
`20 May 2004 (20.05.2004) US
`
`(71) Applicant (for all designated States except US): MEN(cid:173)
`TOR CORPORATION [US/US]; 201 Mentor Drive,
`Santa Barbara, California 93111 (US).
`
`(72) Inventor; and
`(75) Inventor/Applicant (for US only): WANG, Wei [CN/GB];
`85 Currievale Drive, Edinburgh EH14 5RW (GB).
`
`(74) Agent: KAYTOR, PH.D., Elizabeth, N.; Fish & Richard(cid:173)
`son P.C., 3300 Dain Rauscher Plaza, 60 South 6th Street,
`Minneapolis, MN 55402 (US).
`
`(81) Designated States (unless otherwise indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, Fl,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE,
`KG, KM, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA,
`MD, MG, MK, MN, MW, MX, MZ, NA, NG, NI, NO, NZ,
`OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL,
`SM, SY, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC,
`VN, YU, ZA, ZM, ZW.
`
`(84) Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
`ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl,
`
`[Continued on next page}
`-------------------------------------------
`(54) Title: METHODS FOR MAKING INJECTABLE POLYMER HYDROGELS
`
`(57) Abstract: Methods
`for preparing
`injectable hydrogels, particularly hydrogels
`containing hyaluronan, are described herein.
`Also described are hydrogel products made
`by the methods provided herein.
`
`10 I
`
`13
`
`40~
`
`50
`
`iiiiiiii
`
`-
`
`-iiiiiiii
`-----
`iiiiiiii --
`iiiiiiii ----
`
`!!!!!!!!
`iiiiiiii
`
`ALL 2024
`PROLLENIUM V. ALLERGAN
`IPR2019-01505 et al.
`
`

`

`WO 2005/112888 A2
`
`1111111111111111 IIIIII IIIII 11111 IIIII IIII I II Ill lllll lllll lllll lllll lllll llll 1111111111111111111
`
`FR, GB, GR, HU, IE, IS, IT, LT, LU, MC, NL, PL, PT, RO,
`SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG).
`
`Declarations under Rule 4.17:
`as to applicant's entitlement to apply for and be granted
`a patent (Rule 4.17(ii)) for the following designations AE,
`AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ,
`CA, CH, CN, CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE,
`EG, ES, Fl, GB, GD, GE, GH, GM, HR, HU, JD, IL, IN, JS,
`JP, KE, KG, KM, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV,
`MA, MD, MG, MK, MN, MW, MX, MZ, NA, NG, NJ, NO,
`NZ, OM, PG, PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL,
`SM, SY, Tl, TM, TN, TR, TT, TZ, VA, VG, UZ, VC, VN, YU,
`ZA, ZM, ZW, ARJPO patent (BW, GH, GM, KE, LS, MW,
`MZ, NA, SD, SL, SZ, TZ, VG, ZM, ZW), Eurasian patent
`
`(AM, AZ, BY, KG, KZ, MD, RU, Tl, TM), European patent
`(AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl, FR, GB,
`GR, HU, IE, JS, IT, LT, LU, MC, NL, PL, PT, RO, SE, SJ,
`SK, TR), OAPJ patent (BF, BJ, CF, CG, Cl, CM, GA, GN,
`GQ, GW, ML, MR, NE, SN, TD, TG)
`as to the applicant's entitlement to claim the priority of the
`earlier application ( Rule 4.17( iii)) for all designations
`
`Published:
`without international search report and to be republished
`upon receipt of that report
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`Methods For Making Injectable Polymer Hydrogels
`
`CROSS-REFERENCE TO RELATED APPLICATIONS
`
`This application claims priority from U.S. Provisional Application Serial No.
`
`60/572,944, filed May 20, 2004.
`
`5
`
`This document relates to processes for preparing injectable polymer hydrogels.
`
`TECHNICAL FIELD
`
`BACKGROUND
`
`10
`
`Injectable gels often are used for soft tissue augmentation. For example,
`
`injectable gels and be used as facial fillers for wrinkles and folds, lip enhancement and
`
`body contour correction, as well as in arthritis prostheses. Biocompatible polymers such
`
`as alginate acid, chitosan, polyacrylamide, and hyaluronan (hyaluronic acid, HA) have
`
`been used to prepare injectable gels for various applications. Injectable gels often are
`
`15
`
`prepared by covalently crosslinking polymers in solution to form a rubber-like network
`
`structure, which is then mechanically homogenized to form injectable microparticles.
`
`Typically, each operation of these multi-step processes is separated, involving various
`
`pieces of equipment and product transfers.
`
`20
`
`SUMMARY
`
`This document provides simple, rapid and low cost processes for preparation of
`
`injectable hydrogels (e.g., injectable hyaluronan hydrogels). The processes can include
`
`the steps of crosslinking one or more polymers and washing the subsequently formed gel,
`
`followed by purification and homogenization to produce an injectable hydrogel. The
`
`25
`
`processes can be carried out in a single reaction vessel as continuous processes, and thus
`
`can result in elimination of the need to carry out any product transfer. In addition, no
`
`organic solvent or drying step is required. The processes also can provide an easily
`
`controllable and repeatable operation for very quick and low cost production of injectable
`
`gels, with different polymer concentrations and different particle sizes for various
`
`30
`
`applications. One production cycle may take as little as three days.
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`Also provided herein are hydrogels made by the processes described herein. The
`
`hydrogels can have a high degree of cross linking but a very deformable soft structure and
`
`superior biostability. As such, the gels can be used in soft tissue augmentation and
`
`medical prostheses. The swelling degree of the gels in PBS can be about 4000-5000%.
`
`5
`
`The gels can have particle sizes on the order of 500 micrometers, and can be easily
`
`injected through G30 ½ needles (inner diameter 150 micrometer). Injectable hyaluronan
`
`gels produced by the processes provided herein can have superior viscoelasticity. The
`
`elastic modulus G' can be much higher than the viscous modulus G", the complex
`
`viscosity can be from about 2 x 104 Pa.s to 35 Pa.s, and the phase angle delta (8) can be
`
`10
`
`very low (around 10), over a range of 0.01-10 Hz. In addition, the injectable hyaluronan
`
`gels prepared by the processes provided herein can exhibit a large degree of biostability to
`
`hyaluronidase as compared with injectable hyaluronan gels such as Restylane® (Medicis
`
`Aesthetics, Inc., Scottsdale, AZ) and Hylaform® (!named Aesthetics, Santa Barbara, CA).
`
`In one aspect, this document features a process for the preparation of an injectable
`
`15
`
`hydrogel. The process can include the steps of crosslinking one or more polymers to
`
`form a gel, washing the gel, purifying the gel, and homogenizing the gel to produce the
`
`hydrogel, wherein the process is carried out in a single reaction vessel as a continuous
`
`process. The polymer can have one or more reactive groups selected from hydroxyl
`
`groups, carboxyl groups and amine groups. The polymer can be a polysaccharide (e.g.,
`
`20
`
`hyaluronic acid, chitosan, alginate acid, starch, dextran, or salts or water soluble
`
`derivatives thereof), a protein or a synthetic polymer, such as poly(acrylic acid) or
`
`poly(vinyl alcohol).
`
`The crosslinking reaction can be carried out with a bi- or polyfunctional
`
`crosslinking agent, such as an epoxide, aldehyde, polyaziridyl or divinyl sulphone. The
`
`25
`
`crosslinking agent can be 1,4-butanediol diglycidyl ether (BDDE). The process can be
`
`carried out at a pH of 11 or higher. The crosslinking reaction can be carried out at a
`
`temperature of 37-60°C (e.g., 50°C), for at least 4 hours.
`
`The process can further include preparing a solution of the polymer in NaOH and
`
`adding the crosslinking agent with stirring. The process can further include cutting the
`
`30
`
`formed gel into pieces using one or more impellers in the reaction vessel, and washing
`
`and purifying the gel with one or more changes of PBS solution. The washing and
`
`2
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`purifying process can be carried out over 2 to 3 days with at least six changes of PBS
`
`solution.
`
`The polymer can be hyaluronic acid. The process can be carried out with a
`
`solution ofhyaluronic acid in 0.25 M NaOH, at a concentration up to 20% by weight.
`
`5
`
`The initial concentration of hyaluronic acid can be 11-14% by weight. The molar ratio of
`
`crosslinking agent to polymer can be 0.5-2.4.
`
`In another aspect, this document features an injectable hydrogel produced using a
`
`process described herein. In addition, this document features a biomaterial containing an
`
`injectable hydro gel as described herein. The biomaterial can be in the form of a sheet,
`
`1 o
`
`bead, sponge, or formed implant.
`
`Unless otherwise defined, 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 methods and materials similar or equivalent to those
`
`described herein can be used to practice the invention, suitable methods and materials are
`
`15
`
`described below. All publications, patent applications, patents, and other references
`
`mentioned herein are incorporated by reference in their entirety. In case of conflict, the
`
`present specification, including definitions, will control. In addition, the materials,
`
`methods, and examples are illustrative only and not intended to be limiting.
`
`The details of one or more embodiments of the invention are set forth in the
`
`20
`
`accompanying drawings and the description below. Other features, objects, and
`
`advantages of the invention will be apparent from the description and drawings, and from
`
`the claims.
`
`DESCRIPTION OF DRAWINGS
`
`25
`
`FIG. 1 is a drawing a stirrer vessel suitable for use in the continuous processes for
`
`preparing injectable hyaluronan gels as described herein.
`
`FIG. 2 is a graphical representation of the rheological data described in Example 1.
`
`DETAILED DESCRIPTION
`
`30
`
`Hyaluronan is a naturally occurring polysaccharide containing alternating N(cid:173)
`
`acetyl-D-glucosamine and D-glucuronic acid monosaccharide units. As used herein
`
`3
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`"hyaluronan" refers to hyaluronic acid and its hyaluronate salts, including, but not limited
`
`to, sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate and calcium
`
`hyaluronate.
`
`The methods provided herein can include the use of a vessel such as stirrer vessel
`
`5
`
`10 (Figure 1), which can be equipped with motor 13,jacket 16, and stirrers/impellers 20.
`
`Water, NaOH, and hyaluronan can be added into the vessel and stirred. The initial
`
`hyaluronan solution concentration, typically up to 20%, can be important in determining
`
`the properties of the final gel. An initial hyaluronan concentration of 11-14% (e.g., 11 %,
`
`11.5%, 12%, 12.5%, 13%, 13.5%, or 14%) by weight can be particularly useful. If the
`
`10
`
`hyaluronan solution concentration is lower (e.g., 8% or less), only a weak hydrogel may
`
`be obtained. Higher initial hyaluronan concentrations may result in hydrogels with too
`
`large a degree of crosslinking, which in tum can be difficult to homogenize to form
`
`injectable gels and which also may have poor viscoelasticity.
`
`NaOH at a concentration of about 0.2 M to about 0.3 M (e.g., 0.2 M, 0.21 M, 0.22
`
`15
`
`M, 0.23 M, 0.24 M, 0.25 M, 0.26 M, 0.27 M, 0.28 M, 0.29 M, or 0.3 M) can be useful for
`
`dissolving hyaluronan quickly. Further, the inventors have found that crosslinking
`
`reactionscanreadilyproceedatapHhigherthan 11 (e.g., 11, 11.2, 11.4, 11.6, 11.8, 12,
`
`or higher than 12). In a typical process, 3.5 to 4 hours may be required to dissolve
`
`hyaluronan in 0.25 M NaOH at room temperature to produce a homogeneous solution,
`
`20
`
`even at a concentration up to 20%.
`
`A crosslinking agent such as, for example, 1,4-butanediol diglycidyl ether
`
`(BDDE) can be added and the hyaluronan solution can be kept at a temperature between
`
`about 37°C and about 60°C (e.g., 37°C, 40°C, 45°C, 50°C, 55°C, or 60°C) for 3 to 5
`
`hours (e.g., 3, 3.5, 4, 4.5, or 5 hours). A temperature of 50°C and a reaction time of 4
`
`25
`
`hours can be particularly useful. At room temperature, no strong crosslinking is achieved,
`
`and at temperatures over 65°C the hyaluronan can degrade quickly. Shorter times such as
`
`2 hours may not give strongly crosslinked gels, and longer times do not appear to provide
`
`gels with improved properties, but may result in degradation of the hyaluronan. The
`
`molar ratio of crosslinking agent, e.g., BDDE, to hyaluronan also can be an important
`
`30
`
`parameter. A useful molar ratio can be in the range of 1.4: 1 to 2.0: 1 (e.g., 1.4: 1, 1.5: 1,
`
`1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2.0:1). These conditions can result in injectable gels having
`
`4
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`hyaluronan content of 19-23 mg/g and having very good viscoelasticity, injectability and
`
`biostability.
`
`A crosslinking reaction can be stopped by lowering the temperature and PBS can
`
`be added to the formed hydrogel. The formed hydrogel can be exhaustively washed and
`
`5
`
`purified directly with PBS (pH= 7.4) under stirring at room temperature in the stirrer
`
`vessel to remove residual crosslinking agent and unreacted hyaluronan from the gel. The
`
`gel can then be homogenized into small, injectable pieces by operating the impellers at a
`
`high speed. The impellers can have sharpened blades and can be moved in a vertical
`
`plane, which can facilitate homogenization. The distance between the outer edge of the
`
`10
`
`impeller blades and the inner wall of the vessel should be kept to a minimum. Particle
`
`size typically decreases and deformability increases with increasing stirring time. The
`
`particles can be washed with one or more (e.g., one, two, three, four, five, six, seven,
`
`eight, nine, or ten) changes of PBS over a period of 1 to 4 days (e.g., 1, 1.5, 2, 2.5, 3, 3.5,
`
`or 4 days) via a well fitted filter and valve in the vessel (e.g., filter 30 and valve 40 on the
`
`15
`
`bottom of stirrer vessel 10, shown in Figure 1). Washing in PBS for about 2.5 days, with
`
`about 6 changes of fresh PBS in the stirrer vessel, can be useful to obtain a gel that is pure
`
`and is hydrated to an equilibrium weight or volume, such that further washing with PBS
`
`does not increase the weight or volume of the obtained gel. Sah1rated gels stabilized with
`
`PBS typically are highly swollen. Finally, a purified gel can be homogenized by high
`
`20
`
`· speed stirring of the impellers. The gel can be removed from the vessel (e.g., through
`
`valve 50 shown in Figure 1) and packaged (e.g., in vials or syringes) before or after
`
`sterilization (e.g., by autoclaving).
`
`Using the processes described herein, injectable hyaluronan gels with hyaluronan
`
`concentrations of, for example, from 1.0% to 3.5% (e.g., 1.0%, 1. 1 %, 1.2%, 1.25%, 1.3%,
`
`25
`
`1.4%, 1.5%, 1.6%, 1.7%, 1.75%, 1.8%, 1.9%, 2.0%, 2.1 %, 2.2%, 2.25%, 2.3%, 2.4%,
`
`2.5%, 2.6%, 2.7%, 2.75%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.25%, 3.3%, 3.4%, or 3.5%)
`
`can be prepared. Gels with lower concentrations ofhyaluronan may not be sufficiently
`
`stable. Higher concentrations of hyaluronan can offer good stability, but injectability
`
`through G30 needles may be poor, although such gels can be injected through G27
`
`30
`
`needles having an inner diameter of 200 micrometers. Concentrations in the range of 1.9-
`
`5
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`2.3%, i.e., 19-23 mg/g gel, can have sufficient stability and good injectability through a
`
`G30 needle.
`
`If the initial concentrations ofhyaluronan and crosslinking agent are the same
`
`from preparation to preparation, final gels (after washing and purification) of consistent
`
`5
`
`hyaluronan content and swelling degree can be obtained. Thus, the hyaluronan
`
`concentration and degree of swelling of the final injectable gel can be controlled by
`
`means of controlling initial hyaluronan solution concentration and the molar ratio of
`
`crosslinking agent to hyaluronan. In addition, the viscoelasticity, injectability, and
`
`biostability of the final injectable gels can be controlled by crosslinking level and
`
`10
`
`crosslinking density, which are mainly controlled by initial hyaluronan concentration and
`
`molar ratio of crosslinking agent to hyaluronan.
`
`Viscoelasticity can be measured using, for example, a rheometer at room
`
`temperature. Injectability can be tested using G 30 ½ needles, and biostability can be
`
`evaluated by incubation in a hyaluronidase PBS solution at 3 7°C for 24 hours, followed
`
`15
`
`by analysis of degraded glucuronic acid weight using a carbazole assay (Bitter and Muir,
`
`Analytical Biochemistry, 1962, 4:330). For example, the inventors have found that
`
`digestion in PBS solution with 22 units ofhyaluronidase (1 gram injectable gel in 5 ml) at
`
`3 7°C for 24 hours resulted in a weight loss of about 10 percent.
`
`Injectable gels prepared by the processes described herein may be further
`
`20
`
`processed to form a variety ofbiomaterials such as sheets, beads, sponges, and formed
`
`implants. The gels can be used in a variety of pharmaceutical, medical (including
`
`surgical) and cosmetic applications. Thus, they may for example be useful in promoting
`
`wound healing, e.g., as a dermal wound dressing. They may also be useful in preventing
`
`adhesion formation e.g., preventing tissue growth between organs following surgery. The
`
`25
`
`crosslinked gels may also find application in the ophthalmic field, e.g., for vitreous fluid
`
`replacement, as corneal shields for delivery of drugs to the eye, or as lenticules.
`
`The crosslinked gels also may be useful in surgery, for example as solid implants
`
`for hard tissue augmentation e.g., repair or replacement of cartilage or bone, or for soft
`
`tissue augmentation, as breast implants, or as coating for implants intended for long term
`
`30
`
`use in the body, such as breast implants, catheters, cannulae, bone prostheses, cartilage
`
`replacements, mini pumps and other drug delivery devices, artificial organs and blood
`
`6
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`vessels, meshes for tissue reinforcement, etc. They may also be used as joint lubricants in
`
`the treatment of arthritis.
`
`A further use for the injectable gels provided herein can be in the delivery of
`
`therapeutically active agents including in any of the aforementioned applications.
`
`5
`
`Therapeutically active agents may be chemotherapeutic agents or biologically active
`
`factors (e.g., cytokines) and include anti-inflammatory agents, antibiotics, analgesics,
`
`anaesthetics, e.g., lidocaine, wound healing promoters, cytostatic agents,
`
`immunostimulants, immunosuppressants, DNA and antivirals. Such therapeutically
`
`active factors may be bound, either physically or chemically, to the crosslinked gel using
`
`1 o
`
`methods well known in the art.
`
`The present invention will now be illustrated by the following examples, which
`
`are not intended to limit the invention as set forth in the claims.
`
`15
`
`EXAMPLES
`
`Example 1
`
`This example illustrates a procedure for making an injectable hyaluronan gel.
`
`
`1.1 gram hyaluronan (MW: 2.3 x 106) was dissolved in 10 ml 0.25 M NaOH
`
`aqueous solution in a stirrer vessel at room temperature for 4 hours. 1.0 ml BDDE was
`
`added to the hyaluronan solution under stirring, and then the solution was kept at 50°C for
`
`20
`
`four hours. Subsequently 500 ml PBS was added to the stirrer vessel to wash and hydrate
`
`the obtained gel. After 2.5 day washing with six changes of fresh PBS, the gel was
`
`filtered to remove free PBS and stirred into injectable gel by impeller stirring for four
`
`hours. The yield of gel was 55 grams, which was then used to fill syringes for
`
`autoclaving. Figure 2 provides the rheological properties of prepared injectable gels. The
`
`25
`
`percentage degraded by hyaluronidase was less than 10%.
`
`Example 2
`
`This example illustrates the effect of initial hyaluronan solution concentration on
`
`rheology and biostability of the injectable gel.
`
`30
`
`Injectable gels were prepared with four hours crosslinking with BDDE at 50°C
`
`and BODE/HA molar ratio of 1.4: 1 and four hours homogenization, but using different
`
`7
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`initial hyaluronan solution concentrations. The properties of the gels produced are shown
`
`in Table 1.
`
`It is clear that the concentration, viscoelasticity, and biostability of the gels was
`
`increased with increasing initial HA solution concentration, due to an increase in the level
`
`5
`
`of crosslinking. An initial hyaluronan concentration of about 12% was found to be
`
`optimum, resulting in a gel with good viscoelasticity and biostability. Of course, other
`
`concentrations can be utilized according to the desired properties of the final gel.
`
`1 o
`
`Table 1
`Effect of Initial Hyaluronan Concentration on Gel Concentration, Rheology and
`Biostability
`
`Initial HA solution
`concentration (g/ml)
`
`Final gel
`concentration( mg/ g)
`
`Viscoelasticity(0.1-10 Hz)
`G' (Pa)
`G"(Pa)
`Phase angle
`Complex viscosity 11*(Pa.s)
`
`Biostability (degraded
`percent in hyaluronidase at
`37°C for 24 hours)
`
`8%
`
`10%
`
`12%
`
`14%
`
`11
`
`16
`
`22
`
`34
`
`75-113
`14-15
`10-8
`95-2
`
`537-725
`74-76
`8-6
`950-12
`
`1324-1976
`283-371
`12-10
`1326-32
`
`Too dry to
`measure
`
`90
`
`71
`
`12
`
`3.5
`
`Example 3
`
`This example illustrates the effect of molar ratio ofBDDE/hyaluronan on
`
`15
`
`rheology and biostability of the injectable gel.
`
`Injectable gels were prepared with four hour crosslinking with BDDE at 50°C and
`
`10% initial hyaluronan concentration and four hour homogenization, but using different
`
`BDDE/HA molar ratios. The properties of the gels are given in Table 2.
`
`The higher the molar ratio ofBDDE/hyaluronan, the higher the gel concentration
`
`20
`
`and viscoelasticity as well as the biostability, meaning that the degree of swelling
`
`decreased because of increasing crosslinking level and density.
`
`8
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`Table 2
`Effect of BDDEIHA molar ratio on Gel Concentration, Rheology and Biostability
`
`BDDE/HA molar ratio
`
`1.0:1
`
`1.4:1
`
`2.0:1
`
`2.4:1
`
`Final gel
`concentration(mg/g)
`
`Viscoelasticity(0.1-10 Hz)
`G' (Pa)
`G"(Pa)
`Phase angle
`Complex viscosity 11*(Pa.s)
`
`Biostability ( degraded
`percent in hyaluronidase at
`37°C for 24 hours)
`
`14
`
`16
`
`20
`
`75-113
`14-15
`10-8
`95-2
`
`537-725
`74-76
`8-6
`950-12
`
`958-1254
`153-166
`9-5
`1212-20
`
`23
`
`-
`
`81
`
`71
`
`29
`
`23
`
`Example 4
`
`5
`
`This example illustrates the effect of crosslinking time on rheology and
`
`biostability of the injectable gel.
`
`Injectable gels were prepared by crosslinking with BDDE at 50°C and with 10%
`
`initial hyaluronan concentration, a BDDE/hyaluronan molar ratio of 1.0: 1 and four hour
`
`homogenization, but using differing crosslinking times. The properties of the gels are
`
`10
`
`listed in Table 3.
`
`Crosslinking level and density was increased with crosslinking reaction time. A
`
`four hour crosslinking reaction appeared to be optimum. Increasing this to five or six
`
`hours did not significantly change the properties of the resultant gels.
`
`9
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`Table 3
`Effect of Cross/inking Time on Gel Concentration, Rheology and Biostability
`
`Crosslinking time (hour)
`
`Final gel
`concentration(mg/ g)
`
`Viscoelasticity (0.1-10 Hz)
`G' (Pa)
`G"(Pa)
`Phase angle
`Complex viscosity ri*(Pa.s)
`
`2
`
`10
`
`4
`
`14
`
`5
`
`16
`
`6
`
`17
`
`45-85
`7-8
`8-6
`56-2
`
`264-418
`52-50
`11-7
`337-7
`
`253-445
`59-66
`13-8
`326-7
`
`392-608
`70-91
`10-9
`498-10
`
`Biostability ( degraded
`percent in hyaluronidase at
`3 7°C for 24 hours)
`
`Totally
`degraded
`
`81
`
`79
`
`76
`
`Example 5
`
`5
`
`This example illustrates the effect of stirring time on particle size of the injectable
`
`gel.
`
`The gel prepared in Example 1 was homogenized for different times. The final
`
`particle size is shown in Table 4. A four hour homogenization was sufficient to provide
`
`good injectability through a G30 needle. The particle size was about 500 µm or lower.
`
`10
`
`Table 4
`Effect of Homogenization Time on Particle Size and lnjectability
`
`Time (hours)
`
`Particle Size (µm)
`
`2
`
`610
`
`3
`
`550
`
`4
`
`510
`
`Inj ectability
`
`Easy pass through G27 needle
`
`Easy pass through
`G30 needle
`
`15
`
`This example illustrates the effect of crosslinking level on homogenization and
`
`Example 6
`
`particle size.
`
`10
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`Gels were prepared at different initial HA concentrations and molar ratios of
`
`BDDE to hyaluronan. These were homogenized under the same conditions for four hours
`
`with a stirrer, with the resultant particle sizes being shown in Table 5. The lower the
`
`crosslinking level, the softer the gel obtained with resultant easier homogenization and
`
`5
`
`smaller particle size.
`
`Table 5
`Effect of Cross/inking Level on Homogenization and Particle Size
`
`Initial HA concentration(%)
`
`Molar ratio ofBDDE/HA
`
`Crosslin.king time (hour)
`
`Particle Size (µm)
`
`10
`
`1.4:1
`
`2
`
`313
`
`10
`
`1.4: 1
`
`4
`
`501
`
`Injectability through G30 ½
`needle
`
`Too easy
`
`Easy
`
`10
`
`2.0:1
`
`4
`
`520
`
`Little
`harder
`
`11.5
`
`2.4:1
`
`4
`
`603
`
`Hard
`
`10
`
`Example 7
`
`This example compares the properties of the gels made according to the processes
`
`described herein with commercial hyaluronan gels. The injectable gel prepared in
`
`Example 1 was analyzed and the properties evaluated and compared with Restylane® and
`
`Hylaform®, commercial soft tissue augmentation products. The results are shown in
`
`15
`
`Table 6.
`
`11
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`Table 6
`Property Comparison of Injectable Gel with Restylane and Hylaform
`
`Injectable gel
`
`Gel prepared as
`described herein
`
`Restylane®
`
`Hylaform®
`
`Gel concentration (mg/g)
`
`21
`
`20
`
`-
`
`Viscoelasticity
`(over the range of0.1-10 Hz)
`G' (Pa)
`G" (Pa)
`Phase angle
`Complex viscosity 11*(Pa.s)
`
`1559-2198
`274-384
`9-10
`2519-35
`
`666-1042
`132-183
`11-10
`1080-20
`
`114-173
`23-22
`11-8
`185-3
`
`Particle Size(µm)
`
`500
`
`450
`
`589
`
`Biostability ( degraded
`percent in hyaluronidase at
`37 °c for 24 hours)
`
`About 10%
`
`Over 90%
`
`Over60%
`
`5
`
`OTHER EMBODIMENTS
`
`It is to be understood that while the invention has been described in conjunction
`
`with the detailed description thereof, the foregoing description is intended to illustrate and
`
`not limit the scope of the invention, which is defined by the scope of the appended claims.
`
`Other aspects, advantages, and modifications are within the scope of the following
`
`10
`
`claims.
`
`12
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`WHAT IS CLAIMED IS:
`
`1. A process for the preparation of an injectable hydrogel, the process comprising the
`
`steps of crosslinking one or more polymers to form a gel, washing the gel, purifying
`
`the gel, and homogenizing the gel to produce the hydrogel, wherein the process is
`
`carried out in a single reaction vessel as a continuous process.
`
`2. A process as claimed in claim 1, wherein the polymer has one or more reactive groups
`
`selected from hydroxyl groups, carboxyl groups and amine groups.
`
`3. A process as claimed in claim 2, wherein the polymer is a polysaccharide, a protein,
`
`or a synthetic polymer selected from the group consisting of poly(acrylic acid) and
`
`poly(vinyl alcohol).
`
`4. A process as claimed in claim 3, wherein the polysaccharide is hyaluronic acid,
`
`chitosan, alginate acid, starch, dextran, or salts or water soluble derivatives thereof.
`
`5. A process as claimed in any one of claims 1 to 4, wherein the crosslinking reaction is
`
`carried out with a bi- or polyfunctional crosslinking agent.
`
`6. A process as claimed in claim 5, wherein the crosslinking agent is an epoxide,
`
`aldehyde, polyaziridyl or divinyl sulphone.
`
`7. A process as claimed in claim 5, wherein the crosslinking agent is 1,4-butanediol
`
`diglycidyl ether (BDDE).
`
`8. A process as claimed in any one of claims 1 to 7, which is carried out at a pH of 11 or
`
`higher.
`
`9. A process as claimed in any one of claims 1 to 8, wherein the crosslinking reaction is
`
`carried out at a temperature of 3 7-60°C, for at least 4 hours.
`
`10. A process as claimed in claim 9, wherein the crosslinking reaction is carried out at a
`
`temperature of 50°C.
`
`13
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`11. A process as claimed in any one of claims 8 to 10, wherein a solution of the polymer
`
`in NaOH is first prepared, to which is added the crosslinking agent, with stirring.
`
`12. A process as claimed in any one of claims 1 to 11, wherein the formed gel is
`
`subsequently cut into pieces using one or more impellers in the reaction vessel and is
`
`then washed and purified by means of one or more changes of PBS solution.
`
`13. A process as claimed in claim 12, wherein the washing and purification process is
`
`carried out over 2 to 3 days with at least six changes of PBS solution.
`
`14. A process as claimed in any one of claims 1 to 13, wherein the polymer is hyaluronic
`
`acid.
`
`15. A process as claimed in claim 14, wherein the process is carried out with a solution of
`
`hyaluronic acid in 0.25M NaOH, at a concentration up to 20%, by weight.
`
`16. A process as claimed in claim 15, wherein the initial concentration ofhyaluronic acid
`
`is 11-14%, by weight.
`
`17. A process as claimed in any one of claims 1 to 16, wherein the molar ratio of
`
`crosslinking agent to polymer is 0.5-2.4.
`
`18. An injectable hydrogel produced by a process as defined in any one of claims 1 to 17.
`
`19. A biomaterial comprising an injectable hydrogel as claimed in claim 18.
`
`20. A biomaterial as claimed in claim 19 in the form of a sheet, bead, sponge, or formed
`
`implant.
`
`14
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`1/2
`
`Figure 1
`
`10
`
`I
`
`13
`
`20
`
`40~
`
`50
`
`

`

`WO 2005/112888
`
`PCT/0S2005/017641
`
`2/2
`
`Figure 2
`
`WW-72
`
`..... ca
`a. ....
`,,
`C ca
`(!)
`
`(!)
`
`~
`
`10000..-----~---~----~---~-----, 90
`~ G'
`0--£1 G"
`,;;----,;; 6
`
`80
`
`70
`
`1000
`
`..... e...
`
`c,o
`
`60
`
`50
`
`40
`
`30
`
`90
`
`10
`100 .___ _ _ _ ......_~--.-...o.-~--....__~-~~-~-............... 0
`0.001
`0.01
`0.1
`1
`10
`100
`
`Frequency [Hz]
`
`